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OVER-EXPRESSION, PURIFICATION, CHARACTERIZATION AND
CRYSTALLIZATION OF RECOMBINANT SNAPC VARIANTS

presented by

Andrej Hanzlowsky

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OVER-EXPRESSION, PURIFICATION, CHARACTERIZATION AND
CRYSTALLIZATION OF RECOMBINANT SN APc VARIANTS

By

Andrej Hanzlowsky

A DISSERTATION

Submitted to
Michigan State University
In partial fulfillment of the requirements
For the degree of

DOCTOR OF PHILOSOPHY

Department of Chemistry

2006

ABSTRACT

OVER-EXPRESSION, PURIFICATION, CHARACTERIZATION AND
CRYSTALLIZATION OF RECOMBINANT SNAPC VARIANTS

By

Andrej Hanzlowsky

In humans, the small nuclear RNA (snRN A) genes are transcribed by either RNA
polymerase II or III. Different protein-protein and protein-DNA interactions are
responsible for polymerase specificity and a large number of transcription factors are
involved in this process. The small nuclear RNA activating protein complex (SNAPC) is
required for transcription initiation for both classes of snRNA genes. SNAPC is a five
subunit complex responsible for recognition of the proximal sequence element (PSE) on
the DNA and interacts with multiple transcription factors that guide the transcription
process. Co-expression of SNAPC subunits in baculovirus resulted in small quantities of
material that is active for both the DNA binding and transcription. On the other hand,
expression of the subunits in bacteria and reconstitution of the complex resulted in
material capable of DNA binding, but not capable of transcription initiation.

Here we present a method for efficient expression and purification of a mini
SNAPC (mSNAPc) complex, composed of SNAPl90(l—505), SNAPSO, SNAP43, and
SNAP19 in bacteria. The complex was co-expressed in E. coli and was active for both
DNA binding and transcription initiation from snRNA genes. Furthermore, the amount of
recovered material is greatly increased compared to any other previously described

method.

The larger amounts and increased purity of the complex obtained by this new
approach enabled structural studies and analysis of zinc content in mSNAPc. We
determined that the SNAPSO subunit of the complex contains a zinc finger domain that is
involved in DNA binding of the complex. Crystallization conditions were identified,
yielding protein crystals that diffracted both electrons in a transmission electron
microscope and x-rays.

Increased production of this complex is a step forward in understanding how the

SNAPC recognizes DNA, and how it interacts with a multitude of transcription factors.

ACKNOWLEDGMENTS

I sincerely thank my advisor, Prof. James H. Geiger for his guidance and support
throughout the research. I would like to thank our collaborator Prof. Bill Henry for
providing DNA materials and for his countless discussions regarding this research. I
would like to thank my co-workers Blanka, Justin, Suzan, Xiaofei and Fang for their
direct involvement in this project. I would also like to thank all present and past members
of Dr. Geiger’s and Dr. Henry’s research groups for their help and advice. I would like to
thank Dr. Kathleen Foley for introducing me into the world of cloning. I must sincerely
thank Gauri W. Jawdekar for performing all DNA binding and transcription initiation
assays, Dr. Alicia Pastor-Leicha for her help with the transmission electron microscopy,
Dr. Kathryn Severin for her help with flame atomic absorbtion, David Szymanski for
assistance with the ICP-MS, Dr. Aizhuo Liu for his assistance with NMR, and Janet

Haun for her advice on non-research related subjects.

iv

TABLE OF CONTENTS

ACKNOWLEDGMENTS ................................................................................................. iv
TABLE OF CONTENTS .................................................................................................... v
LIST OF FIGURES ......................................................................................................... viii
LIST OF TABLES ............................................................................................................ xv
LIST OF ABBREVIATIONS .......................................................................................... xvi
1. Introduction ................................................................................................................... l
1.1. RNA polymerase and promoters .............................................................................. 2
1.1.1. RNA polymerase I ............................................................................................ 2
1.1.2. RNA polymerase II ........................................................................................... 3
1.1.3. RNA polymerase III .......................................................................................... 6
1.2. Composition of human RNAP transcription initiation complexes .......................... 9
1.2.1. RNA polymerase I transcription initiation complex ......................................... 9
1.2.2. RNA polymerase II transcription initiation complexes .................................. 10
1.2.3. RNA polymerase III transcription initiation complexes ................................. 11
1.3. SNAPC .................................................................................................................... 13
1.3.1. The composition of SNAPC ............................................................................ 13
1.3.2. The SNAP complex and chromatin ................................................................ 15
1.4. Proteins that interact directly with the SNAP complex ......................................... 17
1.4.1. TBP is associated with SNAPC through multiple interactions ........................ 17
1.4.2. Oct-1 POU domain relieves self inhibitory properties of SNAPC towards
DNA binding ............................................................................................................. 18
1.4.3. p53 associates with SNAPC and TBP ............................................................. 19
1.4.4. Rb associates with SNAPSO and SNAP43 ...................................................... 19
2. Results and discussion ................................................................................................ 21
2.1. SNAP subunits - overview ..................................................................................... 21
2.1.1. SNAP19 .......................................................................................................... 21
2.1.1.1. Over-expression, purification and crystallization of SNAP19 ......... 22
2.1.1.2. Cloning, over-expression, purification and crystallization of
SNAP19(1-85) ...................................................................................................... 24
2.1.1.3. Nuclear magnetic resonance analysis of SNAP19 ............................ 25
2.1.2. SNAPl90(1-505) ............................................................................................ 30
2.1.2.1. Over-expression and purification of SNAPl90(l-505) .................... 31
2.1.2.2. Crystallization of SNAPl90(l-505) ................................................. 32
2.1.2.3. Crystallization of SNAP190(1-505)/SNAP19 complex ................... 32
2.2. Over-expression and purification of SNAPC subunits ........................................... 33

2.3. Cloning of plasmids for co-expression .................................................................. 39

2.4. Over-expression of mSNAPc variants ................................................................... 43
2.5. Comparison of the individual expression of SN APc subunits to the co-expression
system ........................................................................................................................... 46
2.6. Purification of mSNAPc ........................................................................................ 48
2.7. DNA binding activity of mSNAPc variants ........................................................... 51
2.8. mSNAPcy4 is capable of recruiting TFIIIB to the promoter ................................. 53
2.9. Transcription initiation mediated by mSNAPc ...................................................... 56
2.10. Zinc finger domain in SNAPSO ........................................................................... 57
2.10.1. ICP-MS and FAAS analysis of mSNAPc variants ....................................... 60
2.10.2. Ab-initio modeling of SNAPSO C-terminus ................................................. 63
2.10.3. Mutagenesis .................................................................................................. 63
2.10.4. DNA binding activity of SN AP50 mutants .................................................. 65
2.10.5. Transcription initiation assay ........................................................................ 66
2.11. Crystallization of mSNAPc variants .................................................................... 67
2.12. X-ray diffraction of mSNAPc crystals ................................................................. 70
2.13. Evaluation of mSNAPc crystals using transmission electron microscopy (TEM)
....................................................................................................................................... 70
2.14. Diffraction of mSNAPc crystals using TEM ...................................................... 73
2.15. Purification optimization of mSNAPcy4 ............................................................. 77
. Methods and materials ............................................................................................... 91
3.1. Preparation of expression plasmids ....................................................................... 91
3.1.1. Cloning of the SNAPSO subunit ..................................................................... 91
3.1.2. Cloning of the SNAP43 subunit ..................................................................... 91
3.1.3. Cloning of the SNAP19 subunit ..................................................................... 92
3.2. Preparation of expression host ............................................................................... 92
3.2.1. Preparation of competent cells for heat shock ................................................ 92
3.2.2. Preparation of competent cells for electroporation ......................................... 93
3.2.3. Preparation and use of a glycerol stock .......................................................... 93
3.2.4. Transformation into the cloning bacterial host ............................................... 93
3.2.5. Transformation into the expression bacterial host Codon Plus RIL ............... 94
3.2.6. Transformation into the co-expression bacterial host ..................................... 94
3.2.7. Electroporation ................................................................................................ 95
3.3. Growth media ......................................................................................................... 95
3.3.1. Preparation of culture plates ........................................................................... 95
3.3.2. LB growth media ............................................................................................ 96
3.3.3. TB growth media ............................................................................................ 96
3.3.4. M9 minimal media .......................................................................................... 96
3.4. Over-expression ..................................................................................................... 97
3.4.1. Over-expression of individual SNAP subunits ............................................... 97
3.4.2. Co-expression of mSNAPcy3 and mSNAPcy4 ............................................... 97
3.5. Purification ............................................................................................................. 98
3.5.1. GST purification ............................................................................................. 98
3.5.2. GST purification in the absence of a detergent ............................................... 99
3.5.3. Ion-exchange purification ............................................................................... 99

vi

3.5.4. Gel filtration purification .............................................................................. 100

3.6. SDS-PAGE .......................................................................................................... 100
3.7. Western blotting ................................................................................................... 101
3.8. DNA purification ................................................................................................. 102
3.9. TEM analysis of mSNAPc crystals ...................................................................... 102
3.9.1. Cross-linking of mSNAPc crystals ............................................................... 102
3.9.2. Preparation of the TEM grid ......................................................................... 103
3.9.3. TEM analysis ................................................................................................ 103
3.10. Quantification of zinc in SNAP50 ..................................................................... 104
3.10.1. Determination of protein concentration ...................................................... 104
3.10.1.1. UV absorbance in 6 M urea ............................................................ 104
3.10.1.2. Standardized Bradford assay ........................................................... 104
3.10.2. Flame atomic absorption ............................................................................. 105
3.10.3. ICP-MS ....................................................................................................... 105
4. References ................................................................................................................... 107

vii

LIST OF FIGURES

Figure 1: The upstream control element (UCE, -180 to -107) and the control promoter
(CP, -45 to +20) are the regulatory regions required for efficient targeting by RNA
polymerase I. ....................................................................................................................... 3

Figure 2: mRN A promoters are often characterized by the presence of a TATA box.
Other promoter regions are often present upstream or downstream of the transcription
start site. .............................................................................................................................. 4

Figure 3: H. sapiens U1 snRNA promoter region determined by the presence of both the
DSE and the PSE, but by the absence of the TATA box. ................................................... 5

Figure 4: A. thaliana snRNA recognized by RNAP H contains the upstream sequence
element (USE) and a TBP recruiting TATA box7. ............................................................. 5

Figure 5: snRN A promoter region from D. melanogaster with two promoter elements
recognized by RNA polymerase H7. ................................................................................... 6

Figure 6: X. laevis SS RNA gene contains three elements in the internal control region

(ICR) which can be divided into box A (+50 to +60), an intermediate element (IE, +67 to
+72) and box C (+80 to +90). A simple run of T residues determines the end of the genes.
............................................................................................................................................. 7

Figure 7: X. laevis tRNA'eu internal promoter region consisted of boxes A (+8 to +19) and
B (+52 to +62)8. .................................................................................................................. 7

Figure 8: H. sapiens U6 snRNA with gene external promoter regions. The DSE (-240 to -
215), the PSE (~65 to -48) and the TATA box (-32 to -25) are all located upstream of the
transcription start site8 ......................................................................................................... 8

Figure 9: The S. cerevisiae U6 gene with a combination of gene internal A-box (+21 to
+31) and gene external promoter elements; The TATA box (-30 to -23) upstream from
the trapscription start site and B-box downstream (+234 to +244) from the U6 coding
region . ................................................................................................................................ 9

Figure 10: H. sapiens RNA polymerase I transcription from an rRNA promoter with SL1,
a TBP containing complex and UBF 1, a multi-subunit transcription factor targeting the
USE and the CP regions respectively. Placement of TFIs on the figure is arbitrary. ....... 10

Figure 11: H. sapiens polymerase II transcription initiation complex: (A) an example of a
mRNA promoter with a TATA box, recognized by the TBP or by the TBP containing

TFIID complex and (B) TATA—less U1 snRNA type of promoter, with the PSE sequence
recognized by the SNAP complex. ................................................................................... 11

viii

Figure 12: H. sapiens polymerase III transcription initiation complex: a U6 snRNA
promoter (A) with the PSE sequence, targeted by the SNAP complex, and the TATA box,
targeted by the TFIIIB like complex, assembled from TBP, Brf2 (BrfU) and del. (B)
TATA-less tRNA type of promoter with boxes A and B targeted by TFIIIC. TFIIIB is
required for transcription initiation. .................................................................................. 12

Figure 13: Protein—protein/DNA contact map of SN APc. The complex is composed of
five subunits SNAP190 interacts with TBP using the TBP recruitment region (TRRl)
from amino-acids 34 to 84 and with Oct-1 using the OIR (Oct-1 interacting region) from
amino-acids 888 to 912. RSRR is the arginine and serine rich area of SNAP190, a target
for phosphorylation. The unusual Myb domain is responsible for DNA binding of
SNAP190 (adopted from Hernandez et al.28). .................................................................. 15

Figure 14: Proposed model of SNAPC recruitment to the U6 promoter. SNAPc is
recruited to the promoter by interaction with Oct-1 and TBP is recruited to the TATA box
by interaction with SNAPC. The N-terminal domain of SNAP190, SNAP43 and SNAP45
are involved in the interaction with TBP. SNAP190 contains three main domains; the N-
terminal region, the Oct-1 interacting region (OIR) and the C-terminal region responsible
for the interaction with SNAP45. SNAP190 and SNAP50 interact with the PSE region of
the DNA. Positioning of the nucleosome is crucial for Oct-l dependent activation. ....... 16

Figure 15: TBP contains a highly conserved C-terminal domain and an N-terminal
domain that can be divided into three regions. Region II is a stretch of glutamines and the
number of glutamine residues varies between organisms ................................................. 18

Figure 16: SNAP19 purified over GSH resin. Protein and beads suspension after 10 min
(CLl), 20 min (CL2), 2 h (CL3) and 6 h (CL4) thrombin cleavage. Cleaved SNAP19
(ELI and EL2, two successive fractions of the eluted material). ..................................... 22

Figure 17: Ion-exchange purification of SNAP19. Consecutive fractions containing
highly purified SNAP19 (IE1, IE2 and IE3). 15 mg of SNAP19 purified by affinity
chromatography were diluted 3 fold, to bring the salt concentration below 50 mM and
loaded onto Source-Q ion-exchange resin (15 mL). The protein material was eluted from
the column by a salt gradient (0-70%, 1 M KCl). 8-10 mg of highly purified SNAP19
were recovered in two fractions (IE2, IE3) ....................................................................... 23

Figure 18: Commassie blue stained SDS-PAGE of GST purified SNAP19(1-85) (lane 2)
and wild type SNAP19 (lane 3). ....................................................................................... 24

Figure 19: 1H NMR spectra of SNAP19. .......................................................................... 26

Figure 20: HSQC (heteronuclear single quantum coherence transfer) spectra of SNAP19.
........................................................................................................................................... 28

Figure 21: 1H NMR spectra of SNAPl90(84-133) peptide, SNAP19 and the complex
obtained by mixing the two proteins ................................................................................. 30

ix

Figure 22: SNAPl90(l-505) purification. GST-SNAPl90(1-505) immobilized on the
GSH agarose (lane 2) and cleaved and eluted material (lanes 3 and 4) ............................ 31

Figure 23: Purification of (A)SNAP190(1-505) and (B)SNAP43 using GST affinity
chromatography. The proteins were purified from equal culture volumes under identical
conditions. The majority of SNAP43 can not be recovered and the material that eluted off
the beads sticks to the reaction vessel in a short time. The material can be recovered by

boiling in the sample buffer for SDS-PAGE. ................................................................... 34

Figure 24: Purification of (A) SNAP50 showing the strong presence of an unidentified
protein that can not be removed by subsequent purification and (B) SNAP19 using GST
affinity chromatography. The proteins were purified from equal culture volumes under
identical conditions. .......................................................................................................... 35

Figure 25: Electro mobility shift assay (EMSA) of the recombinant mSNAPc (mrSNAPc)
assembled from individually expressed and purified subunits, and endogenous SNAPc
obtained from HeLa cells (monoQ fractions). Increasing amounts of mSNAPc that was
assembled from subunits individually expressed in E. coli (approximately 1, 2.5, and 7.5
ng; lanes 5—7, respectively) were added to EMSA reactions containing dsDNA probes
containing a wild-type (wt) or mutant (mu) PSE, as indicated. Reactions loaded in lanes 3
and 4 contained 7.5 uL of partially purified endogenous SNAPC (approximately 0.3 ng
SNAPC/mL). Reactions containing only the probe DNAs are shown in lanes 1 and 2
(experiment performed by Craig Hinkley) 58. ................................................................... 36

Figure 26: In-vitro U6 transcription initiation assay of the recombinant mSNAPc.
Increasing amounts of mSNAPc were added to human U6 in vitro transcription reactions
for which the HeLa whole cell extract (WCE) was treated with oc-SNAP43 antisera to
remove endogenous SNAPC, as shown in lanes 6—8. Lane 4 shows the decreased signal
for the correctly initiated U6 transcription upon removal of endogenous SNAPC. Lane 5
shows the U6 signal dependent upon addition of endogenous SNAPC obtained from
biochemical fractionation of a HeLa cell nuclear extract. Note, this signal is comparable
to the mock depleted WCE (lane 3) or WCE alone (lane 1)“. ......................................... 38

Figure 27: The pGST190(1-505) plasmid with pET origin of replication and ampicillin
resistance. The SNAPl90(l—505) sequence follows the sequence for the GST tag and the
thrombin recognition sequence. ........................................................................................ 40

Figure 28: pCDF43/50-1 plasmid with CDF origin of replication and streptomycin
resistance. SNAP50 gene is in MCSI and SNAP43 is in MCSII ...................................... 42

Figure 29: pRSFl9-l plasmid with RSF origin of replication and kanamycin resistance
with SNAP19 gene in MCSI and un-utilized MCSII. ...................................................... 43

Figure 30: Analysis of expression for SNAPC subunits in the individual expression and
co-expression approach by Western blot. GST labeled protein were visualized using 0t-
GST antibodies and the SN APc subunits were visualized using antibodies directed
against the subunit of interest. .......................................................................................... 47

Figure 31: Purification of co-expressed mSNAPcy4 using the GST affinity resin and
thrombin cleavage. The mSNAPcy4 was purified under the same conditions as the
individual subunits. ........................................................................................................... 49

Figure 32: Recovery of the co-expressed material compared to individually expressed and
purified subunits. SNAP19 improves complex stability and subunit recovery. Subunit
recovery for partial SNAPC assembled without and with co-expressed SNAP19
(mSNAPcy3 and mSNAPcy4, respectively) was estimated by SDS—PAGE and
Coomassie blue staining. Note the increased recovery of SNAP43 and SNAP50 in
mSNAPcy4 (lane 7) compared to mSNAPcy3 (lane 6). Lanes 2—5 contain aliquots of the
individually expressed SNAPC subunits for reference. Note that the unrecovered SN AP43
is used to show the migration of SNAP43. ....................................................................... 50

Figure 33: mSNAPcy4 facilitates TBP promoter recruitment. Increasing amounts of
mSNAPcy4 (3 and 10 ng) were added to DNA binding reactions containing radiolabeled
probes that harbor a wt PSE and wt TATA box (lanes 1—4), mu PSE and wt TATA box
(lanes 5—8), wt PSE and mu TATA box (lanes 9—12), or mu PSE and mu TATA box
(lanes 13—16). Lanes 4, 8, 12, and 16 contain 50 ng of recombinant full-length human

T BP in addition to 10 ng of mSNAPcy4. Reactions containing only the DNA probes are
shown in lanes 1, 5, 9, and 13. Positions of the mSNAPcy4 and mSNAPcy4 lus TBP
complexes are indicated (experiment was performed by Gauri W. J awdekar) 8 .............. 52

