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3 1293 00904 9382

This is to certify that the

thesis entitled
Purification of the Human Basal

Transciption Factor TFIIE Produced in E.coli

Data?

0-7639

presented by
David P. Chavez

has been accepted towards fulfillment
of the requirements for

M.S. Biochemistry

degree in

 

 

  

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c:\clrc\datedmrpm3-p.1

PURIFICATION OF THE
HUMAN BASAL TRANSCRIPTION FACTOR
TFIIE PRODUCED IN BACTERIA

BY

David P. Chavez

A THESIS

Submitted to
Michigan State University
in partial fullment of the requirements
for the degree of
MASTER OF SCIENCE
Department of Biochemistry

1993

Zachary Burton, Advisor

ABSTRACT

PURIFICATION OF THE
HUMAN BASAL TRANSCRIPTION FACTOR
TFIIE PRODUCED IN E.COLI
BY
David P. Chavez

Overproduction and Purification of TFIIE, a basal
transcription factor required for eukaryotic transcription
initiation by RNA polymerase II, is described. Human TFIIE
consists of two subunits of relative molecular mass 34,000
(TFIIE-B) and 56,000 (TFIIE-a) and enters the preinitiation
complex after RNA polymerase and TFIIF. Here we report the
expression of these cDNAs using a T7 RNA polymerase system in
E.coli. Production of human TFIIE-B is efficient using the
expression vector pET16b. TFIIE-B accumulates in inclusion
bodies and is solubilzed using 6M urea. Approximately 15 mg of
highly purified and soluble TFIIE-B can be obtained from one
liter culture of cells. TFIIE-a was produced as a soluble
protein in E.coli and did not require denaturant for
purification. A yield of 6 mg TFIIE-a per liter of cultured
bacteria was obtained. The pET16b vector is designed so that
a stretch of 10 histidines is fused to the NHf‘terminus. This
allows convenient purification using a Niri-affinity resin.
Polyclonal antibodies were raised against both recombinant
subunits. In Western blot analyses, these antibodies react
with the recombinant proteins as well as with RNA polymerase
II - associating proteins of 34 and 56 kDa. The antiserum
raised against the 34 kDa subunit inhibited transcription
effectively (90%) in in Vitro transcription assays, similar to
serum raised against RAP30 (95%), while anti-serum against the
56 kd subunit inhibited slightly less effectively (70%).
Recombinant TFIIE protein and antibodies raised against TFIIE
subunits will be useful in future studies of accurate

transcription in Vitro by RNA polymerase II.

DEDICATION

To my parents, my wife, AnneMarie and

to God who made life.

ii

ACKNOWLEDGEMENTS

I wish to thank Dr. Zach Burton for his guidance,
criticism, patience, tolerance and insight. I would also like
to acknowledge the other members (past and present) of the
Burton Lab for their technical assistance and general
discussion, Ann Finkelstein, Janek Werel, Bo Qing Wang, Shi-
men Fang, Chun-hsiang Chang, Shu-min Lin, Richard Fentzke,
Cory Kostrub and Jenny Jensen. Best of luck to all of you in
your future endeavors. I wish to thank Larry Graham for his
friendship, and I would also like to thank Lee Kroos and Jon
Kaguni for being on my committee and for their constructive
criticism. I also acknowledge the support of the Michigan
Competitive Doctoral Fellowship, the National Institiute of
Health, under the research supplement for minorities program,
the MSU Department of Biochemistry and the College of Natural

Sciences.

TABLE OF CONTENTS

DEDICATION ............ .. ....... . ......... ..... ...... . ..... ii
ACKNOWLEDGEMENTS ..................... . ....... . ............ iii
TABLECH’CONTENTS..................................... ..... iv
LIST OF FIGUES AND TABLES ............. ............ ........ vi
LIST OF ABBREVIATIONS ..................................... viii
1.0 INTRODUCTION . . . . . . . . . . . . . . . . . 1

1.1 Eukaryotic Nuclear RNA Polymerases I, II, and
III . . . . . . . . . . . . . . . . . . . . . 1
1.2 Organization of Class II promoters . . . . 6

1.3. Order-Of—Addition Model for Formation of a
Preinitiation Complex . . . . . . . . . . . . 8
1.4 RNA-Polymerase-II-Associating Proteins . . . 17

1.5 Energy Requirements for Accurate Initiation of
Transcription . . . . . . . . . . . . . . . . 20

1.6 Structure and Properties of Transcription
Factor TFIIE . . . . . . . . . . . . . . . . 23
1.7 Production of Human Protein in E.coli . . . . 28

iv

 

2.0 MATERIALS AND METHODS . . . . . . . . . . .

2.1 Construction of recombinant
pET16b/TFIIE-a and pET16b/TFIIE-B . .

2.2 Growth and Lysis of cells . . . . . . .

2.3 Purification of TFIIE-a (58 kd subunit)

2.4 Purification of TFIIE-B (36 kd subunit)

clones

o

2.5 Production of Anti-TFIIE-a and Anti-TFIIE-B

Anti-serum and Western Blot Analysis .