Figure 34: mSNAPcy4 is capable of recruiting TFIHB to the promoter. Coordinated
DNA binding by SNAPC and TBP facilitates higher order complex assembly with Brf2
and de1. DNA binding reactions were performed with the indicated combinations of
SNAPCy4 and Brf2-TFIIIB subunits. These results suggest that preinitiation complex
assembly follows the order SNAPc>TBP>Brf2>de1 (experiment was performed by
Gauri w. Jawdekar)” ........................................................................................................ 54

Figure 35: SNAPC stimulates DNA binding by Brf2-TFIIIB. Electrophoretic mobility
shift assays were performed using a U6 probe containing a wild-type mouse U6 PSE and
a wild-type TATA-box (AC probe). DNA binding was carried out in the absence (lanes
1-8) or presence (lanes 9-12) of wild type SNAPCy4. Reactions containing individual
TBP, Brf2, and de1 (1-470) subunits are shown in lanes 2-4. Reactions containing pair
wise combinations of TBP with Brf2, TBP with de1 (1-470), and Brf2 with del (1-
470) are shown in lanes 4, 5, and 8. DNA binding by the complete Brf2-TFIIIB complex
in the absence of SNAPC is shown in lane 7. Additional reactions were performed with
SNAPC alone (lane 9) or in combination with Brf2-TFIIIB subunits (lanes 912), as
indicated. Lane 1 shows migration of the probe alone. The relative positions of the
various SN APcy4/Brf2-TFIIIB complexes are shown on the right (experiment was
performed by Gauri W. J awdekar)5 9. ................................................................................ 55

Figure 36: mSNAPcy4 functions for U1 snRNA transcription by RNA polymerase H.
HeLa cell nuclear extract was either mock depleted with a preimmune rabbit sera (lane 2)
or a-SNAP43 antisera (lanes 3—10) to deplete endogenous SNAPC. Extracts were then
used for human U1 in vitro transcription assays. The Ul-specific signal was diminished
upon removal of endogenous SNAPC, as shown in lane 3. Increasing amounts of

xi

mSNAPcy4 (0.08, 0.25, 0.75, 2.5, 7.5, 25, and 75 ng) reconstituted the correctly initiated
transcription from a human U1 reporter, as shown in lanes 4—10. Lanes 1 and 2 show the
U1 signal obtained from either untreated or mock depleted reactions (experiment was
performed by Gauri W. J awdekar)5 8. ................................................................................ 56

Figure 37: mSNAPcy4 functions for U6 snRNA transcription by RNA polymerase III. In
vitro transcription of human U6 snRNA was carried out using HeLa cell nuclear extract
that was treated as in Figure 36. Lane 2 shows the reduced U6 signal upon removal of
endogenous SNAPC. Increasing amounts of mSNAPcy4 (0.08, 0.25, 0.75, 2.5, 7.5, 25,
and 75 ng) reconstituted the correctly initiated transcription from a human U6 reporter as
shown in lanes 3—9. Approximately 75 ng of GST was added to the transcription reaction
shown in lane 10 (experiment was performed by Gauri W. J awdekar)5 8. ........................ 57

Figure 38: Sequence alignment of human SNAP50 C-terminal amino acids (301-411)
with corresponding regions from SN AP50 homologues of other species. Putative zinc
fingers similar to TFIIIA (Hstmex3C) and steroid receptors (szcxzzcsz) are
indicated as region 1 and region 2, respectively. The sequence of highly conserved amino
acids derived from this alignment corresponds to Lx4GX6Hx3CxHx20-23YPx1 1-
1ZCXZCXIng3-4Cx2CFx3Hx1-4G. This alignment was performed using the Clustal W
program. Homo sapiens (NP003075), Cannisfamiliaris (XP853813), Bos taurus
(AAX08912), Mus musculus (NP084225), Rattus norvrgicus (NP001013230),
Drosophila melanogaster (NP724647), Drosophila pseudaoobscura (EAL25490),
Trypanosoma brucei (XP827295), Arabidopsis Ihaliana (AAO30067), Caenorhabditis
elegans-l (NP500819), Caenorhabditis elegans-Z (NP497807), Plasmodium falciparum
(), Danio rerio (XP694501), Leishmania major (XP843572), Anopheles gambiae
(XP310411), Dictyostelium discoideum (XP644064), Entamoeba histolytica (XP653151).
This comparison was prepared by Gauri W. J awdekarS 9. ................................................. 59

Figure 39: Flame atomic absorption spectroscopic analysis of mSNAPcy3 and
mSNAPcy4. Unlabeled data points represent the absorption obtained for standard zinc
solutions. ........................................................................................................................... 61

Figure 40: Mutations in the SNAP50 zinc finger domain do not disrupt SNAPC assembly.
Approximately 20 ng of each of the SNAPcy4 complex containing substitution mutations
in HA-SNAPSO were affinity purified first using glutathione agarose to pull down GST-
SNAP190 (1-505) followed by immunoprecipitation with (it-SNAP43 antibodies.
Associated wild type or mutant HA-SNAPSO was detected by a-HA Western analysis
(lanes 6-22). A titration of wild type SNAPcy4 using 8, 4 and 2% of the input material is
shown in lanes 1-3, respectively. Lanes 4 and 5 contain wild type SNAPcy4 recovered
with the protein-G agarose beads alone or with pre-immune serum, respectively. The
bottom panel represents 4% of the input material directly analyzed by oc-HA Western
analysis (experiment was performed by Gauri W. Jawdekar)”. ...................................... 64

Figure 41: Mutations in the SNAP50 zinc finger domain disrupt DNA binding by
SNAPC. Increasing amounts (3 and 10 ng) of SNAPcy4 with wild type (lanes 2 and 3) or
mutant HA-SNAPSO (lanes 4-35) containing the indicated substitution mutations was
tested in an EMSA for binding to a radiolabeled DNA probe containing a high affinity

xii

PSE and TATA box (AC probe). Lane 1 shows the probe alone with no added proteins.
Mutations H3l3-A, C317-A, and H319-A resulted in reduced DNA binding, whereas
mutations C354-A, C357-A, C380-A, and C383-A completely abolished DNA binding
activity. Mutation H388—A also exhibits weakened DNA binding activity (experiment
was performed by Gauri W. J awdekar)5 9 .......................................................................... 65

Figure 42: U1 and U6 in-vitro transcription initiation assay of wt or mutant HA-SNAPSO
containing mSNAPcy4. Mutations that resulted in reduced DNA binding also showed
reduced transcription initiation activity for both types of promoters, with the exception of
the H388-A mutation, which resulted in weakened U1 activity, but wild type U6 activity
of transcription initiation59. HeLa cell nuclear extract was depleted with u-SNAP43
antibodies to immunodeplete endogenous SN APc. In vitro U1 and U6 transcription was
then tested in the absence (lanes 1) or presence of purified SNAPcy4 (5 ng) containing
wild type SNAP50 (lane 3) or mutant SNAP50 with the indicated alanine substitutions
(lanes 4-19). Addition of GST alone did not reconstitute either U1 or U6 transcription as
shown in lane 2 (experiment was performed by Gauri W. J awdekar)5 9. .......................... 67

Figure 43: Crystals of mSNAPcy4 as observed under the light microscope grown in 9%
PEGSOOOMME, 100 mM Tris pH 8.5, 100 mM NaCl and 100 mM MgC12. ................... 68

Figure 44: Optimized crystals of mSNAPcy4 as observed under the light microscope
grown in 8% PEGSOOOMME, 100 mM Tris pH 8.4, 100 mM NaCl and 100 mM MgC12.

........................................................................................................................................... 69
Figure 45: Needle clustered crystals of mSNAPcy4 under the TEM have a heavily
branched appearance and the branches are even further sectioned ................................... 71
Figure 46: Crystals of mSNAPcy4 under the TEM. Close-up on one of the branches
showing a heavily fragmented formation. ........................................................................ 72
Figure 47: Circular diffraction pattern typical for a powdered material ........................... 73
Figure 48: TEM image of mSNAPcy4 single crystals as observed under TEM. ............. 74

Figure 49: TEM diffraction images obtained from a single crystal. Image B was obtained
from a crystal rotated approximately 0.5 degree with respect to the crystal from image A.

........................................................................................................................................... 75
Figure 50: TEM image of mSNAPcy4/DNA crystals ....................................................... 76
Figure 51: Diffraction pattern from a single branch obtained under TEM. ...................... 77

Figure 52: A representative elution profile for ion-exchange purification (Source-S) of
mSNAPcy4. The solid line represents the relative absorption intensity of the eluted
protein material and the dotted line represents the salt gradient. ...................................... 79

Figure 53: Gel filtration chromatograms of mSNAPcy4 purified with affinity
chromatography (A); mSNAPcy4 rerun fraction 10 (B); mSNAPcy4 rerun fractions 12

xiii

and 13 (C). The first peak in fractions 9 and 10 corresponds to 650 kDa and is also the
void. The second peak corresponds to a protein with an apparent molecular weight of 150
kDa according to the gel filtration standards (Biorad, data not shown). .......................... 83

Figure 54: Gel filtration chromatogram of mSNAPcy4 (A) supplemented with zinc (B),
iron (C) or EDTA (D). ...................................................................................................... 86

Figure 55: Gel filtration of mSNAPcy4 using Sephacryl-300 size exclusion beads. (A)
Biorad molecular weight standards (a=670 kDa, b=158 kDa, c=44 kDa, d=17 kDa), (B)
GST purified mSNAPcy4, (C) fraction 9—11 from the first gel filtration rerun using the
same column, (D) fractions 9-11 from the second gel filtration rerun (e: void peak, f:
mSNAPcy4 heterotetramer). Fractions were 5 mL. .......................................................... 88

Figure 56: Crystals of mSNAPcy4 grown in 100 mM Tris pH 8.5, 10% PEG4000, 100
mM NaCl and 100 mM MgC12. Protein material used to obtain the crystals was prepared
by successive gel filtration purification steps. .................................................................. 90

xiv

LIST OF TABLES

Table 1: Quantification of zinc and nickel using ICP-MS and FAAS for mSNAPcy3 and
mSNAPcy4. Two isotopes “Zn (3) and 68Zn (b) were measured for ICP-MS. Two
independently prepared samples of mSNAPcy4 were used in the analysis: (1) extensively
purified complex used for crystallization and (2) void fraction from gel filtration
purification of the complex. The protein concentration was determined by absorbance
measurement in 6M urea and by the Bradford assay. ....................................................... 62

Table 2: Combinations of antibiotics used for preparation of agar plates. AMP, KAN,
STR and CAM represent ampicillin, kanamycin, streptomycin and chloramphenicol
respectively. ...................................................................................................................... 96

Table 3: Antibiotic concentrations used in individual and co-expression systems .......... 98

XV

A/ADE

Amp

BOG

C
Cam
C/CYT

C terminal

DNA
DNase
DSE
dsDNA

DTT

EDTA

LIST OF ABBREVIATIONS

alanine
adenine

ampicillin

n-octyl-beta-D-glucopyranoside

base pair

cysteine
chloramphenicol
cytosine

carboxy terminal

aspartic acid

Dalton
deoxyribonucleic acid
deoxyribonucleosidase
distal sequence element
double stranded DNA

dithiothreitol

glutamic acid

Diaminoethanetetraacetic acid

xvi

EMSA

F

FAA(S)

G/GUA
GST

GSH

HCl
Hepes

HSQC

ICP—MS

Kan
KCl

kDa

electrophoretic mobility shift assay

phenylalanine

flame atomic absorption (spectroscopy)

glycine
Guanine
Glutathione S-Transferase

glutathione

histidine
hydrochloric acid
N—[2-hydroxyethyl] piperazine-N’-[ethane sulfonic acid]

heteronuclear single quantum coherence transfer

isoleucine
inductively coupled plasma mass spectroscopy

Isopropyl-B-D-Thiogalactopyranoside

lysine
kanamycin
potassium chloride

kilo Dalton

xvii

MCS

mg

118

mL

MW

mRNA

mSNAPc

mrSNAPc

NaCl

"8

NMR

N -terminal

OIR

PCR

leucine

methionine

multiple cloning site

milligram

microgram

milliliter

molecular weight

messenger RNA

mini small nuclear RNA activating protein complex

mini recombinant small nuclear RNA activating protein complex

asparagine

sodium chloride

nanogram

nuclear magnetic resonance

amino terminal

Oct-l interacting region

proline

polymerase chain reaction

xviii

pol
PEG

PMSF

ppb
ppm

PSE

Rb
RNA
RN AP

rRNA

S

siRN A
SNAP
SNAPC
snRNA
SDS-PAGE

Str

polymerase

polyethylene glycerol
phenylmethylsulfonylfluoride
parts per billion

parts per million

proximal sequence element

glutamine

arginine

retinoblastoma protein
Ribonucleic Acid
Ribonucleic Acid Polymerase

Ribosomal RNA

serine

small interfering RNA

small nuclear RNA activating protein

small nuclear RNA activating protein complex

small nuclear RNA

sodium dodecyl sulfate — polyacrylamide gel electrophoresis

Streptomycin

xix

T/T HY

TBP

TEM

TFI

TFII

TFIII

Tris

tRN A

UV

VIS

WCE

threonine

Thymine

TATA (box) Binding Protein

transmission electron microscopy
Transcription Factor I

Transcription Factor 11

Transcription Factor III
2-Amino-2-(hydroxymethyl)-1,3-propendiol

transfer RNA

uracil

ultraviolet

valine

visual

tryptophan

whole cell extract

tyrosine

XX

1. Introduction

Genetic information of all living organisms, with the exception of retro viruses, is
stored on the DNA. Transcription, a process in which the information on the DNA is
transferred to the RNA, is achieved by accurate synthesis of RNA from the DNA
template by different RNA polymerases. One RNA polymerase exists in prokaryotes and
it is solely responsible for the transcription of the genomic information of the organism.
In contrast, eukaryotes contain four classes of polymerases. The four classes differ in
localization, template specificity, and susceptibility to activators and inhibitors. RNA
polymerase I, located in the nucleoli, is responsible for transcription of ribosomal RNA
and transcribes the genes for 188, 5.85 and 28S ribosomal RNA (rRNA). RNA
polymerase 11, located in the nucleoplasm, is responsible for transcription of all
messenger RNA (mRNA) and some small nuclear RNA (snRNA), such as U1 and U2
snRNA. RNA polymerase III, also located in the nucleoplasm, is responsible for
transcription of SS ribosomal RNA, the transfer RNA (tRNA) and also the U6 snRNA.
RNA polymerase IV was recently identified in plants and is responsible for transcription
of short interfering RNAs (siRNAs) which were implicated in gene silencing and also
mitochondrial RNA1’2’3.

Strict control of transcription is required for healthy cell existence and is achieved
by targeting regions of DNA called promoters, located upstream or downstream from
transcription start sites. A promoter is a region on the DNA that is capable of recruiting
transcription factors and eventually the RNA polymerase. An enhancer is a region on the
DNA that is responsible for recruitment of transcription factors that influence

transcription, but do not directly recruit the RNA polymerase. Different transcription

factors bind to the promoter region and can initiate transcription by helping the
polymerase to specifically bind to the DNA and start the transcription process. Other
transcription factors function as repressors and inhibit the binding of the polymerase to
the DNA, and thus reduce or abolish transcription. Some factors, called basal
transcription factors, are always present in the cell4. Some are incapable of DNA binding
and can, only by specific external stimuli, overcome this inability. When the inability to
bind the DNA is eliminated, these factors become active and can then activate or repress
the transcription process. The organism uses such mechanisms to adapt to a change in
external conditions or to a different developmental stage, by expressing the proteins
required to adapt to the new environment. A different type of transcription factor is
expressed only in response to external stimuli and can then regulate transcription. In the
absence of the stimuli these transcription factors are present in only small concentrations

. .4
or are not present at all1 3 .

1.1. RNA polymerase and promoters
1.1.1. RNA polymerase I

RNA polymerase I (RNAP I) is unique among the nuclear RNAs in that it is
responsible for transcription of only one set of genes, the ribosomal RNA (rRNA) and
has to be able to recognize only one promoter structure. There are essentially two
different genes for all the rRNAs in a tandem repeated form. A long precursor for rRNA
is transcribed by RNA polymerase I and it is subsequently cleaved and processed into the
individual ribosomal RNAs. Two control regions make up the promoter for this

polymerase; the core promoter (CP) extending from -45 to +20 and the upstream control

element (UCE) extending from -180 to -107 which increases the efficiency of the core
promoter. Both promoter regions are unusually GC-rich. SL1 (H. sapiens), TIF-IB (M.
muscullus) or CF (S. cerevisiae) are TBP associated multi-subunit complexes responsible
for binding to the core promoter and are essential for RNAP I transcription. The upstream
control element recruits another multi-subunit factor called the upstream binding factor or
UBF (UBF-1, H. sapiens) and this factor greatly enhances SL1 recruitment and its

. . . ‘3
specrficrty1 .

-180 -45 +1

l" e
—[]—l I l

UCE CP

 

 

Figure 1: The upstream control element (UCE, -l80 to —107) and the control
promoter (CP, -45 to +20) are the regulatory regions required for efficient

targeting by RNA polymerase 1.

1.1.2. RNA polymerase II

RNA polymerase 11 (RN AP II) is responsible for transcription of genes coding for
all messenger RNAs (mRNAs) and most small nuclear RNA (snRNA). The RNAP 11
promoter region is the most diverse compared to other polymerases recognized by other
promoters. Many different factors bind to this promoter region and influence the
transcription of the genes. This is understandable because of the need for a living cell to
be able to quickly increase or decrease the expression of particular proteins in response to

changes in the environment or progress in the developmental stage of the cell. A variety

of promoter elements have been identified including the TATA box (TATAAAA),
upstream elements such as the CCAAT box, the GC box (GGGCGG) and downstream
elements. RNAP II promoters have one or more of these elements in different numbers

and combinations (Figure 2)3.

+1

l'"
W

TATA

Figure 2: mRNA promoters are often characterized by the presence of a TATA
box. Other promoter regions are often present upstream or downstream of the

transcription start site.

Human U1 small nuclear RNA promoter is an example of a TATA box-less
promoter and differs from the U6 snRN A promoter by the absence of the TATA box. The
two promoters serve as an interesting model in the understanding of RNAP specificity for
transcription of genes with similar promoter regions. The U1 promoter contains two
regions; a distal sequence element (DSE) and the proximal sequence element (PSE,

4.5.6

Figure 3)

+1

n I”
i] u l

DSE PSE

 

Figure 3: H. sapiens U1 snRNA promoter region determined by the presence of

both the DSE and the PSE, but by the absence of the TATA box.

The upstream sequence element (USE) in A. thaliana (Figure 4) and the
PSEA in D. melanogaster (Figure 5) are similar to the human PSE. Factors that
are recruited to these promoter regions are not well characterized, but it is
possible that the polymerase specificity in these systems is determined by
different conformations of the same factor, which are defined by the USE or
PSEA sequence. Different conformations of a factor can than recruit other factors

that can than bind to the promoter and regulate transcription7.

+1

r"
El—[l—I

USE TATA

 

Figure 4: A. thaliana snRNA recognized by RNAP 11 contains the upstream

sequence element (USE) and a TBP recruiting TATA box7.

+1

r1 FT
u [H

PSEA PSEB

 

Figure 5: snRNA promoter region from D. melanogaster with two promoter

elements recognized by RNA polymerase 117.

1.1.3. RNA polymerase III

RNA polymerase III (RNAP III) transcribes genes that encode structural or
catalytic RNA and are in general shorter than 400 base pairs (bp), which is also consistent
with the elongation properties of RNA polymerase III. The RNA molecules transcribed
by RNA polymerase III are involved in fundamental metabolic processes. These RNAs
are components of the protein synthesis, mRN A splicing and tRNA processing apparatus.

RNA polymerase 111 uses three main types of promoters. The type 1 RNA polIII
promoter has only one example; the X. laevis SS promoter, which contains an internal
control region (ICR). This promoter region consists of three elements; the A box (+50 to
+60), the intermediate element (IE; +67 to +72) and the C box (+80 to +90; Figure 6). In

S. cerevisiae SS genes, only C box is required for efficient transcription3‘4‘8’9.

+1 +120

ICR

I 7 7 , 7 I
LRHJ W
A IE c

 

 

 

l—_

Figure 6: X. laevis SS RNA gene contains three elements in the internal control
region (ICR) which can be divided into box A (+50 to +60), an intermediate
element (IE, +67 to +72) and box C (+80 to +90). A simple run of T residues

determines the end of the genes.

Type 2 promoters are represented by the genes of Ad2 VAl and most tRNAs. The
main hallmark of this promoter type is the presence of gene internal A and B boxes.
These regions are conserved in tRNA genes between various species. The A-boxes of
type 1 and 2 are interchangeable in X. laevis (Figure 7)”). This reflects the similarity in
sequence rather than a conserved function, since the A-boxes of SS RNA and tRNA

genes bind different transcription factorsl 1.

+1 +8 +52 +80

1"
E U TTTT
A B

 

 

Figure 7: X. laevis tRNAlcu internal promoter region consisted of boxes A (+8 to

+19) and B (+52 to +62)8.

The H. sapiens U6 snRNA gene contains a type 3 promoter and is characterized

by the presence of gene external promoter regions only. The distal sequence element

(DSE), proximal sequence element (PSE), and the TATA box are located upstream from
the transcription start site. Representative members of this promoter type are the U6
snRNA genes, coding for the U6 snRNA component of the spliceosomelz‘l3 ‘14, the 78K
gene whose RNA product was implicated in the regulation of the CDK9/cyclin T
complex”, and the HI RNA gene, which codes the RNA component of hRNaseP and the
RNA component of hRNase MRPIG'”. Interestingly in the vertebrate snRNA promoters,
RNA specificity can be switched between RNAPH and RNAPIII and vice versa by

deletion or generation of the TATA box element (Figure 8)”.

-240 -65 -32 +1 +106

i] EJ—[l-l W"

DSE PSE TATA

 

 

Figure 8: H. sapiens U6 snRNA with gene external promoter regions. The DSE (-
240 to -215), the PSE (-65 to -48) and the TATA box (-32 to -25) are all located

upstream of the transcription start sites.

The S. cerevisiae U6 snRNA gene is a type 4 RNAPIII promoter and is a hybrid
consisted of gene internal and gene external regions. The TATA box is located upstream

of the transcription start site and the B-box is located downstream from the coding

region. The A-box is located gene internally (+21 TO +31, Figure 9). All three of these

. . . . . 9
promoter elements are required for transcrrptron 1n vrvo1 .

-30 +1 +113 +234

I'"
mu m. l]—

TATA A B

 

 

Figure 9: The S. cerevisiae U6 gene with a combination of gene internal A-box
(+21 to +31) and gene external promoter elements; The TATA box (-30 to -23)
upstream from the transcription start site and B-box downstream (+234 to +244)

from the U6 coding regiong.

1.2. Composition of human RNAP transcription initiation complexes
1.2.1. RNA polymerase I transcription initiation complex

The TATA box binding protein (TBP) is the only known transcription factor that
seems to be almost universally required for transcription regardless of the promoter type
and polymerase used to transcribe a gene. Interestingly TBP is required for transcription
whether or not the TATA box element is present in the promoter. In humans and in the
case of RNA polymerase I the necessary transcription factor SL1 (selectivity factor) is a
TBP containing multi-subunit complex and is responsible for the recognition of the USE
region of the promoter, while the upstream binding factor (UBFl) is responsible for

recognition of the CP region (Figure 10) 1‘3,

TF IS

su RNAP I

 

 

TBP UBF1 THs
TFIS
E::::}-——-{::::}-——-—
use CP

Figure 10: H. sapiens RNA polymerase I transcription from an rRNA promoter
with SL1, a TBP containing complex and UBFl, a multi—subunit transcription
factor targeting the USE and the CP regions respectively. Placement of TFIs on

the figure is arbitrary.

1.2.2. RNA polymerase II transcription initiation complexes

mRNA type promoter, which is transcribed by RNA polymerase II and contains
the TATA box, can be recognized by TFIII) or TBP. Once TFIID or TBP are
successfully recruited to the TATA box, other TAFus are then recruited to the promoter.
First to follow TFIID is TFIIB, then the TFIIF-RNAP H complex, TFIIE and TFIIH.
TFIIA can enter the initiation complex at any stage of the assembly and its main function
is counteracting repressors that associate with TBP and reduce or prevent its association

with the DNA (Figure 11)2°'2"22'23‘2“.

10

  

pse . RNAP II

Figure 11: H. sapiens polymerase II transcription initiation complex: (A) an
example of a mRNA promoter with a TATA box, recognized by the TBP or by
the TBP containing TFIID complex and (B) TATA-less U1 snRNA type of

promoter, with the PSE sequence recognized by the SNAP complex.

1.2.3. RNA polymerase III transcription initiation complexes

The key player in polymerase III transcription initiation is the TFIIIB complex,
because it contacts the polymerase directly. H. sapiens Brfl-TFHIB is composed of three
subunits, the TATA-box binding protein, Brfl and de1. An example of Brfl-TFIIIB
dependent initiation is transcription from the tRNA type of promoters, where the TBP

containing TFIIIB is recruited to the DNA by positioning of TFIIIC to boxes A and B.

TBP is required even though the promoter is TATA-less. In the case of U6 snRNA
promoters, the Brf2-TFIIIB complex, is assembled from TBP, Brf2 and de1. This
complex is recruited by binding of the SNAPC complex to the proximal sequence element

upstream of the TATA box (Figure 12)1'3’25 '26.

 

 

 

 

Figure 12: H. sapiens polymerase III transcription initiation complex: a U6
snRNA promoter (A) with the PSE sequence, targeted by the SNAP complex, and
the TATA box, targeted by the TFIIIB like complex, assembled from TBP, Brf2
(BrfU) and del. (B) TATA-less tRNA type of promoter with boxes A and B

targeted by TFIIIC. TFIIIB is required for transcription initiation.

12

1.3. SNAPC
1.3.1. The composition of SNAPC

The small nuclear RNA activating protein complex (SNAPC) is a basal
transcription factor responsible for transcription initiation for polymerase II and III
snRNA systems. This transcription factor is at least a five subunit complex, composed of
SNAP190, SNAP50, SNAP43, SNAP45 and SNAP196'27'28. TBP was found to often co-
purify with the SNAP complex purified from HeLa cells”.

The largest subunit of SNAPC, SNAP190, is the backbone of the complex. This
subunit interacts with all other subunits of the complex. The N-terminal part of SNAP190
is the most important region and interacts with SNAP50, SNAP43 and SNAP19 (Figure
13). The N-terminus is responsible for the DNA binding activity of the protein and also
interacts with TBP. The unusual DNA binding domain of SNAP190 resembles the Myb
DNA binding motif and is composed of four full (Ra, Rb, Rc and Rd) and one half (Rh)
repeats. An exceptionally serine rich area following an arginine rich region in the N-
terminal part of SNAP190 was shown to be a target for phosphorylation. This
modification had an important impact on the ability of the complex to bind DNA and to
initiate transcription by changing the electrostatic properties of this region of
SNAP1903O’3 '. Oct-1 is recruited to the complex by interaction with SNAP190 in the area
encompassing amino acids 869-91232‘33'34‘3SSNAP43 recruits SNAP50 to the largest
subunit of the complex and was shown to also interact with SNAP19 and TBP (Figure
13)6. SNAP50 is, like SNAP190, responsible for DNA binding of this transcription factor
judged from the DNA cross-linking experiments. An unusual zinc binding domain was

predicted in the C-terminal part of SNAP50. This zinc-containing domain is believed to

13

be involved in the DNA binding properties of SNAP506. The smallest subunit of the
SNAP complex is SNAP19. An exceptionally charged C-terminal tail and a leucine
zipper motif characterize this subunit. SNAP19 is not required for the formation of the
complex between SNAP190, SNAP43 and SNASO at higher concentrations, but it seems
to be needed for efficient complex formation at lower, physiologically relevant
concentrations”. SNAP45 interacts with the C-termini of SNAP190 and TBP. A SNAP
complex missing SNAP45 (SNAPC—SNAP45) showed a diffusive band in the EMSA,
which migrated slower than complete SNAPC, suggesting that the C—terminal domain of
SNAP190 in a SNAP complex missing SNAP45 may assume multiple conformations.
Addition of recombinant SNAP45 to the SNAPC-SNAP45 resulted in a band that
migrated at the same position as the complete SNAPC. This suggests that SNAP45 was
incorporated into the complex and due to direct protein-protein interactions, the C-
terrninal domain of SNAP190 assumed a more rigid conformation”.

The stoichiometry of the subunits in the complex is unknown and so is the size of

the complex”.

14

 

 

 

 

 

 

 

 

1 I SNAP50 411
I ; """"""
I 164 263
1L ISNAP43 I I368 1.90 1I SNAP45 am
1 eeeee ‘ ~~~~~~ ‘\.\

 

 

 

 

 

 

 

l E I! . ‘w L L
MYI’ a) "0'3 ' 1409
LE . ‘ m 7'13: 7 7
503 1281 1393

73484133 263

 

ITBPI

 

Figure 13: Protein-protein/DNA contact map of SNAPC. The complex is
composed of five subunits SNAP190 interacts with TBP using the TBP
recruitment region (TRRI) from amino-acids 34 to 84 and with Oct-1 using the
OIR (Oct-1 interacting region) from amino-acids 888 to 912. RSRR is the
arginine and serine rich area of SNAP190, a target for phosphorylation. The
unusual Myb domain is responsible for DNA binding of SNAP190 (adopted from

Hernandez et al.28).

1.3.2. The SNAP complex and chromatin

The two critical promoter elements on the DNA, the proximal sequence element
(PSE) and the distal sequence element (DSE) are separated by 150 base pairs. The
separation of the two promoter elements is conserved in several organisms. SNAPC binds
the PSE and the transcription activator Oct-1 binds to the DSE, resulting in transcription

activation. It was also shown that SNAPC dependent Oct-1 activation is not observed

15

when naked DNA with separated DSE and PSE is used in in-vitro assays”. The Oct-1
POU domain is capable of increasing SNAPc recruitment to the promoter if DNA
between the DSE and PSE is spaced closer together. It was also observed that
reconstruction of the nucleosome, by addition of purified histones, leads to increased
activation of the Oct-1 induced SNAPc recruitment and transcription activation. DNA
foot printing revealed that a nucleosome core is positioned between the two regions. The
localization of the DNA wrapped around the histones is defined by the presence of the
PSE and the DSE and the proteins that bind to the two regions, SNAPc and Oct-l.
Interestingly, the two proteins come in close contact with the nucleosome core, since 146
base pairs of the DNA are needed to form the nucleosome38'39’40. It is thus possible that
SNAPc or Oct-1 come into direct contact with the nucleosome, not only with the PSE and
the DSE regions, hence stabilizing the formation of the pro-initiation complex (Figure

14).

  
 

 
    
    
   

  

  

    
    

Pg; SNAP19(\\
/“ Y f::i::‘:-(""’<>IR J ‘\31‘e‘”"/‘
\—r~”” ,/ ”‘\ \ l
\ /.,,-/+~~\ e / \ISNAP45 /
NUCLEOSCIDME 319‘ SNAP43 /
It. I) VCORl; \x \ /</
\. SNAP190 % 7// V” \\

Figure 14: Proposed model of SNAPc recruitment to the U6 promoter. SNAPc is

recruited to the promoter by interaction with Oct-1 and TBP is recruited to the TATA box

16

by interaction with SNAPc. The N-terminal domain of SNAP190, SNAP43 and SNAP45
are involved in the interaction with TBP. SNAP190 contains three main domains; the N-
terminal region, the Oct—1 interacting region (OIR) and the C-terminal region responsible
for the interaction with SNAP45. SNAP190 and SNAP50 interact with the PSE region of

the DNA. Positioning of the nucleosome is crucial for Oct-1 dependent activation.

1.4. Proteins that interact directly with the SNAP complex
1.4.1. TBP is associated with SNAPc through multiple interactions

Multiple subunits of the SNAP complex interact with TBP and the protein was
co-immuno purified with antibodies to different SNAP subunits. SNAPc was shown to
improve TBP binding to the DNA by specific interactions with the N-terminal region of
TBP”. TBP contains an extremely conserved C-terminal domain and a nonconserved N —
terminal domain. The C-terminal domain is capable of performing all TBP functions like
DNA binding and interaction with transcription factors. In EMSA the high affinity mouse
U6 DNA was used because the human U6 DNA binds with the SNAP complex only
weakly. Interestingly when full length h-TBP and SNAPc are used together, the two can
bind to the human U6 probe strongly, though SNAPc or TBP alone bind only weakly“.
The N-terminal region of TBP can be divided into three parts; regions I, II and III.
Region I is required for SNAPc recruitment and U6 transcription. This region is also
involved in down-regulation of the binding to the TATA box. Region II is made out of a
run of glutamine residues and the number of glutamines varies drastically between

different organisms. This region appears to be involved in SNAPc recruitment. Region III

17

has no apparent function, since a deletion mutant without this region is fully active and

capable of SNAPc dependent DNA binding and U6 transcription (Figure 15)“.

 

 

1 55 96 159 335
I l I II I III I J
N-terminal domain C-terminal domain

Figure 15: TBP contains a highly conserved C-terminal domain and an N-terminal
domain that can be divided into three regions. Region II is a stretch of glutamines and the

number of glutamine residues varies between organisms.

h-TBP was shown to interact strongly with SNAP190, SNAP43 and SNAP45,
weakly with SNAP50, but not with SNAP19. Interestingly, when the U6 probe with
mutant TATA box was used, no change in migration was observed in EMSA by addition
of TBP. Thus, although TBP interacts with SNAPc subunits, the intact U6 promoter with
both the PSE and the TATA box is needed for cooperative binding of SNAPc and

TB P41 .42.43

1.4.2. Oct-l POU domain relieves self inhibitory properties of SNAPc towards DNA
binding
Complete SNAPc does not bind efficiently to the human PSE region of the U6
DNA, but binding is enhanced drastically in the presence of Octl-l bound to the DSE.
mSNAPc, composed of only SNAPl90(1-505), SNAP50 and SNAP43, binds efficiently

to the PSE, even though it does not interact with Oct-l. SNAP190 interacts with Oct-1

18

POU domain with its OIR (869-912), which is located in the C-terminal half of the
largest subunit of SNAPc, SNAP190. This suggests that the C-terminal domain of
SNAP190 has a built-in DNA-binding damper, which is deactivated by Oct-l POU

- 32. .44
domain 33 .

1.4.3. p53 associates with SNAPc and TBP
p53 is a tumor suppressor protein that plays a critical role in preventing
uncontrolled cell proliferation and was shown to be responsible for the selection of

45,415.47. p53 is capable of repressing

cellular outcome, such as apoptosis or cell cycle arrest
RNAPI and RNAPIII transcription. RNAP III transcription is elevated in several cancer-
derived cell lines that lack active p53“. Ultimately, the function of this protein is to
prevent the passage of mutations to daughter cells by inducing apoptosis or cell cycle
arrest as a response to DNA damage.

Furthermore, p53 was also shown to repress RNAP II transcription, along with
RNAP III transcription of human snRNA genes. p53 was shown to interact with

SNAP190 and SNAP43 subunits of SNAPc and also with TBP, but no significant

interaction was observed for SNAP45, SNAP50 and SNAP19”.

1.4.4. Rb associates with SNAP50 and SNAP4350

The retinoblastoma (Rb) protein is also a tumor suppressor that controls cell
proliferation by its influence on cell cycle progression, differentiation, apoptosis and
growth. Mutations of the Rb coding gene are found in several human cancers, as are

mutations in the promoter regions that are targeted by Rb. The function of Rb as a tumor

19

suppressor is linked to its ability to down-regulate transcription and therefore gene
expression“‘52‘53’54‘55.

Rb is capable of inhibiting transcription of polymerase I, II and III and regulates
rRNA, mRNA, tRNA and snRNA expression. Clearly the activity of Rb thru the
repression of different RNAs plays an important role in cell cycle progression and
apoptosis, since these RNAs are essential for cell viability.

In the context of U6 snRNA expression Rb was shown to be capable of repressing
transcription by targeting the SNAP complex and TFIIIB. RB was shown to strongly
interact with SNAPc through SNAP43 and also through SNAP50, as determined by co-

immunoprecipitation and Western analysis“.

20

2. Results and discussion

2.1. SNAP subunits - overview

The SNAPc is made out of five subunits: SNAP190, SNAP50, SNAP43,
SNAP45, and SNAP19. The main focus of my research was the crystallization of SNAP
subunits. SNAP19 being the smallest subunit was a perfect candidate for crystallization
and the expression levels were significant. A crystal form was also identified.
SNAPl90(1-505) that is capable of forming an active mSNAPc, was also successfully
over-expressed and purified and a few crystal forms were identified. SNAP43 was
expressed in significant levels, but the recovery of the material was notoriously
inefficient. SNAP50 was over-expressed, partially purified and low quality crystals were
obtained”. SNAP45 was over-expressed and partially purified, but the cleavage of the

GST tag resulted in protein over-digestion.

2.1.1. SNAP19

SNAP19 is the smallest subunit of the complex and is responsible for enhancing
the interaction between SNAP190 and SNAP43. The protein contains a leucine zipper
motif at its N-terminus and a glutamic acid rich C-terminus. The only known function of
the protein lies in its ability to bind to the N-terminal domain of SNAP190 and recruit

SNAP43 into the complex.

21

2.1.1.1. Over-expression, purification and crystallization of SNAP19

The protein was over-expressed in E. coli as a GST fusion peptide and purified
over GSH (glutathione) agarose (Figure 16, see Methods and materials for expression and
purification protocols). The protein material was further purified on Source-Q ion-
exchange column and the resulting highly purified material was estimated to be at least

95% pure by SDS-PAGE and Commassie stain (Figure 17).

 

2
11
<
Z
"P
'03 :3 S 3 :5 3 £3
2 O 0 o o 0 LU LLI
78.5 kDa
39 w w * -GST—SNAP19
32 _ -GST
16 -SNAP19
6

 

 

 

12345678

Figure 16: SNAP19 purified over GSH resin. Protein and beads suspension after
10 min (CLl), 20 min (CL2), 2 h (CL3) and 6 h (CL4) thrombin cleavage. Cleaved

SNAP19 (ELI and EL2, two successive fractions of the eluted material).

22

 

‘5
2 E Q Q l1!)
39kDa
32
16
6

 

 

 

Figure 17: Ion-exchange purification of SNAP19. Consecutive fractions
containing highly purified SNAP19 (IE1, IE2 and IE3). 15 mg of SNAP19 purified by
affinity chromatography were diluted 3 fold, to bring the salt concentration below 50 mM
and loaded onto Source-Q ion-exchange resin (15 mL). The protein material was eluted
from the column by a salt gradient (0-70%, 1 M KCl). 8-10 mg of highly purified

SNAP19 were recovered in two fractions (IE2, IE3).

The high expression levels and complete recovery of SNAP19 enabled
crystallization studies, and a crystal form was identified in a PEG containing condition.
Crystals were of extremely poor quality and optimization of crystallization did not
improve the crystal quality or morphology. The crystals obtained had an appearance of a
quasi-crystal, multi crystal cluster. Seeding experiments resulted in reduced time needed
for crystal formation and growth, but the morphology remained poor. Using X-ray

radiation, no diffraction pattern was observed for these crystals (data not shown).

23

 

2.1.1.2. Cloning, over-expression, purification and crystallization of SNAP19(1-85)
Since SNAP19 contains a glutamic acid rich region on its C-terminus with 10
successive glutamic acid residues, we hypothesized that this region might be responsible
for the low crystal quality due to this glutamic acid stretch, which is likely to be
unstructured. The C-terminal domain was removed by quick change PCR and the
resulting truncated version of SNAP19 was over-expressed and purified under identical
conditions to the wild—type (see Methods and materials) with similar recovery levels

(Figure 18).