2.6 Preparation of protein-affinity fractions for

Western analysis . . . . . . . . . .
2.7 In vitro transcription Assay . . . .
RESULTS . . . . . . . . . . . . . . . . .

3.1 Production and Purification of TFIIE-a (58 kDa

subunit) . . . . . . . . . . . . . . .

3.2 Production and Purification of TFIIE-B (36 kDa

subunit) . . . . . . . . . . . . . . .

3.3 Western blot Analysis of recombinant TFIIE

DISCUSSION . . . . . . . . . . . . . . . . .

APPENDIX. . . . . . . . . . . . . . . . . .

BIBLIOGRAPHY . . . . . . . . . . . . . . . .

34

34

34

36

37

37

38

39

42

42

47

48

54

60

66

Table

Figure

Figure

Figure

Figure

Figure

Figure

Figure

1.3

1.3

1.6

1.7

2.6

List of Figures and Tables

page
Summary of Basal Transcription Factors ...... 9
Proposed Model for the Order of Addition
of Basal Transcription Factors...... ....... 11
structural Features of TFIIE Subunits
a and B... ............ ....... ...... ......26
Schematic of Bacterial Expression Vector
pET16b.. ...... . ...... ............ .......... 31
Schematic of TFIIE-a and TFIIE-fl over-
production clones ................... . ...... 35

Schematic of In Vitro Transcription Assay..40

Overproduction of the Large Subunit of Hw-
TFIIE-a in bacteria ........................ 43
Overproduction of the Small Subunit of Hm-

TFIIE-B in bacteria ........................ 44

vi

Figure

Figure

Figure

Figure

Figure

Figure

Figure

3.1C

3.2

3.3A

3.3B

3.3C

3.4

4.1A

Purified and Partially Purified ............ 45

One Step Purification of Recombinant

Proteins Hm-TFIIE-a and 6....... ...... .....46

Western Blot Analysis for detection of

the large subunit (TFIIE-a)................49

Western Blot Analysis for detection of

the small subunit (TFIIE-B) ................ 50

Western Blot Analysis of RAP Fractions ..... 51

Inhibition of Accurate Transcription by

Anti—Serum raised against Basal Subunits...53

Current Model of the Preinitiation Complex.55

Abs

AdMLP

ATP

ATPase

BTF

CTD

CDNA

DNA

DEAE

DTT

EDTA

EGTA

E.coli

IPTG

LIST OF ABBREVIATIONS

Antibodies

Adenovirus Major Late Promoter

Adenosine 5’-triphosphate

Adenosine 5’-triphosphatase

Basal transcription factor

(Hg-terminal domain

Complementary DNA

Deoxyribonucleic acid

Diethylaminoethyl

Dithiothreitol

Ethylenediamine tetraacetic acid

Ethyleneglycol-bis-(amino-ethyl ether)

tetraacetic acid

Escherichia coli

isopropyl-Beta-D-thiogalactopyranoside

viii

kDa

KCl

nt

PAGE

SDS

TAFS

TBP

TF

Tris

kilodalton

Potassium chloride

nucleotide

polyacryliamide gel electrophoresis

Polymerase

precipitation

Ribonucleic acid

RNA polymerase II

RNA polymerase II-associating proteins

Sodium dodecyl sulfate

Tata binding protein-associating factors

Tata binding protein

Transcription Factor

Tris(hydroxylmethyl)aminomethane

ix

1.0 INTRODUCTION

1.1 Eukaryotic Nuclear RNA Polymerases I. II. and III

In eukaryotic cells, there are three distinct nuclear RNA
polymerases. RNA polymerases I, II, and III, respectively,
transcribe genes encoding ribosomal RNA, pre-messenger RNA (or
protein-coding RNA ) and various short RNAs including transfer
RNAs and SS ribosomal RNA.