 

 

 

 

 

a
33 92
e e
0. 2
g ”3
2 (I) E
:nga u”
M.
16 w,
6 I
It
1 2 3

Figure 18: Commassie blue stained SDS-PAGE of GST purified SNAP19(1-85)

(lane 2) and wild type SNAP19 (lane 3).

Crystallization of SNAP19(1-85) in sparse matrix screens (C51, C82, Mystic72,
Peg/pH) yielded crystals similar in appearance to the wild-type SNAP19. Further

optimization, by varying crystallization buffer ingredients and concentrations, of the

24

crystallization conditions including additive screening failed to improve the quality of

crystals (data not shown).

2.1.1.3. Nuclear magnetic resonance analysis of SNAP19

Crystallization of this protein was proven to be problematic as no crystals of
diffraction quality were obtained. SNAP19 with the molecular weight of 11 kDa is
sufficiently small to be a suitable candidate for NMR analysis. The high solubility and
high expression levels make SNAP19 an even better candidate for such studies.
Preliminary studies on this protein using dynamic light scattering (DLS) methods
suggested the protein to be a tetramer and therefore at the limit of practical structural
analysis by NMR (data not shown). In order to test whether the NMR spectra of SNAP19
would be of sufficient quality lH spectra of this protein were collected. The width of the
peaks was determined to be sufficiently narrow to allow for NMR structure determination
(Figure 19). Next, the cells expressing GST-SNAP19 were grown in M9 minimal media
supplemented with ”N ammonium chloride, to facilitate the expression of 15N labeled
protein. Although the expression levels of the protein in M9 media were severely
reduced, compared to the growth in LB medium, enough purified 15N labeled material

was obtained for NMR analysis.

25

 

 

 

 

 

 

 

ITTIIIIIIIIITIIIITIIIITIIIIIIIIIIITIIIIIIIIIII

9 8 7 6 5 4 3 2 1 0

 

 

PPm

Figure 19: lH NMR spectra of SNAP19.

15N Labeled SNAP19 was subjected to a 'SN-detected 'H-‘SN correlation
experiment; HSQC (heteronuclear single quantum coherence transfer) to assess for the
viability of the structural analysis of this protein using NMR techniques, since 15N
ammonium chloride is relatively inexpensive compared to 13C labeled carbon sources.
Although the peaks were relatively sharp, the dispersity of the amide proton (8-8.5 ppm)
peaks was low, suggesting that the protein is not very well folded. Folded proteins have a
high level of dispersity (6.5-10 ppm) of the amide protons due to different environments
of the proton. Unfolded proteins or peptides have low dispersity (8-8.5 ppm) due to

similar environments of the amide protons. According to these results it seems that

26

SNAP19 has very little tertiary structure and structure determination of such a protein is

at best complicated due to poor separation of the signals (Figure 20).

27

 

11.:
l

 

 

 

 

(ppmfi ’ '
1125 .
s. «r it
1142
E :
116f . '-
5 ,3 t
118; t. ;
120—:
122%
~a‘425-«za. .
E '0 '-)'°
126‘: ', f.
—: -..- ,
128% =7
130% . -'
J‘fll'Il'l'fl II II II [T I'TIITITTI

8.5 8.0 7.5 7.0 6.5 6.0

F2 (ppm)

Figure 20: HSQC (heteronuclear single quantum coherence transfer) spectra of

SNAP19.

28

We hypothesized that SNAP190 is required for efficient folding of the short
SNAP19 and that SNAP19 should, when mixed with the partnering region of SNAP190,
assume a more ordered tertiary structure. To test this, an excess of SNAP190 (84-133)
peptide was mixed with SNAP19. SNAP19, the SNAP190 peptide, and a mixture of the
two were all analyzed by NMR. Efficient binding and presumed folding of SNAP19
should be evident from the dispersity of the amide proton peaks in the 1H spectra.
Unfortunately, the spectra obtained for the mixture had similar dispersion of peaks to
those of SNAP19 and SNAP190 peptide alone, showing that SNAP19 does not have
much ternary structure even in the presence of SNAPl90(84-133), based solely on the

dispersion of the proton signal (Figure 21).

29

 

 

SNAPl90(84-l 33) M

SNAP19 W—J
Complex ,_/

Figure 21: 1H NMR spectra of SNAPl90(84-133) peptide, SNAP19 and the

 

 

 

complex obtained by mixing the two proteins.

2.1.2. SNAPl90(l-505)

SNAP190(1~505) was a second candidate for structural analysis from the SNAPc
family of proteins. The protein was expressed in sufficient quantity and efficiently
purified to high homogeneity. Crystallization trials were performed and a few

crystallization conditions were identified.

30

2.1.2.1. Over-expression and purification of SNAPl90(1-505)

The pGST190(1-505) plasmid was transformed in to E. coli BL21(DE3) Codon
Plus RIL cells and expressed at 16°C for 12-16 h. The protein was purified using the GST
purification protocol and the tag was removed by thrombin cleavage (Figure 22). The
protein was further purified using ion-exchange chromatography (Source Q).
Approximately 1.5-2 mg of protein was recovered in the last purification step per 1 L
culture. Greater than 95% purity was achieved as determined by SDS-PAGE and

Commassie staining.

Cleaved SNAP190(1 r.05) F1
Cleaved SNAP190(1-505) F2

GST—SNAP190(1—SOS)

 

200kD v—v
131 '

78.5

39 ‘3 1‘

32

 

 

 

Figure 22: SNAPl90(1-505) purification. GST-SNAPl90(1-505) immobilized on

the GSH agarose (lane 2) and cleaved and eluted material (lanes 3 and 4)

31

2.1.2.2. Crystallization of SNAPl90(1—SOS)

The protein was buffer exchanged in to a buffer (10 mM Hepes pH 7.5, 100 mM
KC], 3 mM DTT and 10% glycerol), concentrated to 4-5 mg/mL and sparse matrix
crystallization solutions were used to find a crystallization condition (30% 2-propanol,
0.1 M Hepes, 0.2 M Mng, pH 7.5) that resulted in formation of crystals with needle
cluster appearance (data not shown). The crystals passed the poke test and were labeled
as protein crystals. However, extensive screening of the condition did not result in an

improved crystal form.

2.1.2.3. Crystallization of SNAPl90(1-505)/SNAP19 complex

SNAPl90(1-505) was mixed with purified SNAP19 in a 1 to 1.2 molar ratio,
buffer exchanged in to a buffer (10 mM Hepes pH 7.5, 100 mM KC], 3 mM DTT and
10% glycerol) and concentrated to 3 mg/mL. Sparse matrix screening (C31, C82,
Mystic72, PEG/pH) was performed in search of a crystallization condition and a few
conditions yielding potential protein crystals were identified (30% 2-propanol, 0.1 M
Hepes, 0.2 M Mng, pH 7.5 and 30% PEG4000, 0.1 M Tris HCl, 0.2M MgClz pH 8.5).
The crystals obtained had the appearance of needle clusters and were highly fragmented.
Extensive optimization screening failed to find a condition with improved crystal size and

morphology (data not shown).

32

2.2. Over—expression and purification of SNAPc subunits

To facilitate crystallization and X-ray structural studies, individual SNAPc
subunits (SNAPl90(l-505), SNAP19, SNAP43 and SNAP50) were over-expressed as
GST fusion peptides in E. coli and purified on an affinity column using glutathione resin.
Although the expression of the proteins was satisfactory, the recovery of the protein
material from the affinity beads was often troublesome at best. Most SNAPc subunits
were inherently prone to aggregation, over—digestion by thrombin and precipitation.
SNAP19 was the only subunit that was straightforwardly recovered from the affinity
resin and further purified (Figure 24). Full length SNAP190 can not be expressed in E.
coli, but the truncated version SNAPl90(1-505) can. After successful immobilization of
this protein on the glutathione affinity resin, great care must be taken when thrombin is
used to cleave the protein material off the beads. SNAPl90(1-505) is extremely prone to
thrombin over-digestion, but was successfully recovered from the beads and further
purified using ion-exchange chromatography (Figure 23). In E. coli SNAP43 was over-
expressed in large quantities and immobilized on the GSH resin. The tag was successfully
removed by thrombin cleavage. Only minuscule amounts of this protein were recovered
from the beads and the recovered protein precipitated and adhered to purification beads
or reaction vessels (Figure 23). SNAP50 was also expressed in large quantities and
successfully immobilized to the resin. SDS-PAGE analysis of bound material revealed a
strong presence of another protein that appears to be about 60 kDa in size (Figure 24).
Removal of the tag and elution of the SNAP50 resulted in non-homogenous material with
the strong presence of the 60kDa protein. Further purification methods were unsuccessful

in removal of this contamination. Great care had to be taken when the tag was being

33

removed with thrombin cleavage as SNAP50 is also prone to ever-digestion and the
apparent single sized GST-fusion protein often resulted in several products smaller in size

compared to SNAP50, which were probably degradation products of SNAP50.

 

 

 

 

 

 

 

 

A a a B
A 0 UN
LA in .—
8 5 ‘3"
.- o ,_ m
25 ,0: a. Q X
e .. a z: .. a
2‘ g V‘ <1: <2: (I)
2 V‘ >' z m >'
(I) 5 8 (I) > 8
O ' O
p. U 2 I— U e
(n a) t: (11 cu c
2 (9 c: 3 2 (9 t: D
ZSOkD -+ ZSOkD F—
150 ' ~ 150 -—-
100 ’1- 100 a
75 -U 75 -"
- —
I".-
37 .-
37 -
‘ 2 3 4 1 2 3 4

Figure 23: Purification of (A)SNAP190(1-505) and (B)SNAP43 using GST
affinity chromatography. The proteins were purified from equal culture volumes
under identical conditions. The majority of SNAP43 can not be recovered and the
material that eluted off the beads sticks to the reaction vessel in a short time. The

material can be recovered by boiling in the sample buffer for SDS-PAGE.

34

 

 

 

 

 

 

 

 

o 8 0x
A g 22 9: a
W < Z " < Z
0. Z W CL 2 V)
< . < .
Z A > 2 J3 >
v: > 8 V.» > 8
I; 3 .7.
Q)
2 o a: D E o a: D
zsoko — $2ng a
150 "" ,00 u
100 .— 75 -
75 a... .. -GST—SNAPSO so -
SO - — -' ' -SNAPSO h
25 . -- - 'GST
37 -
20 4.
- 'SNAP19
’ 1s a "'
1 2 3 4 1 2 3 4

Figure 24: Purification of (A) SNAP50 showing the strong presence of an
unidentified protein that can not be removed by subsequent purification and (B)
SNAP19 using GST affinity chromatography. The proteins were purified from

equal culture volumes under identical conditions.

It is apparent that the isolated subunits share a few common characteristics. They
are prone to thrombin degradation, which is most likely due to structurally unstable
regions like exposed loops. Secondly, some subunits like SNAP43 and SNAP50 and to
some extent SNAP190( 1-505), were hard to recover after thrombin cleavage. SNAP43 is
virtually impossible to recover from the glutathione resin and different approaches were
tested to cope with this problem, but were unsuccessful. Only small amounts of SNAP43

can be successfully eluted from the beads and the quantity of the protein is only sufficient

35

for biological, but not for structural studies and the majority of the cleaved material
remained immobilized on the resin or adhered to the reaction vessel.

The mini SNAP complex, made from SNAPl90(1-505), SNAP50 and SNAP43
(mSNAPc) can be assembled from individually expressed and purified subunits.
Although the production of such material is at best inefficient, the amounts obtained and
the quality of the material were sufficient to perform DNA binding studies using U1 and
U6 DNA probes. The complex was capable of binding to the PSE region of the U1 or U6
DNA, as can be judged by the retardation of the mobility of radiolabeled DNA as was the
endogenous SNAPc isolated from HeLa cells. DNA binding was specific since it was

abolished when mutant PSE was used (Figure 25)”.

 

 

monoQ mtS NAPC
I 1 I fl r I
wt mu W1 mu wt ————’ mu
w - S NAPc
”an- - mtS NAPC

.7 Free
Jprotn

 

 

 

Figure 25: Electro mobility shift assay (EMSA) of the recombinant mSNAPc

(mrSNAPc) assembled from individually expressed and purified subunits, and

36

endogenous SNAPc obtained from HeLa cells (monoQ fractions). Increasing
amounts of mSNAPc that was assembled from subunits individually expressed in
E. coli (approximately 1, 2.5, and 7.5 ng; lanes 5—7, respectively) were added to
EMSA reactions containing dsDNA probes containing a wild-type (wt) or mutant
(mu) PSE, as indicated. Reactions loaded in lanes 3 and 4 contained 7.5 uL of
partially purified endogenous SNAPc (approximately 0.3 ng SNAPc/mL).
Reactions containing only the probe DNAs are shown in lanes 1 and 2

(experiment performed by Craig Hinkley) 58.

This complex was also tested for the ability to restore transcription from SNAPc
depleted nuclear extract. Surprisingly, the complex was unable to restore transcription
significantly for either U1 or U6 types of promoters (Figure 26). Endogenous SNAPc
isolated from HeLa cells (monoQ fractions) was in contrast capable of both DNA binding
and transcription initiation. The amounts of the material obtained from this expression
system are significantly lower than the amounts that can be obtained form bacterial

expression”.

37

 

E
5 a
S w
L ‘0
g 5 WCE a—SNAPC depleted
O r l
+ E g
w L” L” c + mrS NAP:
s—’ .w u o
? 5‘: '5 - E

 

 

-~ --—-a -U6 5'

   

 

 

 

Figure 26: In-vitro U6 transcription initiation assay of the recombinant mSNAPc.
Increasing amounts of mSNAPc were added to human U6 in vitro transcription
reactions for which the HeLa whole cell extract (WCE) was treated with on-
SNAP43 antisera to remove endogenous SNAPc, as shown in lanes 6—8. Lane 4
shows the decreased signal for the correctly initiated U6 transcription upon
removal of endogenous SNAPc. Lane 5 shows the U6 signal dependent upon
addition of endogenous SNAPc obtained from biochemical fractionation of a
HeLa cell nuclear extract. Note, this signal is comparable to the mock depleted

WCE (lane 3) or WCE alone (lane 1)”.

We hypothesized that the SNAPc components are unable to fold properly when
expressed separately and have exposed surfaces that render the material unstable and
sticky. Also, since co-expression using the baculo virus expression system yielded fully

active mSNAPc, we thought that co-expressing the subunits in E. coli could potentially

38

yield quantities sufficient for biological and structural studies. We also hypothesized that
the material obtained from a co-expression system would, unless specific secondary
modifications are needed, yield fully active complex similar to the baculovirus system,

but with increased expression levels.

2.3. Cloning of plasmids for co-expression

The goal of simultaneously expressing 3 or 4 subunits in the same cell can be
achieved by either using 3 or 4 plasmids with different antibiotic resistance, or by using a
polycistronic plasmid with several expression cassettes. We decided to use a combination
of the two approaches, since the number of compatible plasmids with different antibiotic
resistance is limited. Also, the strategy to ligate several genes in to the same plasmid,
targeting many different multiple cloning sites (MCS) is complicated. First, a theoretical
purification plan was devised that would enable rapid and efficient extraction and
purification of the co-expressed complex. Since all subunits were previously cloned in a
GST expression plasmid we decided to keep the existing plasmid with SNAPl90(1-505)
in the cloning site following the GST and thrombin recognition sequences (Figure 27).
Other subunits would be placed on different plasmids with different antibiotic resistance.
First, SNAP43 was cloned into pCDFDuet-l to form pCDF43-1 and SNAP50 was cloned
into pRSFDuet-l to form pRSFSO-l. Since pCDF43-l and pRSFSO-l carry the
information for streptomycin and kanamycin resistance. respectively, it is possible to
transform these two plasmids into the same E. coli cell with the pGST-190 plasmid
carrying the ampicillin resistance already present. pCDFDuet-l was also used to insert

the SNAP50 gene in to the multiple cloning site I (MCSI) and following that the SNAP43

39

gene was ligated into the second multiple cloning site (MCSII), resulting in the
pCDF43R/50—l expression plasmid (Figure 28). The SNAP19 gene was cloned in to
pRSFDuet-l to yield pRSF19-l (Figure 29).

Plasmids were sequentially transformed into E. coli, following calcium treatment
to make the cells competent (to make mSNAPcy3 pGSTl90(l-505) was transformed
first, then pCDF43R/50-l, and finally, to make mSNAPcy4 pRSFl9-l was transformed).
After successful insertion of the plasmid in to the bacterial host, the expression was
performed at 16°C and the proteins were extracted, purified using the GST affinity

column and analyzed (see Methods and materials for details).

mm a,
T7 7%

pGST190(1-505)

’6!
019d

 

Figure 27: The pGST190(l-505) plasmid with pET origin of replication and
ampicillin resistance. The SNAPl90(l-505) sequence follows the sequence for

the GST tag and the thrombin recognition sequence.

40

Different combinations of which gene is present in which cloning site and the
type of plasmid used were used and tested to check for the maximum efficiency of the co-
expression system. And although all subunits were expressed well in almost every case,
the different co-expression systems varied in the apparent stoichiometry of the subunits.

Good expression and recovery levels were obtained using the pGSTl90(l-505)
and pCDF43/50-l, with ampicillin and streptomycin resistances, respectively. The
complex (SNAPl90(l-505), SNAP50, and SNAP43) obtained using this co-expression
system is referred as mSNAPcyB. Better expression and recovery levels were obtained
using the pGSTl90(l-505), pCDF43/SO-1 and pRSFl9-1 with ampicillin, streptomycin
and kanamycin resistances, respectively. The complex (SNAPl90(1-505), SNAP50,
SNAP43, and SNAP19) obtained using this co-expression system is referred to as

mSNAPcy4.

41

Noo I BamH I Sac I

m del

T7 Sac |

(BamH l/Bgl ll)
Kpn I
pCDF43/50-1
6104 bp

800.00

 

Figure 28: pCDF43/50-l plasmid with CDF origin of replication and streptomycin

resistance. SNAP50 gene is in MCSI and SNAP43 is in MCSII.

42

T7

pRSF19—1
4092 bp

'00

 

Figure 29: pRSF19-l plasmid with RSF origin of replication and kanamycin

resistance with SNAP19 gene in MCSI and un-utilized MCSII.

2.4. Over-expression of mSNAPc variants

Once the plasmids were sequentially inserted in to the E. coli BL21-
CodonPlus(DE3)-RIL competent cells, the cultures were grown to the desired density and
over-expression was induced at different temperatures to determine the V optimal
expression conditions. 16°C was determined to be the temperature that yielded the most
material. The expression at increased temperatures resulted in a drastic reduction of the
target protein material. Both the mSNAPcy3 and mSNAPcy4 systems expressed all the

desired proteins as was determined by SDS-PAGE and Western blot analysis.

43

Different strains of E. coli were tested in search of the best co-expression host,
like BL21(DE3), CodonPlus-RIL, CodonPlus-RP, and pLysS (Stratagene). The
BL21(DE3) competent cells (genotype: E. coli B F' dcm ompT hst(rB” 1113—) gal 7L(DE3))
are an all-purpose strain for high-level protein expression. The BL21(DE3) pLysS
competent cells (genotype: E. coli B F' dcm ompT hst(rB" m3“) gal 1(DE3) [pLysS
Cam']) contain a pLysS plasmid with chloramphenicol resistance and provide tighter
control of protein expression of toxic proteins. The BL21-CodonPlus(DE3)-RIL
competent cells (genotype: E. coli B F' onsz hst(rB’ m3") dcm+ Tetr gal MDE3) endA
Hte [argU ileY leuW Cam']) contain a plasmid encoding rare codons for arginine,
isoleucine and leucine (argU (AGA, AGG), ileY (AUA) and leuW (CUA)) and provides
chloramphenicol resistance. The BL21-CodonPlus(DE3)-RP competent cells (genotype:
E. coli B F” ompT hst(rB' m3-) dcm+ Tetr gal MDE3) endA Hte [argU proL Cam'])
contain a plasmid encoding rare codons for arginine and proline (argU (AGA, AGG),
proL (CCC)) and provides chloramphenicol resistance.

mSNAPcy4 was successfully expressed in all types of E. coli with similar
amounts of the protein obtained, but with the highest amount of the mSNAPc being
expressed in the CodonPlus-RIL cells, as judged by immobilization and recovery of the
complex using the glutathione resin. It was observed from the amino-acid sequence and
the codon analysis that several subunits, especially SNAPl90(1-505) and SNAP50,
contain rare codons and could potentially lead to lower expression levels due to codon
bias.

Next, different cell culture growth media were tested in order to further optimize

the expression levels of partial SNAP complexes. LB and TB media, as well as M9

44

minimal media were tested by immobilization and recovery of the protein from the
glutathione resin. M9 minimal media resulted in hardly detectable levels of the complex,
as judged by Commassie blue staining of proteins separated by SDS-PAGE. LB and TB
yielded the highest levels of expressed material. The over-expression in TB had a two
fold increase compared to the expression levels for the cells grown in LB medium. The
density of the cells grown in TB media was about twice that of the cells grown in LB
media. Overall, the expression levels per cell are comparable for the two growth medias
(data not shown).

The time of [PT G induction was also studied to further improve the
expression levels of the mSNAP complexes. Different times of induction, defined by the
cell density in the growth culture, were tested in the range of 0.1 to 1.4 optical density
(ODooo). After the induction the cell culture was grown for 12—16 h at 16°C and the
amount of the expressed material was evaluated using the GST purification scheme. It
was determined that the expression levels of the protein are in direct proportion to the cell
density at the time of induction. When the cell culture was induced with IPTG at an
OD600 of 1.4, almost double amount of the mSNAPc was obtained as compared to the cell
culture induced at an ODooo of 0.7. The amount of cells obtained, when the culture was
induced at an OD600 of 1.4 was also doubled (data not shown). It seems that the mSNAPc
components are non-toxic to E. coli and the expression level per cell is constant when
grown at identical conditions and independent of the induction point. The only
consequence of the different induction point is the final cell density and therefore the
final amount of the produced material and not the actual amount of protein per cell as is

usually the case when the proteins are even slightly toxic to the cell.

45

The temperature of induction was studied. And the complex over-
expressed only at 16°C. Expression at 24°C or 38 °C resulted in diminished expression

levels of mSNAPc.

2.5. Comparison of the individual expression of SNAPc subunits to the co-expression

system

Cell cultures expressing the mSNAPcy4 or individual subunits were grown under
identical conditions and the expressed material was analyzed by Western blotting method
using the antibody directed against the specific subunit of interest. The SNAP50 and
SNAP43 subunits expressed well in both the individual and co-expression systems.
Interestingly the amount of leaky expression for these two proteins is relatively high in
the co-expression system. The individual expression systems yielded no expressed
protein before induction (Figure 30, panels A and B). The level of the protein in the
insoluble or soluble fractions is similar for the two systems. The relatively high amount
of the material in the insoluble fraction is most likely due to insufficient rinsing of the
material and may not be a measure of the total material actually in the insoluble form.
The amount of the target proteins is also comparable between the two expression
systems, suggesting that expression was not drastically improved. Nevertheless, the
expression levels for both SNAP43 and SNAP50 are elevated in the co-expression system

compared to the individual expression.

46

 

 

 

 

 

 

 

 

 

8 8 a:
0 Q 0 U _
a i? :3 % § 8 3 g
,5 a o 2 'E 8 8 a
5 2 a a a E ‘-= w
r ‘ - -J -GST-SNAP43 l - -—-GST-SNAP50
T CW ' ‘ "‘_—iw".—T_‘
‘- O - -i - SNAP43 in L“: - - -l-SNAP50 in
‘ “’“WW‘” " mSNAPc./4 mSNAPc/4
A B
3 8
8 8 g 2 3 8 £3 2
‘0 o .2 D “O o 2 .o
.9 2 o .2 E 3 0 2
5 E E 8 5 E ‘5” (1°)
l m‘osr-SNAmgom-wm i .. -u- -GST—SNAP19
[1 __-L________ ~ _ GST-SNAP190(1-505) in i _
l : ___. z} mSNAP“ . -SNAP19In
--—~~- ** mSNAPcy4
C D

Figure 30: Analysis of expression for SNAPc subunits in the individual
expression and co-expression approach by Western blot. GST labeled protein

were visualized using Ot-GST antibodies and the SNAPc subunits were visualized

using antibodies directed against the subunit of interest.

The expression levels of GST-SNAPl90(l-505) are to some extent elevated in the
co-expression system compared to individual expression. Leaky expression was not
observed for this protein and the material is mostly in the soluble fraction of the cell
extract (Figure 30, panel C). In contrast the amounts of SNAP19 in the co-expression
system were not detectable in the cell extract using Western blot. The levels of this
protein are significantly reduced compared to the individual expression system (Figure

30). SNAP19 is the smallest of the subunits and to achieve complex formation where the

47

subunits are equimolar, a smaller amount of SNAP19 is needed compared to the other

subunits, so a complete mSNAPcy4 complex could still be assembled.

2.6. Purification of mSNAPc

Since the SNAPl90(l-505) contains a cleavable linker and a GST tag, the soluble
culture extract harboring mSNAPCy3 or mSNAPcy4 was clarified and allowed to bind to
the glutathione resin to immobilize the expressed complex. Other subunits were not
tagged, yet they were efficiently immobilized thru the GST-190(l—505). Different
purification conditions were tested to explore different approaches to achieve highest
purity of the material in the first step of purification. Parameters like salt, detergent and
glycerol concentration were varied and the purity of the immobilized material was
evaluated with SDS-PAGE and silver staining. It was determined that the detergent
(Tween-20) is absolutely critical for the extraction of the complex from E. coli since
virtually no material was immobilized when Tween-20 was not present as judged by
SDS-PAGE and Commassie blue staining. The Tween-20 could be replaced with n-octyl-
beta—D-glucopyranoside (BOG) with slightly reduced extraction levels in this purification
step. The salt concentration was varied from 150 to 500 mM with the highest purity
achieved when higher salt is used. Lower salt concentration leads to increased presence
of impurities. The bound material can be efficiently removed from the beads by
glutathione elution or by thrombin cleavage of the linker connecting the SNAPl90(l-
505) and the GST tag. In comparison to the GST purification step of some SNAPc
subunits, like SNAP43 or SNAP50, the material was almost fully recovered from the

resin (Figure 31). Furthermore, the relative intensities of the bands observed on the

48

Coomassie stained SDS-PAGE suggest that the subunits are equimolar, although in some
cases the intensity of SNAP50 was weaker than the intensities of SNAPl90(1-505) or
SNAP43. The co-expressed material is also more stable during thrombin cleavage as
relatively high concentration of thrombin can be used for extended periods of time
without ill effects on the complex composition. When the optimal cleavage conditions
were investigated, no truncated products were observed even after three-day cleavage
with 200 units of thrombin at 4°C (6 L cell culture). On the other hand, the individually
expressed subunits were extremely sensitive during thrombin cleavage and prone to over
digestion. More importantly, the co-expressed material was successfully recovered from
the resin, which was not the case for SNAP43 where relatively small amounts of the

protein were recovered (Figure 32).

 

 

 

 

w;
*3 J
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Y. 5’ <
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100 ‘5'."
so -7", - ' :SNAPSO
SNAP43
37 -
25 - .— -csr
20 -
-SNAP19
IS .
I 2 3 4

Figure 31: Purification of co-expressed mSNAPcy4 using the GST affinity resin
and thrombin cleavage. The mSNAPcy4 was purified under the same conditions

as the individual subunits.

49

SNAP190(1-5051
unrecov SNAP43
mSNAPC/B
mSNAPcyd

SNAP50
SNAP19

 

- - ~ - —- SNAP 'l 90(‘1—505)

~ "0 -' SNAP50
- . —SNAP43

- -- SNAP 19

 

 

 

Figure 32: Recovery of the co-expressed material compared to individually
expressed and purified subunits. SNAP19 improves complex stability and subunit
recovery. Subunit recovery for partial SNAPC assembled without and with co-
expressed SNAP19 (mSNAPcy3 and mSNAPcy4, respectively) was estimated by
SDS—PAGE and Coomassie blue staining. Note the increased recovery of
SNAP43 and SNAP50 in mSNAPcy4 (lane 7) compared to mSNAPcy3 (lane 6).
Lanes 2—5 contain aliquots of the individually expressed SNAPc subunits for
reference. Note that the unrecovered SNAP43 is used to show the migration of

SNAP43.

The recovered material was further purified using ion-exchange chromatography.
Cation and anion (SourceQ and SourceS, Pharmacia) exchange resins were tested in a
wide array of purification conditions. The complex had the tendency to bind to the ion-
exchange resin, but the recovery of the protein material was often accomplished only

with severe losses (data not shown). Optimal purification conditions were eventually

50

determined and the detergent Tween-2O was again essential for this purification.
Surprisingly, addition of magnesium ions to the purification buffer resulted in even better
recovery rates. The protein does, however, elute off the column over a broad range of salt
concentrations and the purity is not greatly improved compared to the GST purification
step alone.

The co-expression and GST purification scheme using the GSH resin works well
with both mSNAPcy3 and the mSNAPcy3 and is a major improvement compared to
individually expressed and purified subunits or the baculo—virus co-expression system,

which resulted in low amounts of material or material of poor purity.

2.7. DNA binding activity of mSNAPc variants

The mSNAPcy4 was tested for DNA binding activity for both U1 (Figure 33 lane
11) and U6 (Figure 33 lanes 2, 3) promoters in an electrophoretic mobility shift assay
(EMSA) and was similar to the assembled recombinant mSNAPc active for DNA binding

to both types of promoters”.

51

PSE [TATA was JAIA lPSE‘w-mu-mw—W PROBE

r 1

 

---+---+---+---+TBP

- 4‘-.4 'A'AmSNAPC‘M

 

«mSNAPcy4/TBP
4—mSNAPcy4

   

 

 

 

12 3 4 5 6 7 8 910111213141516

Figure 33: mSNAPcy4 facilitates TBP promoter recruitment. Increasing amounts
of mSNAPcy4 (3 and 10 ng) were added to DNA binding reactions containing
radiolabeled probes that harbor a wt PSE and wt TATA box (lanes 1—4), mu PSE
and wt TATA box (lanes 5—8), wt PSE and mu TATA box (lanes 9—12), or mu
PSE and mu TATA box (lanes 13-16). Lanes 4, 8, l2, and 16 contain 50 ng of
recombinant full—length human TBP in addition to 10 ng of mSNAPcy4.
Reactions containing only the DNA probes are shown in lanes 1, 5, 9, and 13.
Positions of the mSNAPcy4 and mSNAPcy4 plus TBP complexes are indicated

(experiment was performed by Gauri W. J awdekar)5 8.

The complex was also tested for the ability to recruit TBP to the promoter and
was capable of doing so in the case of the U6 promoter (Figure 33 lane 4) but was unable

to recruit TBP in the absence of the TATA box, as is the case in a U1 type of snRNA

52

promoter (Figure 33 lane 12). TBP alone was not capable or binding to the DNA in the
absence of the SNAP complex, under the experimental conditions, regardless of TATA
box presence. mSNAPcy4 binds to the promoter region of the DNA specifically and

mutations in the PSE region abolish the interaction”.

2.8. mSNAPcy4 is capable of recruiting TFIIIB to the promoter

The co-expressed mSNAPcy4 was tested for its ability to recruit Brf2-TFIIIB to
the U6 promoter. Human TBP, Brf2 and de1(l-470) were mixed with mSNAPc and the
labeled U6 DNA probe. In the absence of mSNAPc, Brf2-TFIIIB did not bind to the
DNA (Figure 34, lanes 2-7) or did so weakly (Figure 35, lanes 5 and 7). The Brf2-TFIIIB
complex bound the probe in the presence of mSNAPc. Migration of mSNAPc was altered
in the presence of TBP (Figure 34, lane 9), but not by addition of Brf2 or de1 alone
(Figure 34, lanes 10 and 11), suggesting that TBP is essential for subsequent recruitment
of Brf2 and del and that mSNAPc through direct protein-protein interactions stabilizes
the TBP/DNA interaction. A supershift was also observed when Brf2 was added to the
mSNAPc/TBP/DNA complex, but not when del was added in the absence of Brf2
(Figure 34, lanes 12 and 13). The presence of both Brf2 and del(l-470) resulted in a
band migrating slower than mSNAPc/TBP/DNA and mSNAPc/TBP/Brf2/DNA (Figure
34, lane 15, Figure 35, lane 12 ). Specific protein-protein contacts are involved in the
formation of the mSNAPc/Brf2-TFIIIB/DNA complex and the complex is assembled in

the order mSNAPc-DNA-TBP-Ber-de15°.

53

mSNAPcy4"'-"'++++++++

de1 (1-470) ‘ ' ' + ' + + ' ' ' + ' + + +
8er - + - + + - + - + + +
TBP + - + + + + - + + - +

 

mSNAPc/TBP/Ber/del

— mSNAPchBP/Brf2
. ‘ —\—mSNAPcffBP
_\- mSNAPc

’g

 

 

Free
Probe

12 3 4 5 6 7 8 9101112131415
Figure 34: mSNAPcy4 is capable of recruiting TFIIIB to the promoter.
Coordinated DNA binding by SNAPc and TBP facilitates higher order complex
assembly with Brf2 and de1. DNA binding reactions were performed with the
indicated combinations of SNAPCy4 and Brf2-TFIHB subunits. These results
suggest that preinitiation complex assembly follows the order

SNAPc>TBP>Brf2>del (experiment was performed by Gauri W. J awdekar)”.

54

- mSNAPcy4 + mSNAPcy4

 

de1(1-47o)--- +- +++ ---+
Br12 - - + - + - + + - - + +
TBP ' + ' ' + + + ' ' + + +7

 

mSNAPc/TBP/BerIdel
TBP/Brf2/de1 mSNAPcrrBP/Brf2
" mSNAPcfl'BP
TBP/Ber

mSNAPc

 

 

 

     

12345678 9101112

Figure 35: SNAPc stimulates DNA binding by Brf2-TFIIIB. Electrophoretic
mobility shift assays were performed using a U6 probe containing a wild-type
mouse U6 PSE and a wild-type TATA-box (AC probe). DNA binding was carried
out in the absence (lanes 1-8) or presence (lanes 9-12) of wild type SNAPCy4.
Reactions containing individual TBP, Brf2, and de1 (1-470) subunits are shown
in lanes 2-4. Reactions containing pair wise combinations of TBP with Brf2, TBP
with de1 (1-470), and Brf2 with de1 (1-470) are shown in lanes 4, 5, and 8.
DNA binding by the complete Brf2-TFIIIB complex in the absence of SNAPc is
shown in lane 7. Additional reactions were performed with SNAPc alone (lane 9)
or in combination with Brf2-TFIIIB subunits (lanes 9-12), as indicated. Lane 1

shows migration of the probe alone. The relative positions of the various

55

SNAPcy4/Brf2-TFIIIB complexes are shown on the right (experiment was

performed by Gauri W. J awdekar)”.

2.9. Transcription initiation mediated by mSNAPc

mSNAPcy4 was then tested for its ability to initiate transcription from both U1
and U6 promoters in an in-vitro transcription initiation assay and was, in contrast to the
recombinant mSNAPc (assembled from individual subunits), active for transcription

initiation from U1 (Figure 36) and U6 (Figure 37) promoterssg.

 

 

 

 

U
(D
.‘g a-SNAPC depleted
a) F I
"C? +mSNAPCy4
- E - F
«*4 M * H...“ «who H
1 2 3 4 5 6 7 8 9 10

Figure 36: mSNAPcy4 functions for U1 snRN A transcription by RNA polymerase
II. HeLa cell nuclear extract was either mock depleted with a preimmune rabbit
sera (lane 2) or a-SNAP43 antisera (lanes 3—10) to deplete endogenous SNAPc.
Extracts were then used for human U1 in vitro transcription assays. The U1-
specific signal was diminished upon removal of endogenous SNAPc, as shown in
lane 3. Increasing amounts of mSNAPcy4 (0.08, 0.25, 0.75, 2.5, 7.5, 25, and 75
n g) reconstituted the correctly initiated transcription from a human U1 reporter, as

shown in lanes 4—10. Lanes 1 and 2 show the U1 signal obtained from either

56

untreated or mock depleted reactions (experiment was performed by Gauri W.

J awdekar)5 8.

Ot-SNAPC depleted

r I

_ - +mSNAPcy4 -
a *“*G'I

12 3 4 5 6 7 8 910

 

+ GST

 

-U6 5'

 

 

Figure 37: mSNAPcy4 functions for U6 snRNA transcription by RNA polymerase
III. In vitro transcription of human U6 snRNA was carried out using HeLa cell
nuclear extract that was treated as in Figure 36. Lane 2 shows the reduced U6
signal upon removal of endogenous SNAPc. Increasing amounts of mSNAPcy4
(0.08, 0.25, 0.75, 2.5, 7.5, 25, and 75 ng) reconstituted the correctly initiated
transcription from a human U6 reporter as shown in lanes 3—9. Approximately 75
ng of GST was added to the transcription reaction shown in lane 10 (experiment

was performed by Gauri W. J awdekar)5 8.

2.10. Zinc finger domain in SNAP50

The C-terminal region of SNAP50 is cysteine and histidine rich and is a potential
Zn finger domain based on amino-acid sequence, which might be involved with protein-
DNA or protein-protein contacts. DNA binding domains sometimes consist of multiple
repeats of related zinc finger motifs. The SNAP50 zinc finger domain however contains

an unusual arrangement of six histidine and nine cysteine residues that can be grouped

57

into region I that resembles a TFIIIA—like C2H2 zinc finger and region 11 that resembles a
glucocorticoid-like C2C2 zinc finger.

Sequence alignment of the C-terminal region (301-411) of SNAP50 with the
SNAP50 homologues from different mammals, fish, insects, worms, plants, slime mold
and parasites, revealed two highly conserved potential zinc finger domains (Figure 38).
Interestingly the residues from region I are highly conserved, with the exception of C312,
which is conserved in mammals, but not in lower organisms. The fact that SNAP50 is
highly conserved in lower organisms suggests that this subunit plays an important role in
the transcription initiation mechanism. Region 2 is invariantly conserved in all SNAP50
homologues and the four conserved cysteine residues are similar to steroid receptor
(szszszzC) type zinc fingers. Overall the consensus motif Lx4Gonx3CxHx20-
23YPx1I-1szsz18Px3-4Cx2CFx3Hx1-4G is unlike any other family of zinc fingers

described”.

58

 

 

 

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r....m......m.m.o.593:¢ ...... e ......... we ............ 0:0 .................. m..30..nm..o=....m ...................

Figure 38: Sequence alignment of human SNAP50 C-terminal amino acids (301-

411) with corresponding regions from SNAP50 homologues of other species.

59

Putative zinc fingers similar to TFIIIA (Hstmex3C) and steroid receptors
(szszszzC) are indicated as region 1 and region 2, respectively. The sequence
of highly conserved amino acids derived from this alignment corresponds to
LX4GX6HX3CXHX20-23YPX1l—12CX2CX18PX3-4CX2CFX3HXl-4G- This alignment was
performed using the Clustal W program. Homo sapiens (NPOO3075), Cannis
familiaris (XP853813), Bos taurus (AAX08912), Mus musculus (NP084225),
Rattus norvrgicus (NP001013230), Drosophila melanogaster (NP724647),
Drosophila pseudaoobscura (EAL25490), Trypanosoma brucei (XP827295),
Arabidopsis thaliana (AAO30067), Caenorhabditis elegans-l (NP500819),
Caenorhabditis elegans-2 (NP497807), Plasmodium falciparum (), Danio rerio
(XP694501), Leishmania major (XP843572), Anopheles gambiae (XP310411),
Dictyostelium discoideum (XP644064), Entamoeba histolytica (XP653151). This

comparison was prepared by Gauri W. J awdekarsg.