A common feature of all three eukaryotic RNA polymerases
is the requirement for numerous auxiliary transcription
factors in addition to the polymerase enzyme. Collectively,
RNA polymerase and the basal transcription factors constitute
the basal transcriptional apparatus. Basal factors required
for in vitro transcription have been identified, purified and
in some cases cloned for all three systems. RNA polymerase I
requires at least the two basal factors, UBF and SL1, and
additional peptide components that have not yet been described
(Sollner-Webb and Mougey, 1991; Reeder 1992). There are at
least seven basal RNA polymerase II factors (for review, see
Zawel and Reinberg, 1993; Weinmann, 1992). Finally, RNA
polymerase III requires at least three factors TFIIIA, TFIIIB,
and TFIIIC (Gabrielson and Sentenac, 1991; Geiduschek and
Kassavetis, 1992). The factors TFIIIB and TFIIIC are composed

of multiple peptide components.

2

The three polymerase systems are not completely distinct.
For example, five subunits are shared between the pol I, pol
II, and pol III enzymes (for review, see Sentenac, 1985;
Wolychik and Young, 1990). Second, at least one factor, the
TATA-binding protein (TBP) is required for accurate
transcription by all three polymerases (Comai et a1. 1992;
Cormack and Struhl 1992; Schultz et al. 1992, White et al.
1992). Biochemical studies using recombinant TFIID (TBP) have
supported earlier observations (Reinberg et al. 1991) which
had suggested that in vivo TFIID is part of a large molecular
weight complex . It was later demonstrated (Dynlacht et al.
1991) that endogenous TFIID from Drosophila embryo extracts is
tightly associated with at least six polypeptides ranging in
size from 32 kDA to 150 kDa called TAFs (TATA binding protein—
associating-factors). In the RNA polymerase III (pol III)
system TBP has been shown to participate in transcription of
vertebrate U6snRNA genes for which promoters are exclusively
upstream of the coding sequence (Lobo et al. 1991). TBP is
also required for pol III tRNA and SSrRNA promoters, in which
the promoters are located primarily within the transcribed
sequence of the gene (White et al. 1992; Schultz et a1. 1992).
Since many of these promoters lack an identifiable TATA-box,
their transcription appears analogous in some ways to TATA—

less pol II promoters.

3

A second complex that contains TBP is the pol I basal
factor SL1, which contains 3 TAFs and elutes from
phosphocellulose columns in the high salt D fraction (PC-D;
0.6-1.0M KCL). There is evidence that shows a strong and
specific interaction between the C-terminal domain of TBP and
TFIIIB (white and Jackson, 1992). The precise polypeptide
composition of TFIIIB has not been conclusively determined. It
has been suggested that different TBP-TAF complexes are not
only specific for different classes of RNA polymerases and
that distinct sets of TAFs in each complex confer specificity,
but the TAFs for one enzyme are antagonistic to the activities
of the other enzymes. (Comai et al. 1992, White and Jackson
1992, Taggert et al. 1992).

A TFIIB homolog called PCF4 or TSD4 recently isolated
from S.cerevisiae appears to function in RNA polymerase III-
mediated transcription (Willis et a1. 1992; Buratowski and
Zhou, 1992). Since the SUA7 gene encodes the related RNA
polymerase II factor, each polymerase may have its own TFIIB
homolog to direct entry of the relevant RNA polymerase into
the preinitiation complex. Since each polymerase mechanism
requires TBP, different TAFs likely determine which TFIIB
related function is chosen during assembly. The sequence
similarity is strong (first 300 residues out of a 596 residue
protein). These results suggest a strong conservation of
fundamental transcription_mechanisms for RNA polymerases I, II

and III. Perhaps additional polymerase-specific basal factors

4
will be found to have homologs in other RNA polymerase
systems.

The parallels between the pol II and pol III systems (and
possibly pol I) indicate the possibility of a common
initiation mechanism that was established before the
divergence of the three eukaryotic polymerases. Detailed
mechanistic comparisons of initiation by each of the
eukaryotic RNA polymerases and their associated regulatory
factors will allow definition of the very complex molecular
systems that are key instruments in expressing the genome.

Clues to the functions of the eukaryotic RNA polymerase
subunits are based on sequence similarities with prokaryotes
and with each other. RNA polymerase I purifies into multiple
polypeptides, at least five of which have been cloned from
yeast (Reeder, 1992). The largest polypeptide is a 190 kDa
subunit that shows homology with the largest subunits of RNA
polymerases II and III, and with the 3' subunit of E.coli RNA
polymerase (Sollner-Webb and. Mougey, 1991). Three of the
thirteen polypeptides of yeast RNA polymerase I are shared by
all three RNA pols and two others are shared by RNA polymerase
I and pol III.