2.10.1. ICP-MS and FAAS analysis of mSNAPc variants

SNAP50 contains a potential zinc finger domain. Since SNAP50 is the only
subunit of SNAPc containing a Zn finger motif, the SNAP50-containing mSNAPcy4 and
mSNAPc-73 were used in the metal analysis study to confirm the presence of a zinc atom.
The complex was co-expressed in E. coli and extensively purified. The material was
digested in 2% nitric acid for the inductively coupled plasma mass spectrometry (ICP-
MS) or ashed for flame atomic absorption (FAAS) analysis. 1 zinc atom per molecule of
the complex was determined with the ICP-MS method, where two isotopes 66Zn and 68Zn

were quantified. The FAAS determination of zinc was in good agreement with the ICP—

60

MS study. Both mSNAPcy4 and mSNAPcy3 complexes were determined to contain 1 Zn
atom per molecule of the complex, as they yielded 0.90 and 0.89 moles of zinc per mol of

the complex respectively (Figure 39, Table 1).

1200 .

1000 -

800 .

600d

Ila“) (ppb)

400 r

200 ~

 

 

 

0 0.005 0.01 0.015 0.02 0.025

absorbance

Figure 39: Flame atomic absorption spectroscopic analysis of mSNAPc'y3 and
mSNAPcy4. Unlabeled data points represent the absorption obtained for standard zinc

solutions.

Two separately prepared samples were used for ICP-MS analysis and yielded 1.1
and 1.2 equivalents of zinc and are in agreement with previously determined zinc content
that utilized the flame atomic absorption. Nickel was also measured during this

experiment and the signal for this metal was on the level of the background noise and

61

insignificant. Nickel is therefore not present in the protein sample. Although two
potential metal binding domains were identified in the C-terminal region of SNAP50 by
ab-initio modeling, the presence of only one zinc atom was determined experimentally. It
is possible that a second metal is still present and required when the complex interacts

with the DNA (Table 1).

Table 1: Quantification of zinc and nickel using ICP-MS and FAAS for
mSNAPcy3 and mSNAPcy4. Two isotopes 66Zn (3) and (’an (b) were measured for ICP-
MS. Two independently prepared samples of mSNAPcy4 were used in the analysis: (1)
extensively purified complex used for crystallization and (2) void fraction from gel
filtration purification of the complex. The protein concentration was determined by

absorbance measurement in 6M urea and by the Bradford assay.

 

 

 

 

Sample [Zn] ppb [Ni] ppb [fig/{:13} n(Zn)/n(prt) n(Ni)/n(prt)
mSNAPcy4 (1) 31250.4 - 2.62 1.16 -
b1268.5 - 2.62 1.14 -
g - 17.9 2.62 - 0.02
E: mSNAPcy4(2) a1415.5 - 2.90 1.18 -
b1443.6 - 2.90 1.17 -
- 35.5 2.90 - 0.03
‘2 mSNAPcy3 1519.7 - 3.86 0.89 -
E mSNAPcy4 1827.3 - 4.95 0.90 —

 

62

2.10.2. Ab-initio modeling of SNAP50 C-terminus

Since an experimental structure of SNAP50 is not available, computational
methods were employed by Dr. Michael Feig to predict the structure of the C-terminal
domain of SNAP50 so that the mechanism of SNAP50 function could be better
understood. Because of the unavailability of a structural homolog, an ab-initio approach
using the predicted secondary structure and the amino acid sequence was taken. This
method is possible because the SNAP50 finger domain region is sufficiently short.
Interestingly, one of the structures produces by this ab-initio method had a convincing
arrangement of cysteine and histidine residues for coordination of one zinc atom,
however some of the conserved residues could, although unlikely, also coordinate

another metal atom like zinc, nickel or iron”.

2.10.3. Mutagenesis

In order to examine the function of the zinc finger domain in SNAP50 the
cysteine and histidine residues were changed to alanine. The presumption that the zinc
finger domain is responsible for the DNA interacting function of SNAP50, was tested by
the DNA binding assay. HA-SNAPSO with selected cysteine or histidine single point
mutation to alanine was co-expressed with GST-SNAPl90(l-505), full length SNAP43
and SNAP19. The recombinant complexes were immobilized to the glutathione resin,
washed extensively and released from the beads by the cleavage of the linker with
thrombin. Since the presumption was that the mutation would only affect the binding of
the complex to the DNA and not the actual formation of the complex, GST pulldown and

co-immuno precipitation experiments were performed to confirm the existence of the

63

mSNAP complex. The complex was purified using the glutathione beads in the first step
of the purification. Since only SNAPl90(l-505) contains a GST tag, all the other
subunits were obtained through this subunit. Next, the partially purified complex,
containing SNAPl90(1-505), SNAP43, HA-SNAPSO and SNAP19, was treated with
anti-SNAP43 antibody cross-linked to agarose beads, thus allowing specific
immobilization of the complex through SNAP43 only. The obtained material was tested
for the presence of HA-SNAPSO by western blot targeting the HA tag on SNAP50. The
presence of HA-SNAPSO, proves that different point mutations of the C-terminus of

SNAP50 did not change the consistency of the complex (Figure 40) 59.

rr-SNAPAS 1P

 

 

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1 2 3 4 5 6 7 8 9 10 11 12 13 .14 15 16 17 18 19 20 21 22

Figure 40: Mutations in the SNAP50 zinc finger domain do not disrupt SNAPc
assembly. Approximately 20 ng of each of the SNAPcy4 complex containing substitution
mutations in HA-SNAPSO were affinity purified first using glutathione agarose to pull
down GST-SNAP190 (1-505) followed by immunoprecipitation with a-SNAP43
antibodies. Associated wild type or mutant HA—SNAPSO was detected by OL-HA Western
analysis (lanes 6-22). A titration of wild type SNAPcy4 using 8, 4 and 2% of the input
material is shown in lanes 1-3, respectively. Lanes 4 and 5 contain wild type SNAPcy4

recovered with the protein-G agarose beads alone or with pre-immune serum,

64

respectively. The bottom panel represents 4% of the input material directly analyzed by

OL-HA Western analysis (experiment was performed by Gauri W. J awdekar)”.

2.10.4. DNA binding activity of SNAP50 mutants

The mSNAPcy4 harboring HA-SNAPSO with different point mutations was tested
for DNA binding activity. A PSE-containing DNA probe was used for the assay. Point
mutations in the evolutionarily conserved ngCxH motif, H3l3—A, C317-A and H3l9-A
resulted in reduced DNA binding as compared to the wild type. Interestingly point
mutations C354-A, C357-A, C380-A and C383-A in the conserved CXZCx23Cx2C domain
exhibited complete abolition of the DNA binding activity of the complex. Furthermore
the mutation of the last evolutionarily conserved residue in the C-terminal area of

SNAP50 the H388-A also resulted in reduced DNA binding activity (Figure 41) 59.

mSNAPcy4
1 fl

C-xg-C-----H-x3-C--x--H-xic-H---H-x2--C-\'12-H-x6-C-x2-C-x:9-C-x2~C-x2-C ----- x4 ----- H

m C3028 C312-A 14313». C317-A P3197? 433371 H331-A casd-A H347-A c.354-A C357~A C377-A C380—A 3383A Rae‘s-A 1—358-A
AAA‘AA‘AA‘AAAA‘A A

 

 

“a...“ - qm 1* fl "

 

 

12 3 4 5 6 7 6 91011'213‘4151617181920212223242525272829333132333435

 

Figure 41: Mutations in the SNAP50 zinc finger domain disrupt DNA binding by

SNAPc. Increasing amounts (3 and 10 ng) of SNAPcy4 with wild type (lanes 2 and 3) or

65

mutant HA-SNAP50 (lanes 4-35) containing the indicated substitution mutations was
tested in an EMSA for binding to a radiolabeled DNA probe containing a high affinity
PSE and TATA box (AC probe). Lane 1 shows the probe alone with no added proteins.
Mutations H313-A, C317-A, and H319-A resulted in reduced DNA binding, whereas
mutations C354-A, C357-A, C380-A, and C383-A completely abolished DNA binding
activity. Mutation H388-A also exhibits weakened DNA binding activity (experiment

was performed by Gauri W. J awdekar)”.

2.10.5. Transcription initiation assay

Next, the wild type or mutant HA-SNAPSO containing mSNAPcy4 was tested in
an in-vitro transcription initiation assay. HeLa cell nuclear extract was first depleted of
mSNAPc with anti-SNAP43 antibodies and then transcription was tested in the presence
of purified mSNAPCy4 containing either wild type or mutant HA-SNAPSO. As expected
the mutations that showed abolished DNA binding activity also resulted in abolished
transcription initiation for both U1 and U6 types of promoters. The addition of the
mSNAPc with SNAP50 point mutants C354-A, C357-A, C380-A and C383-A in the
conserved szCx23Cx2C domain were completely unable to restore transcription for both
types of promoters (Figure 42; lanes 13, 14, 16 and 17). Interestingly, the mutations on
the Hx3CxH motif, H313-A, C317-A and H319-A, resulted in complete inability to
restore transcription from the U1 snRNA promoter. Transcription, albeit weakened, was
still evident for the U6 system (Figure 42; lanes 6, 7 and 8). The SNAP50 H388-A
mutant was capable of wild type transcription from the U6 promoter, but its activity was
severely reduced in the U1 system (Figure 42; lane 19; experiment was performed by

Gauri W. J awdekar)5 9.

66

mSNAPc/4

 

 

 

 

 

 

 

 

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1 2 3 4 5 6 7 8 9 10 11 12 1314 15 1617 1819

Figure 42: U1 and U6 in-vitro transcription initiation assay of wt or mutant HA-
SNAP50 containing mSNAPcy4. Mutations that resulted in reduced DNA binding also
showed reduced transcription initiation activity for both types of promoters, with the
exception of the H388-A mutation, which resulted in weakened U1 activity, but wild type
U6 activity of transcription initiation59. HeLa cell nuclear extract was depleted with 01-
SNAP43 antibodies to immunodeplete endogenous SNAPc. In vitro U1 and U6
transcription was then tested in the absence (lanes 1) or presence of purified SNAPcy4 (5
ng) containing wild type SNAP50 (lane 3) or mutant SNAP50 with the indicated alanine
substitutions (lanes 4-19). Addition of GST alone did not reconstitute either U1 or U6

transcription as shown in lane 2 (experiment was performed by Gauri W. Jawdekar)”.

2.11. Crystallization of mSNAPc variants

The mSNAPcy3 and mSNAPcy4 complexes obtained from the GST and ion
exchange purification schemes was each concentrated to approximately 4.5 mg/ml and
screened for crystallization using various crystallization screens, namely the Crystal
Screens I and 11 (Hampton Research) and the PEG/pH grid screen. At room temperature,

needle clusters were obtained in a variety of crystallization conditions. Most conditions

67

had magnesium chloride present as a salt additive and a PEG as a precipitating agent. The
crystals grew in a wide range of pH; from pH 7 to pH 10. The crystals did not appear to
be single under the light microscope and the size was insufficient for X-ray diffraction

studies (Figure 43).

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Figure 43: Crystals of mSNAPcy4 as observed under the light microscope grown

in 9% PEGSOOOMIVIE, 100 mM Tris pH 8.5, 100 mM NaCl and 100 mM MgCl2.

68

 

Figure 44: Optimized crystals of mSNAPcy4 as observed under the light
microscope grown in 8% PEGSOOOMME, 100 mM Tris pH 8.4, 100 mM NaCl

and 100 mM MgC12.

Optimizations of the crystallization condition eventually lead to a condition where
the crystals were larger, but the quality was not sufficient for X-ray crystallographic
studies due to small size and morphology (Figure 44). Additive screening and
replacement of all components of the crystallization condition with their analogs were
tried. The effect of temperature on the crystallization was also investigated, by attempting
to grow the crystals at 4°C. The crystals failed to appear at lower temperature even after
extended periods of time. Further crystallization optimizations were unsuccessful,

therefore micro and macro seeding experiments were performed, again without success.

69

2.12. X-ray diffraction of mSNAPc crystals

A needle cluster was cryogenically protected and tested for diffraction at the
synchrotron facility. Rings at low resolution were observed, similar to a powder
diffraction pattern. The result was surprising, since the crystals forming the needle cluster
were relatively small. The conclusion that can be drawn from this experiment is that the
crystals, albeit small and non-single, still diffract strongly (data not shown). A single
crystal of larger dimensions and better quality could potentially diffract strongly and to

high resolution.

2.13. Evaluation of mSNAPc crystals using transmission electron microscopy (TEM)

Since all crystallization optimization efforts did not result in improved crystal size
and quality, and neither did the seeding experiments, we decided to observe the crystals
under the TEM and investigate the morphology of crystals. The crystal structure of a
protein of this size can not be determined using electron diffraction, but some interesting
crystal parameters, like the unit cell dimensions, could.

The crystals were loaded on the EM grid and observed under TEM. Some crystal
like formations were observed, but they were destroyed by the powerful electron beam.
In order to be able to observe the crystals under the TEM, they were first cross linked
with a formaldehyde and glutaraldehyde mixture, washed with water and then evaluated

with the electron microscope.

7O

The crystals observed were surprisingly not needle like, but appeared rectangular
in nature and needle formations were built out of the small rectangles. What appeared to
be needle clusters under the light microscope were actually large aggregates of small

crystals (Figure 45, Figure 46).

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Figure 45: Needle clustered crystals of mSNAPcy4 under the TEM have a heavily

branched appearance and the branches are even further sectioned.

71

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Figure 46: Crystals of mSNAPcy4 under the TEM. Close—up on one of the

branches showing a heavily fragmented formation.

72

 

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Figure 47: Circular diffraction pattern typical for a powdered material.

When observed in diffraction mode, these crystal clusters diffracted with powder

diffraction like pattern as can be seen by the appearance of the circular pattem (Figure

47).

2.14. Diffraction of mSNAPc crystals using TEM

A few small single crystals were found under the TEM (Figure 48) and the

diffraction aperture was set to select only one crystal. These crystals diffracted like a

73

single crystal and a different diffraction pattern was observed when the grid with the

crystal was rotated for approximately 0.5 degrees (Figure 49).

  
 
  
  
 

 
  
   
   
 

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Figure 48: TEM image of mSNAPcy4 single crystals as observed under TEM.

 

74

 

A B

Figure 49: TEM diffraction images obtained from a single crystal. Image B was
obtained from a crystal rotated approximately 0.5 degree with respect to the

crystal from image A.

Next, mSNAPcy4 was mixed with PSE-containing DNA and
crystallized under similar conditions. The crystals had a similar appearance as the crystals
of the complex alone as observed under the light microscope. Crystals were cross-linked
and analyzed with electron microscopy. The appearance of these crystals was improved
compared to the crystals of mSNAPcy4 alone, but the crystals were still fragmented and
not single. Branch like single crystals were observed, emerging from a stalk (Figure 50).
When the electron beam was focused on a single branch, a diffraction pattern, typical for

a single crystal, was obtained (Figure 51).

75

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Figure 50: TEM image of mSNAPcy4/DNA crystals.

76

III

 

Figure 51: Diffraction pattern from a single branch obtained under TEM.

2.15. Purification optimization of mSNAPcy4

Although the material that was used for crystallization and activity assays is
relatively pure according to the SDS-PAGE stained with Commassie, the inability to
grow high quality crystals suggests that further purification of the complex is required.
Both anion and cation exchange resins were used in the attempts to prepare a more
homogeneous material. The success of such purification was rather disappointing, since
the protein eluted in an extremely broad range of the salt gradient. Modifications to the

buffer used in this purification, with variations in pH of the buffer and additives, were

77

made, without much improvement in the purity of the complex. Due to the fact that the
complex is eluted from the column in such a broad range, the efficiency of the ion-
exchange purification is low. The complex is eluted in approximately 50% of the

fractions, or more specifically in a salt gradient from 150 mM to 650 mM (Figure 52).

 

 

78

 

 

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Figure 52: A representative elution profile for ion-exchange purification (Source-
S) of mSNAPcy4. The solid line represents the relative absorption intensity of the eluted

protein material and the dotted line represents the salt gradient.

79

Since ion exchange chromatography using SourceQ or SourceS resin (Pharmacia)
failed to accomplish an improvement in the purity of the material, gel filtration
chromatography was employed next. Partially purified mSNAPcy4 obtained from the
affinity purification was concentrated either by centrifugation or by ammonium sulfate
precipitation to enable efficient detection during the purification run. The complex was
purified in the absence of the detergent using the size exclusion gel filtration and two
peaks were observed in the chromatogram. The first peak that eluted in the void
contained mSNAPcy4. Interestingly, the second peak with the apparent molecular weight
of 150 kDa contained the complex as well. The majority of the protein material submitted
to gel filtration eluted in the first peak and only a fraction (approximately 5%) of the total
material was eluted in the second peak. SDS-PAGE analysis of all fractions confirmed
the presence of the complex, with some impurities being equally represented in both
peaks (data not shown).

The presence of two peaks confirmed that the complex is not homogenous and an
experiment was designed to determine how the removal of components from one peak
would affect the behavior of the other. The significant difference in the apparent size of
the two peaks and the fact that the first peak eluted close to the void volume suggested
that the complex might be forming aggregates. Several gel filtration runs were performed
and fractions were combined from the runs and concentrated to approximately 5 mg/mL.
Crystallization experiments were set up with these fractions to determine which of the
complexes is required for successful crystallization. Interestingly crystals formed first in
the material from fraction 12, which corresponds to the second peak with the material of

the apparent size of 150 kDa. mSNAPcy4 obtained only with the use of affinity

80

chromatography was used as a positive control and also yielded crystals, but slightly later
than fraction 12. More interestingly fractions 9 and 10 never yielded crystals and fraction
13 yielded crystals that were of better quality than those obtained from fraction 12 or the
control. It is obvious that the material with the apparent size of 150 kDa (second peak) is
required for crystal formation. Unfortunately this material is also a minor part of the total
material obtained from the affinity chromatography, which consisted mostly of the
aggregated material. Furthermore the separation of the two peaks is poor, since the
second peak is only a shoulder of the first peak on the gel filtration run (Figure 53; A).

In order to obtain homogeneous material for crystallization, large quantities of
mSNAPcy4 would need to be purified and then the two major components would have to
be separated on the gel filtration column. The separation would also be further
complicated due to the fact that the two peaks are not well resolved and several
sequential gel filtration experiments, each enriching the complex in the second peak,
would need to be performed. Such a purification scheme would be relatively complicated
and would yield little material suitable for crystallization from a large amount of the
mixture. A pilot experiment was performed to determine the efficiency of the separation
of the two peaks and also to check whether the two forms of the complex are in
equilibrium. Fraction 10, which corresponds to the peak in the void and is presumably an
aggregated form of the complex was concentrated and analyzed on the gel filtration
column. The material eluted in the void volume as before, without any apparent presence
of the seCond peak. Fractions 12 and 13, which correspond to the second peak that is
presumed to be a hetero tetramer, were pooled and analyzed in the same manner. This

material yielded a chromatogram where the first peak is under-represented compared to

81

 

 

the second peak resulting in material which is highly enriched compared to the previous
gel filtration separations (Figure 53). Removal of the fractions containing the aggregate

again would most likely yield a highly homogenous hetero tetramer mSNAPcy4.

82

 

 

 

 

 

 

C

 

 

I I I I I 1 I I I I I I » 1“
WM 7 8 9 1011121314151617

Figure 53: Gel filtration chromatograms of mSNAPcy4 purified with affinity
chromatography (A); mSNAPcy4 rerun fraction 10 (B); mSNAPcy4 rerun fractions 12

and 13 (C). The first peak in fractions 9 and 10 corresponds to 650 kDa and is also the

83

void. The second peak corresponds to a protein with an apparent molecular weight of 150

kDa according to the gel filtration standards (Biorad, data not shown).

Due to the fact that the un-aggregated mSNAPcy4 is under-represented
(approximately 5% of injected protein material) in the starting mixture and the laborious
purification scheme yielding low amounts of homogenous material (approximately 5% of
the total injected protein material), the amount of un-aggregated mSNAPc must be
increased.

In order to change the ratio of the two peaks a few approaches were taken. First,
since the ICP-MS metal analysis of the gel filtration fraction 9, which corresponds to the
aggregate peak, yielded 0.5 mol of iron per 1 mol of protein for fraction 9 and 0.3
equivalents of iron for the mixture (data not shown), it seems plausible that the iron is
responsible for aggregate formation. The aggregate fraction is enriched in iron and it is
possible that the iron binds to the putative second metal binding site in SNAP50. Since
the mixture contains less iron, it is also possible that the un-aggregated material contains
less iron. The ab-initio modeling suggested that zinc, nickel or possibly iron could
occupy the tentative metal binding site in SNAP50. Therefore, supplementing iron into
the mixture of the two forms of the complex could result in complete aggregation of the
complex and in contrast supplementation with zinc or nickel could result in a reduction of
the aggregated material. Chelating the iron with EDTA could also potentially increase the
amounts of the desired form of the complex. To test this assumption, mSNAPcy4 samples
obtained from affinity chromatography were incubated with different metal ions or with

EDTA and then analyzed with size exclusion gel filtration. Interestingly, none of the

84

 

 

additives tested had any relevant effect of the ratio of the two peaks. The effect of the
nickel supplementation could not be tested, since nickel was reduced upon mixing with
the sample. The resulting elementary nickel could potentially damage the gel filtration
column and was therefore not tested. 10 mM and 100 mM metal ions or EDTA were
tested, but again resulted in no visible effect on the ratio of the two peaks suggesting that

the formation of the aggregate is not related to metal content G:igure 54).

85

.Oh’—._.—
—O—'—.

-
-

    

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V(mL) 6 7 8 910111213141516

Figure 54: Gel filtration chromatogram of mSNAPcy4 (A) supplemented with

zinc (B), iron (C) or EDTA (D).

86

 

 

None of the additives used in the gel filtration separation had any effect on the
relative ratio between the aggregate and the hetero-tetramer. In order to efficiently
separate the two peaks, a gel filtration matrix with larger pores (Sephacryl-300) was used.
All aspects of the purification remained the same as with Sephadex-ZOO gel filtration
media used previously. The pores on this gel filtration matrix are approximately 50%
larger compared to Sephadex-ZOO and the hypothesis was that the complex should
migrate slower and therefore separate from the void peak. The hetero-tetramer peak
actually separated and the purification yielded almost baseline separation. Fractions
containing the hetero—tetramer were pooled, concentrated and injected to the same gel
filtration column again. This time the two peaks were separated completely and the
aggregate peak was present as a minor component of the binary mixture. It should be
noted that both gel filtration runs were performed in a buffer without the detergent
Tween-20 and the material could be efficiently concentrated using the Centriprep spin
concentrators. The peak that corresponds to the void could only be concentrated in the

presence of the detergent (Figure 55).

87

 

 

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Fraction 123456 7891011121314151617181920

Figure 55: Gel filtration of mSNAPcy4 using Sephacryl-3OO size exclusion beads.
(A) Biorad molecular weight standards (a=670 kDa, b=158 kDa, c=44 kDa, d=17 kDa),
(B) GST purified mSNAPcy4, (C) fraction 9-11 from the first gel filtration rerun using
the same column, (D) fractions 9-11 from the second gel filtration rerun (e: void peak, f:

mSNAPcy4 heterotetramer). Fractions were 5 mL.

88

 

 

Highly purified and homogenous material that was obtained by two successive
runs on Sephacryl—3OO gel filtration matrix and was concentrated in the absence of the
detergent with high recovery rates. Crystallization trials were started using the sparse
matrix formulations and many conditions were found where the protein crystallized.
Compared to previous preparations of the complex, the new approach resulted in protein
material that gave crystals of improved quality. Although the crystals were not
significantly larger the quality was greatly improved. Crystals no longer formed needle
clusters but resembled single needle-like or orthogonal crystals (Figure 56). The number
of successful crystallization hits was also increased drastically compared to the previous
preparations of mSNAPCy4.

Furthermore, the purification protocol was modified and Tween-2O is not required
in any step of the purification. Although the amount of protein material that is
immobilized on the GSH resin is reduced compared to the purification scheme where
Tween-2O is used, the homogeneity of the complex is improved. The relative amounts of
heterotetramer are comparable in the two purification approaches, but the amount of the
aggregated material is somewhat reduced when no Tween—20 is used. Due to improved
separation of the two peaks in the gel filtration step when Sephacryl-BOO is used the

recovery of the heterotetramer is also improved.

89

 

 

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100 mM NaCl and 100 mM MgC12. Protein material used to obtain the crystals was

prepared by successive gel filtration purification steps.

90

3. Methods and materials

3.1. Preparation of expression plasmids
3.1.1. Cloning of the SNAP50 subunit

Using the primers 5’-TCAGCCATGGCTGAAGGAAGC-3’ and 5’—
AGAGCTCTTAATFAAAGGTFCCAGG-3’, which correspond to the 5’- and 3’-
noncoding region of human SNAP50, respectively, we amplified the SNAP50 DNA from
a pGSTSO plasmid by standard PCR reaction. The PCR product corresponding to
SNAP50 was cleaved and inserted into the first multiple cloning site of the pCDFDuet-l
polycistronic expression vector (Novagen) via Ncol and Sac] cloning sites. The

expression vector pCDFSO-l was transformed into E. coli DHSOt competent cells, and

plated on LB plates supplemented with streptomycin (20 ug/mL).

3.1.2. Cloning of the SNAP43 subunit

Using the primers 5’-GGAATTCCATATGGGGACTCCTCCCGGCCTGCA-3’
and 5’-GAGGATCCTCAGTGTT'ITCTCCTCTTC’ITGGATGC -3’, which correspond
to the 5’— and 3’-noncoding region of human SNAP43, respectively, we amplified the
SNAP43 DNA from a pGST43 plasmid by standard PCR reaction. The resulting PCR
product corresponding to SNAP43 was digested with NdeI and BamHI and inserted into
the pRSFSO-l expression vector via NdeI and Hg”! cloning sites. The expression vector

pCDF43/50-1 was transformed into E. coli DHSOt competent cells, and plated on LB

plates supplemented with streptomycin (20 ug/mL).

91

3.1.3. Cloning of the SNAP19 subunit

Using the primers 5’-CTCACCATGGTGAGCCGGCTTC-3’ and 5’-
AAGGATCCTTAGGAATCTGATTCTTC-3’ which correspond to the 5’- and 3’-
noncoding region of human SNAP19, respectively, we amplified the SNAP19 DNA from
a pGSTl9 plasmid by standard PCR reaction. The resulting PCR product corresponding
to SNAP19 was inserted into pRSFDuet-l (Novagen) via Neal and BamHI cloning sites.

The expression vector pRSF 19-l was transformed into E. coli DHSOL competent cells,

and plated on LB plates supplemented with kanamycin (SO ug/mL).

3.2. Preparation of expression host
3.2.1. Preparation of competent cells for heat shock

The E. coli BL21-CodonPlus(DE3)-RIL were chosen for the over-expression of
SNAP subunits and for co-expression. The cells were made competent by calcium
treatment and the procedure is done on ice; 50 mL culture was grown until the OD600
(optical density) was 0.3 to 0.4 and the culture was cooled on ice for 5 minutes. The cell
pellet was collected by centrifugation, resuspended in 4 mL ice-cold FSB (Frozen
Storage Buffer; 10 mM potassium acetate, 100 mM KCl, 50 mM CaC12 and 10%
glycerol; pH 6.2) and kept on ice for 20 minutes. The cell pellet was again collected by
centrifugation and resuspended in 3 mL FSB. Aliquots of the desired volume were

prepared, frozen on dry ice and stored at -80°C.

92

 

3.2.2. Preparation of competent cells for electroporation

The procedure for preparation of electro competent cells was done on ice. Cells
were spun at 3000xg. 50 mL cell culture was grown until the OD600 of 0.5. The cell pellet
was collected by centrifugation and resuspended in 25 mL of milliQ water twice. The
cells were harvested by centrifugation and resuspended in 10 mL 10% glycerol in milliQ.
The cells were spun again and resuspended in 0.25 mL of 10% glycerol in milliQ. The

resulting suspension of cells was stored in aliquots and frozen at -80°C.

3.2.3. Preparation and use of a glycerol stock

Cell colonies harvested from freshly grown plates were used to inoculate 50 mL
of LB media supplemented with the appropriate antibiotic. The cells were grown for 12-
16 h at 37°C. Glycerol was added to the culture to bring the concentration of glycerol to
20%. Cells were incubated for l h at 37°C, aliquoted and stored at -80°C for long term
storage.

To use a glycerol stock, an aliquot was thawed at room temperature and 100 uL to
lmL of the cells were used to start a 50 mL LB culture, which was then grown for 12-16
h for further use.

Glycerol stocks prepared this way are viable up to at least 2 years.

3.2.4. Transformation into the cloning bacterial host
DHSa competent cells were thawed on ice and 1-5 uL of the DNA was added,

mixed gently and the cells were placed on ice for 10 minutes. The cells were then placed

93

 

in a heated bath at 37°C for 40 seconds and then immediately placed on ice for 2 minutes.
900 uL of LB was added and the cells were incubated at 37°C for l h while shaking. 100
uL of this mixture was plated on agar plates supplemented with the appropriate

antibiotic.

3.2.5. Transformation into the expression bacterial host Codon Plus RIL

BL21(DE3) Codon Plus RIL competent cells were thawed on ice and 1-2 uL of
the DNA were added, mixed gently and the cells were placed on ice for 20 minutes. The
cells were then placed in a heated bath at 42°C for 20 seconds and then placed
immediately on ice for 2 minutes. 900 uL of LB was added and the cells were incubated
at 37°C for 1 h while shaking. 100 uL of this mixture was plated on the agar plates
supplemented with the appropriate antibiotic. Chloramphenicol at 50 ug/mL is required

for the cells to retain the Codon Plus Rm plasmid.

3.2.6. Transformation into the co-expression bacterial host

The cells that were used for co-expression contained a single plasmid after the
first transformation and were made competent by calcium treatment. Next, the cells were
thawed on ice, mixed with 1-2 uL of plasmid DNA and placed on ice for 15 minutes. The
cells were placed on a heated plate at 42°C for 45 seconds and then immediately returned
on ice for 2 minutes. 900 uL of LB was added and the cells were incubated at 37°C for 1
hour and then plated on agar plates supplemented with the appropriate antibiotic mixture.
The order in which the plasmids are transformed into the cells is irrelevant and

eXpression levels are not dependant on the order in which the plasmids were transformed

94

 

 

(data not shown). Usually the pGSTl90(l-505) was transformed first, followed by
pCDF43R/50-l (to make mSNAPcy3 producing cells) and finaly pRSTl9-1 (to make

mSNAPcy4 producing cells).

3.2.7. Electroporation

Electro-competent cells were thawed on ice and 20 uL of the cells were mixed
with 1-2 uL of the DNA. The mixture was transferred into a sterile electroporation
cuvete. The cells were then pulsed with 2000 V using a pulse electroporator (Biorad), 480
uL of LB was added and the cells were incubated at 37°C for one hour and plated on agar

plates supplemented with the appropriate antibiotic mixture.

3.3. Growth media
3.3.1. Preparation of culture plates

To make 1 L of agar media for plates, 15 g of Tryptone, 5 g of yeast extract, 5 g
of NaCl and 15 g of agarose were mixed and dissolved in 1 L of water. The solution was
autoclaved. When the autoclaved solution was sufficiently cooled (60°C) the appropriate
antibiotic was added and the media was poured into Petri-dishes and allowed to cool and

harden. Prepared culture plates were stored at 4°C until use.

95

 

 

Table 2: Combinations of antibiotics used for preparation of agar plates. AMP, KAN,
STR and CAM represent ampicillin, kanamycin, streptomycin and chloramphenicol

respectively.

Concentration of antibiotics used for preparation of agar plates [uglmL]
Antibiotic

 

used Ampicillin Chloramphenicol Streptomycin Kanamycin

AMP 100 - - -
KAN - - - 100
STR - - 20 -
AMP/CAM 100 50 - -
AMP/CAM/

STR 50 50 15 -
AMP/CAM/

KAN 50 50 - 15
AMP/CAM/

KAN/STR 50 50 10 10

3.3.2. LB growth media
To make 1 L of LB media, 15 g of Tryptone, 5 g of yeast extract and 5 g of NaCl
were mixed and dissolved in 1 L of water. The solution was autoclaved. Before use

appropriate antibiotic was added (Table 2).

3.3.3. TB growth media

To make 1 L of TB media, 12 g Tryptone, 24 g yeast extract and 4 mL glycerol

 

was mixed and dissolved in 900 mL of water and autoclaved. Before use 100 mL of salt

solution (2.31 g KH2PO4, 12.54 g K2HPO4) was added to the broth.

3.3.4. M9 minimal media
To make 1 L of M9 minimal media 4 g of (NH4)ZSO4 or 1 g NH4Cl, 13.4 g of

NazHPO4, 6 g KH2PO4, 4 g NaCl and MgSO4 were dissolved in water. 200 uL of the

96

 

trace elements solution (10 mM FeCl3, 10 mM ZnClz and 100 mM CaC12) was added and
the mixture was autoclaved. Before use 20 mL of 20% autoclaved glucose, 20 mL of the

Basal Eagle vitamin medium and the appropriate antibiotic were added.

3.4. Over—expression
3.4.1. Over-expression of individual SNAP subunits

Cells harboring the plasmid with the sequence for the desired protein were grown
in LB medium supplemented with the appropriate antibiotic (100 ug/mL ampicillin). The
50 mL cultures were started from a freshly plated plate or from 3 glycerol stock and were
allowed to grow at 37°C for 12-18 h. The cell culture was then transferred to 1 L LB
containing flasks and allowed to grow at 37°C until 0.6 to 0.8 optical density. The
cultures were induced by the addition of 1 mM IPT G and grown at the appropriate
temperature (16°C for SNAPl90(l-505), SNAP50 and SNAP43, and 23°C for SNAP19).
The cells were grown for 12-18 h. The cells were collected by centrifugation and stored

at -20°C until needed.

3.4.2. Co-expression of mSNAPcy3 and mSNAPcy4
Co-expression was performed virtually the same way as the expression of
individual subunits. The only difference was the amount and the type of antibiotic used

(Table 3).

97

 

 

Table 3: Antibiotic concentrations used in individual and co-expression systems

Concentrations of antibiotics used for growth in LB or TB medium [uglmL]
Protein construct in

BL2 1 (DE3) Codon

Plus RIL Ampicillin Chloramphenicol Streptomycin Kanamycin
GST-SNAP190( 1 -505) 100 - - _
GST-SNAP50 100 - - _
GST-SNAP43 100 - - -
GST-SNAP19 100 - - _
MSCy3 50 50 20 -
MSCy4 50 50 20 20

 

3.5. Purification
3.5.1. GST purification

3L of cells harboring the over-expressed protein material were thawed on ice,
resuspended in 80 mL TEMGT-250 (25 mM Tris pH 7.9, 2 mM EDTA, 12.5 mM Mng,
10% glycerol, 0.1% Tween-20, 250 mM KCl and 3 mM DTT) and sonicated (3x 1
minute pulse, 1 minute pause). The cell extract was clarified by centrifugation, mixed
with the glutathione (GSH) agarose beads (Amersham Pharmacia) and allowed to shake
for 12 h at 4°C. GSH beads were washed with TEMGT-250 by centrifugation, transferred

to a glass column equipped with a frit and washed with TEMGT-250 until the flow-thru

 

showed no detectable protein matter by Bradford assay. 20 mL of TEMGT-250 were
mixed with the resin and 50 u of thrombin were added to remove the GST tag. Cleavage
was allowed to proceed to completion (12-20 h) and the protein material was eluted off
the beads by several consecutive TEMGT—250 washes. This method was used to purify

the individual subunits and the co-expressed material.

98

3.5.2. GST purification in the absence of a detergent

3L of cells harboring the over-expressed protein material were thawed on ice,
resuspended in 80 mL TEG-250 (25 mM Tris pH 7.9, 2 mM EDTA, 10% glycerol, 250
mM KCl and 3 mM DTT) and sonicated (3x 1 minute pulse, 1 minute pause). The cell
extract was clarified by centrifugation, mixed with the glutathione (GSH) agarose beads
(GE) and allowed to shake for 12 h at 4°C. GSH beads were washed with TEG-250 by
centrifugation, transferred to a glass column equipped with a frit and washed with TEG-
250 until the flow-thru showed no detectable protein matter by Bradford assay. 20 mL of
TEG-250 were mixed with the resin and 50 u of thrombin were added to remove the GST
tag. Digestion was allowed to proceed for 6-8 h, while monitoring the cleavage progress
by SDS-PAGE. Once the protein was completely digested 1 mM PMSF was added to
stop the digest and the protein material was eluted off the beads by several consecutive

TEG-250 washes.

3.5.3. Ion-exchange purification

The protein material obtained after the GST purification step was diluted with
water, until the salt concentration was below 50 mM (5 fold) and loaded to ion exchange
resin (Source-Q or Source-S, GE). The protein was then eluted with a 50 mM to l M
NaCl salt gradient (50 mM or 1 M NaCl, 20 mM Tris pH 7.5, 10% glycerol, 2 mM
EDTA, 12.5 mM MgC12, 0.1 % Tween—20 and 3 mM DTT). The purification was
performed with 2 mL of resin, 5-8 mg of protein material, flow rate was 4-6 mIJmin, and

the FPLC instrument used was by Amersham Pharmacia.

99

”'1'.
-LJ

 

 

3.5.4. Gel filtration purification

The partially purified protein material was concentrated to a desired concentration
(0.5-5 mg/mL) and injected onto the gel filtration matrix. A variety of buffers were tested
for this purification with relatively successful results. The formulation of a gel filtration
buffer that works well is 10 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol, 0.05%
Tween-20 and 3 mM DTT. For the Superdex-200 (GE) and Sephacryl-300 (GE) the flow

rates were 1 mIJmin and 0.5 mUmin respectively.

3.6. SDS-PAGE

Sample solution was mixed with an equal volume of 2xSPRS buffer (30 mM Tris
HCl, 2.5% glycerol, 6% SDS, 0.005% Bromophenol Blue, 0.025% B-mercaptoethanol)
and placed into a bath with boiling water for 1-2 minutes to denature the proteins. The
samples were loaded in the well portion of the stacking gel (4% acrylamide) and
separated at 220 V for 50-60 minutes. The resolving portion of the gel was 9-15 %
acrylamide, 1% SDS in Tris HCl pH 7.5.

Proteins on the gel were stained with Commassie (4% methanol, 10% acetic acid
and 1% Commassie blue in water; Commassie blue must be dissolved in methanol first,
then mixed with acid and finally diluted with water due to the low solubility of the dye in
water).

The gel was destained to reveal the positions of the protein material with

DESTAIN (5% acetic acid, 20% methanol in water).

100

 

Destained gels were soaked in water for 30 minutes, then soaked in 5% glycerol
for 30 minutes and dried between cellophane sheets for 24-48 h, to preserve the gels for

long term storage.

3.7. Western blotting

Protein samples were separated using SDS-PAGE. Two filter paper sheets were
placed on the bottom of the transfer cell (Biorad). Semi-dry buffer (50 mM Tris, 40 mM
glycine, 0.037% SDS and 20% w/v methanol) was added and any air was removed by
rolling with a cylinder. The gels were then placed on the prepared filter paper and more
semi-dry buffer was added. The membrane was cut to fit the gel, soaked in the semi-dry
buffer and placed on top of the gel carefully. 6 sheets of filter paper were soaked in semi-
dry buffer and placed on the membrane. Any air was removed by rolling the cylinder.
The transfer was allowed to continue for 50 minutes at 12 V. The membrane was
removed and blocked with 4% milk solution in TBST (20 mM Tris pH 7.6, 0.15 M NaCl
and 0.2 % v/v Tween-20) buffer. The membrane was then transferred to the primary
antibody solution (antibody for the protein of interest in 4% milk solution in TBST) for 1
h. The membrane was rinsed with TBST buffer twice by soaking the membrane in the
buffer for 10 minutes. The membrane was then transferred to the secondary antibody
solution (secondary antibody in 4% milk solution in TBST) for l h and again rinsed with
TBST as before. Solutions 1 and 2 were mixed in equal ratio and the membrane was
soaked in this solution for 1 minute. The membrane was then placed on a filter paper,

wrapped in Saran wrap and the bands were visualized on a photographic paper.

101

 

 

 

 

Primary antibody solution was prepared by a 1000 fold dilution of the antibody
stock in 4% milk solution in TBST. The secondary antibody solution was prepared by a
3000 fold dilution of the stock antibody.

Rabbit and mouse antibodies were used for SNAPc subunits and GST,
respectively, as primary antibodies. Horse a-rabbit and OL-mouse were used as secondary

antibodies. Antibodies a-SNAP190 #234, OL-SNAPSO CSH303, OL-SNAP43 C848, 0t-

SNAP19 CS543, a-mouse and oz-rabbit were a generous gift from Dr. R. W. Henry.

3.8. DNA purification

DNA was separated on 1% agarose gel, stained with ethydium bromide and
observed under UV light. Bands corresponding to the desired DNA product were excised
with a spatula and the DNA was extracted from the gel slice with QIAGEN gel extraction

kit, following the manufacturer’s instructions.

3.9. TEM analysis of mSNAPc crystals
3.9.1. Cross-linking of mSNAPc crystals

A 1:1 mixture of paraformaldehyde and glutaraldehyde was added directly to the
hanging drop containing protein crystals to a final concentration of 1% aldehyde and 1%
glutaraldehyde. The drop was returned on t0p of the well for 1 h. Cross-linked crystals
were then washed with water by addition of water and removal of the solvent with a

pipette, to remove buffer, salt and precipitant components of the crystallization reagent.

102

 

 

3.9.2. Preparation of the TEM grid

Glass strips were thoroughly washed with a detergent and wiped dry. A few drops
of 25% Formvar solution were added to the glass strip to form a thin film, without
creating thick areas. The prepared film was allowed to dry completely (2-5 min). A razor
blade was used to mark and cut the edges on the film. The glass strip with film was then
carefully placed in a bowl with water and if successful the film was lifted from the glass
and floated on the surface. 300 mesh copper grids were then carefully placed on the
floating film and a paper was placed on top to sandwich the grids between the film and

the paper. The prepared grids on the paper were removed from the water and dried.

3.9.3. TEM analysis

Cross-linked crystals were crushed with small crystallization tools to create a
suspension with a large number of small crystals. A drop (1-5 ul) of the crushed and
cross-linked crystals was placed on a Formvar grid (300 mesh copper) and excess liquid
was removed with a filter paper. The grid was then placed in the sample holder and
inserted in the instrument.

Samples were analyzed with JEOL 100CX transmission electron microscope.
Voltage on the electron gun was 100,000 V. Condenser aperture #2 and objective
aperture #2 was used. In diffraction mode diffraction aperture #2 or #3 was used.

The grid was viewed in low magnification mode (magnification 5,000) to identify
an area on the grid with desired properties such as intact membrane, absence of

precipitate, absence of dark areas, and presence of crystalline material. The crystals were

103

 

 

then observed at increasingly higher magnification and focus was adjusted at every
magnification increase until the desired images were obtained. Images were recorded
with a digital camera installed in the electron microscope and controlled with a
MegaViewII digital camera controller. Images were viewed and manipulated with the

AnalySIS software package.

3.10. Quantification of zinc in SNAP50
3.10.1. Determination of protein concentration
3.10.1.1. UV absorbance in 6 M urea

The approximate protein concentration was first determined with Bradford assay
as per manufacturer’s directions. The sample was then mixed with urea in such manner
that the measured absorbance was in the range of 0.1 to 0.6 where the assay is linear. In
the protein concentration range of approximately 2 to 4 mg/mL (for mSNAPc) the
expected absorbance was in the linear range if 50 uL of the sample was mixed in 450 uL

of 6.5 M urea.

3.10.1.2. Standardized Bradford assay

Working Bradford solution (20%) was prepared fresh before use, by diluting the
Bradford Assay stock in water. To measure the concentration, the protein (1-20 uL) is
mixed in 1 mL of the working Bradford solution and absorbance at 600 nm was
measured. A linear plot was obtained with the BSA standard solution (Biorad) in the
range of concentrations from 0.05 to 0.8 mg/mL. The protein concentration was then

determined from the plot. It is important that the absorbance of the sample in the

104

 

 

Bradford solution is within 0.1 and 0.6 to remain in the linear range. The plot points were

prepared in duplicate and the protein was measured in triplicate.

3.10.2. Flame atomic absorption

The concentration of the protein sample was determined by UV absorbance
measurement or by the standardized Bradford assay. The sample was transferred to a
crucible, dried and ashed at 260°C in an oven and concentrated nitric acid was added to
the brown powder and heated until all liquids evaporated. The process was repeated until
only white powder remained in the crucible. Solution was reconstituted by the addition of
a known volume of 5% nitric acid. To prepare the zinc standards, the mass of zinc metal
(Spectrum, 99.9 %) was measured carefully and the zinc was dissolved in concentrated
nitric acid and brought to the desired volume. Standards in the concentration range from
10 to 1000 ppb were made by serial dilution of the stock zinc solution with 5% nitric
acid. The samples and standards were analyzed on the flame atomic absorption
instrument (Varian SpectrAA-200) equipped with a zinc hollow cathode lamp operating

at 213.9 nm. The flow rate was 1 mL/min.

3.10.3. ICP-MS

The protein concentration was determined with the standardized Bradford Assay
using BSA as a standard. The sample solutions (protein, buffer) were transferred to a
Teflon vial and brought to dryness on a hot plate. Concentrated nitric acid was added and
the sample was placed on the hot plate and allowed to hydrolyze for 30 min. After

cooling the sample was diluted with water to bring the acid concentration to 2% and 2%

105

 

 

nitric acid was added to bring the solution to the desired volume. Standards containing
nickel and zinc in the concentration range 0-1000 ppb were prepared in 2% nitric acid
from a 1000 ppm stock solution (Trace grade). The samples and standards were mixed
with indium and bismuth solution as internal standards. The samples were run on an ICP-
MS instrument (Micromass) with flow rate 0.5 ml/min. 66Zn, 68Zn and 60Ni isotopes
were measured and quantified. The responses of zinc and nickel were corrected according

to indium and bismuth response.

106

 

 

4. References

 

1. Paule M. R., White R. J., Survey and summary: transcription by RNA polymerases I
and III, Nucleic Acids Research, 28, 1283 (2000).

2. Herr A. J ., Jensen M. B., Dalmay T., Baulcombel D. C., RNA Polymerase IV Directs
Silencing of Endogenous DNA, Science, 308, 118 (2005).

3. Lee T. 1., Young R. A., Regulation of gene expression by TBP-associated proteins,
Genes Dev., 12, 1398 (1998).

 

4. Lobo S., Hernandez N., in Transcription, Mechanisms and Regulation (Conway R. C.,
and Conway J. W., eds) pp. 127-159, Raven Press, Ltd., New York (1994).

5. Bai L., Wang 2., Yoon J .-B., R. G. Roeder., Cloning and characterization of the beta "1;
subunit of human proximal sequence element-binding transcription factor and its
involvement in transcription of small nuclear RNA genes by RNA polymerases H and

111, Mol. Cell. Biol., 16, 5419 (1996).

 

6. Henry R. W., Ma B., Sadowski C. L., Kobayashi R., Hernandez N., Cloning and
characterization of SNAP50, a subunit of the snRNA-activating protein complex
SNAPc, EMBO J., 15, 7129 (1996).

7. Wang Y., Stumph W. E., RNA Polymerase II/III Transcription Specificity Determined
by TATA Box Orientation, Mol. Cell. Biol, 18, 1570 (1998).

8. Schramm L., Hernandez N., Different human TFIIIB activities direct RNA polymerase
1H transcription from TATA-containing and TATA-less promoters, Genes Dev., 16,
2593 (2002).

 

9. Challice J. M., Segall J ., Transcription of the 5 S rRNA gene of Saccharomyces
cerevisiae requires a promoter element at +1 and a l4-base pair internal control
region, J. Biol. Chem, 264, 20060 (1989).

10. Ciliberto G., Raugei G., Costanzo F., Dente L., Cortese R., Common and
interchangeable elements in the promoters of genes transcribed by RNA polymerase
111, Cell, 32, 725 (1983).

11. Braun B. R., Kassavetis G. A., Geiduschek E. P., Bending of the Saccharomyces
cerevisiae SS rRNA gene in transcription factor complexes, J. Biol. Chem, 267,

22562 (1992).

107

 

12.

l3.

14.

15.

l6.

l7.

18.

19.

20.

21.

22.

23.

Krol A., Carbon P., Ebel J. P., Appel B., Xenopus tropicalis U6 snRNA genes
transcribed by Pol III contain the upstream promoter elements used by Pol II
dependent U snRNA genes, Nucleic Acids Res., 15, 2463 (1987).

Das G., Henning D., Wright D., Reddy R., Upstream regulatory elements are
necessary and sufficient for transcription of a U6 RNA gene by RNA polymerase III,
EMBO J., 17, 503 (1988).

Kunkel G. R., Pederson T., Upstream elements required for efficient transcription of a
human U6 RNA gene resemble those of U1 and U2 genes even though a different

 

polymerase is used, Genes Dev., 2, 196 (1988). M
Murphy 5., Tripodi M., Melli M., A sequence upstream from the coding region is

required for the transcription of the 78K RNA genes, Nucleic Acids Res., 14, 9243

(1986).

Baer M., Nilsen T. W., Costigan C., Altman S., Structure and transcription of a a

human gene for H1 RNA, the RNA component of human RNase P, Nucleic Acids
Res., 18, 97 (1989).

Topper J. N., Clayton D. A., Characterization of human MRP/T h RNA and its nuclear
gene: full length MRP/T h RNA is an active endoribonuclease when assembled as an
RNP, Nucleic Acids Res., 18, 793 (1990).

Lobo S., Hernandez N., A 7 bp mutation converts a human RNA polymerase II
snRNA promoter into an RNA polymerase III promoter, Cell, 58, 55 (1989).

Echenlauer J. B., Kaiser M. W., Gerlach V. L., Brow D. A., Architecture of a yeast
U6 RNA gene promoter, Mol. Cell Biol, 13, 3015 (1993).

Orphanides G., Lagrange T., Reinberg D., The general transcription factors of RNA
polymerase II, Genes Dev., 10, 2657 (1996).

 

Kuhlman T. C., Cho H, Reinberg D., Hernandez N., The General Transcription
Factors 11A, 11B, IIF, and [IE Are Required for RNA Polymerase II Transcription
from the Human U1 Small Nuclear RNA Promoter, Mol. Cell. Biol, 19, 2130 (1999).

Hampsey M., Molecular Genetics of the RNA Polymerase II General Transcriptional
Machinery, Microbiol. Mol. Biol. Rev., 62, 465 (1998).

Smale S. T., Kadonaga J. T., The RNA polymerase 11 core promoter, Annu. Rev.
Biochem, 72, 449 (2003).

108

 

25.

26.

27.

28.

29.

30.

31.

32.

33

34.

. Wu S. —Y., Kershnar E., Chiang C. —M., TAFII-independent activation mediated by

human TBP in the presence of the positive cofactor PC4, EMBO J., 17, 4478 (1998).

Nikitina T. V., Tishchenko L. 1., RNA polymerase HI transcription machinery:
Structure and transcription regulation, Mol. Biol, 39, 161 (2004).

Ghavidel A., Schultz M. C., Casein kinase II regulation of yeast TFIIIB is mediated
by the TATA-binding protein, Genes Dev., 11, 2780 (1997).

Yoon J. B., Roeder R. G., Cloning of two proximal sequence element-binding
transcription factor subunits (gamma and delta) that are required for transcription of
small nuclear RNA genes by RNA polymerases II and III and interact with the
TATA-binding protein, Mol. Cell. Biol, 16, l ( 1996).

Hernandez N., Small Nuclear RNA Genes: a Model System to Study Fundamental
Mechanisms of Transcription, J. Biol. Chem, 276, 26733 (2001).

Henry R. W., Sadowski C. L., Kobayashi R., Hernandez N., A TBP-TAF complex
required for transcription of human snRN A genes by RNA polymerase II and III,
Nature, 374, 653 (1995).

Wong M. W., Henry R. W., Ma B., Kobayashi R., Klages N., Matthias P., Strubin M.,
Hernandez N ., The large subunit of basal transcription factor SNAPc is a Myb
domain protein that interacts with Oct-1, Mol. Cell. Biol, 18, 368 (1998).

Gu L., Esselman J ., Henry R. W., Cooperation between Small Nuclear RN A-
activating Protein Complex (SNAPC) and TATA-box-binding Protein Antagonizes
Protein Kinase CK2 Inhibition of DNA Binding by SNAPC, J. Biol. Chem, 280,
27697 (2005).

Mittal V., Ma 8., Hernandez N., SNAPc: a core promoter factor with a built-in DNA-
binding damper that is deactivated by the Oct-l POU domain, Genes & Dev., 13,
1807 (1999).

. Ford E., Strubin M., Hernandez N., The Oct—1 POU domain activates snRN A gene

transcription by contacting a region in the SNAPc largest subunit that bears sequence
similarities to the Oct-1 coactivator OBF-l, Genes & Dev., 12, 3528 (1998).

Hovde S., Hinkley C. S., Strong K., Brooks A., Gu L., Henry R. W., Geiger J.,
Activator recruitment by the general transcription machinery: X-ray structural

analysis of the Oct-l POU domain/human U1 octamer/SNAP190 peptide ternary
complex, Genes & Dev., 16, 2772, (2002).

109

 

 

 

 

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

Sytina E. V., Pankratova E. V., Transcription Factor Oct-1: Plasticity and Multiplicity
of Functions, Mol. Biol, 37, 637 (2003).

Henry R. W., Mittal V., Ma B., Kobayashi R., Hernandez N., SNAP19 mediates the
assembly of a functional core promoter complex (SNAPc) shared by RNA
polymerases II and III, Genes & Dev., 12, 2664 (1998).

Sadowski C. L., Henry R. W., Kobayashi R., Hernandez N., The SN AP45 subunit of
the small nuclear RNA (snRNA) activating protein complex is required for RNA

polymerase H and III snRNA gene transcription and interacts with the TATA box
binding protein, Proc. Natl. Acad. Sci., 93, 4289 (1996).

 

Kunkel G. R., Hixsom J. D, The distal elements, OCT and SPH, stimulate the
formation of preinitiation complexes on a human U6 snRN A gene promoter in vitro,
Nucl. Acids Res., 26, 1536 ( 1998).

Zhao X., Pendergrast P. S., Hernandez N., A positioned nucleosome on the human U6
promoter allows recruitment of SNAPc by the Oct-1 POU domain, Mol. Cell, 7, 539
(2001).

Spector D. L., The dynamics of chromosome organization and gene regulation, Annu.
Rev. Biochem, 72, 573 (2003).

Hinkley C. S., Hirsch H. A. Gu L., LaMere B., Henry R. W., The Small Nuclear
RNA-activating Protein 190 Myb DNA Binding Domain Stimulates TATA Box-
binding Protein-TATA Box Recognition, J. Biol. Chem, 278, 18649 (2003).

Mittal V., Hernandez N., Role for the amino-terminal region of human TBP in U6
snRN A transcription, Science, 275, 1136 (1997).

 

Das D., Scovell W. M., The Binding Interaction of HMG-l with the TATA-binding
Protein/TATA Complex, J. Biol. Chem, 276, 32597 (2001).

Ford E., Hernandez N., Characterization of a Trimeric Complex Containing Oct-1,
SNAPc, and DNA, J. Biol. Chem, 272, 16048 (1997).

Jin S., Levine A. J., The p53 functional circuit, J. Cell Sci., 114, 4139 (2001).
K0 L. J ., Prives C., p53: puzzle and paradigm, Genes Dev., 10, 1054 ( 1996).

Levine A. J ., p53, the Cellular Gatekeeper for Growth and Division, Cell, 88, 323
(1997).

110

 

48.
49.
50.

51.
(1995).

52.

53.
54.
55.
56.

57.

Stein T., Crighton D., Boyle J. M., Varley J. M., White R. J., RNA polymerase III
transcription can be derepressed by oncogenes or mutations that compromise p53
function in tumours and Li-Fraumeni syndrome, Oncogene, 21, 2961 (2002).

Gridasova A. A., Henry R. W., The p53 Tumor Suppressor Protein Represses Human
snRN A Gene Transcription by RNA Polymerases H and III Independently of
Sequence-Specific DNA Binding, Mol. Cell Biol, 25, 3247 (2005).

Hirsch H. A., Gu L., Henry R. W., The Retinoblastoma Tumor Suppressor Protein
Targets Distinct General Transcription Factors To Regulate RNA Polymerase HI
Gene Expression, Mol. Cell Biol, 20, 9182 (2000).

 

Weinberg R. A., The retinoblastoma protein and cell cycle control, Cell, 81, 323

 

Horowitz J. M., Park S. H., Bogenmann E., Cheng J .C., Yandell D.W., Kaye D. J.,
Minna J .D., Dryja T. P., Weinberg R. A., Frequent Inactivation of the Retinoblastoma
Anti-Oncogene is Restricted to a Subset of Human Tumor Cells, Proc. Natl. Acad.
USA, 87, 2775 (1990).

Horowitz J. M., Yandell D.W., Park S. H.,Canning S., Whyte P., Buchkovich K.,
Harlow E., Weinberg R. A.,Dryja T. P., Point mutational inactivation of the
retinoblastoma antioncogene, Science, 243, 937 (1989).

Novitch B. G., Mulligan G. J ., Jacks T., Lassar A. B., Skeletal muscle cells lacking
the retinoblastoma protein display defects in muscle gene expression and accumulate
in S and G2 phases of the cell cycle, J. Cell Biol, 135, 441 (1996).

Onadim Z., Hogg A., Baird P. N., Cowell J. K., Oncogenic Point Mutations in Exon
20 of the RBI Gene in Families Showing Incomplete Penetrance and Mild Expression
of the Retinoblastoma Phenotype, Proc. Natl. Acad. USA, 89, 6177 (1992).

Hirsch H. A., Gu L., Henry R. W., The Retinoblastoma Tumor Suppressor Protein
Targets Distinct General Transcription Factors To Regulate RNA Polymerase III
Gene Expression, Mol. Cell Biol, 20, 9182 (2000).

Hovde S. L., personal communication.

58. Hanzlowsky A., Jelencic B., J awdekar G. W., Hinkley C. S., Geiger J. H., and Henry

R. W., Co-expression of multiple subunits enables recombinant SNAPC assembly
and function for transcription by human RNA polymerases II and III, Protein Expr.
Purif., 48, 215 (2006).

111

 

59. Jawdekar G. W., Hanzlowsky A., Hovde S. L., Jelencic B., Feig M., Geiger J. H.,
Henry R.W., The unorthodox SNAP50 zinc finger domain contributes to co-
operative promoter recognition by human SNAPc, J. Biol. Chem, 281, 31050 (2006).

 

 

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