The amino acid sequences of the RNA. polymerase II
subunits reveal that the two largest subunits, RPB1 and RPB2,
are clearly related to the two largest subunits of E.coli RNA
polymerase, B and 6'(Young et al. 1987). The sequence

similarity occurs in eight or nine segments that span the two

5
large subunits. In addition, the two large subunits of
prokaryotic and eukaryotic RNA polymerases are functional
homologs. The B’(RPB1) subunit binds DNA, and the 3 subunit
(RPB2) binds nucleoside triphosphate substrates and interacts
with the transcription factor sigma.

In yeast the RPBl subunit has a unique C-terminal domain
(CTD) that is not shared with its prokaryotic homolog. This
CTD contains 26 or 27 repeats, depending upon the strain, of
the consensus sequence YSPTSPS. In mammals, the CTD consists
of 52 repeats of the consensus heptamer sequence, YSPTSPS
(Allison et al. 1985), which exists in phosphorylated and
unphosphorylated forms that are distinguishable by apparent
molecular mass on SDS-polyacrylamide gels (IIO - 240 kDa and
IIA - 214 kDA respectively). A third form, IIB, arises from
proteolytic cleavage of the C-terminal domain, resulting in
the formation of a subunit with an apparent molecular weight
of 180,000 (Allison et al. 1985). The CTD is required for cell
viability, but the presence of this structure and its
extensive modification by serine-threonine phosphorylation has
yet to be correlated with a definite function (for review, see
Dahmus and Dynan, 1992). Both large subunits contribute to the
active site for catalysis.

Several points of evidence indicate that subunit RPB3 and
E.coli a polymerase subunit are probably functional homologs.
Both are similar in size and both occur twice in the RNA

polymerase molecule. Also, mutations in both subunits affect

 

6
assembly of the enzyme (Kolodziej and Young, 1990). These
identical polypeptides, together' with ‘the large subunits,
constitute a core polymerase that may be the functional
equivalent of the prokaryotic core enzyme. Detailed sequence
comparisons between the RNA polymerases of archaebacteria,
eubacteria and eukaryotes indicate that RNA polymerases II and
III are more closely related to each other than to RNA

polymerase I (Sentenac et al. 1988).

1.2 Organization of Class II promoters

DNA sequence elements within promoters for RNA polymerase
II can be divided into two classes: 1) basal elements, which
are recognized by the basal transcription factors, and 2)
promoter-specific elements, which are recognized by promoter-
specific regulatory proteins. Two basal promoter elements,
the initiator (Inr) (Smale and Baltimore, 1989), which
encompasses the transcription start site, and the TATA, which
is located approximately 30 nucleotides upstream, constitute
the most common minimal promoter elements required in order
for specific transcription to occur. Both of these DNA
elements position the assembly of the basic transcription
machinery at the +1 position of a given gene. Promoter-
specific elements are sequence elements which are recognized
by DNA specific binding proteins. These may be proximal to the

basal promoter or at distal sites. The promoter is required

7
for accurate and efficient initiation of transcription,
whereas enhancers increase the rate of transcription from
promoters. The distinctive characteristic of enhancers is that
they can act on cis-linked promoters at great distances
upstream in an orientation-independent manner and can also
function downstream from the transcription unit.

The types of regulation observed with cellular enhancers
indicate that enhancers can be divided into two categories:
those that respond to changes in the environment (inducible
enhancers) and those that are active only at specific times
during development or only in specific tissues (temporal and
tissue-specific enhancers). The general principle to emerge
from the study of promoters is that both positive and negative
regulation may be controlled by the modification of factors
that interact with promoters and enhancers. Furthermore, if
inducible or tissue-specific gene activation involves the
modification of one or a few limiting factors that are
required for the assembly of a transcription complex, it may
be difficult to mimic in vivo conditions that may not occur in

vitro.

8
1.3. Order-Of-Addition Model for Formation of a Preinitiation

m

In contrast to prokaryotes, in which the RNA polymerase
holoenzyme is often sufficient for promoter recognition and
accurate initiation (for review see Goodrich and McClure,
1991), accurate initiation by RNA. polymerase II requires
assembly of a multiple factor complex. This entails the
ordered interaction of an array of basal factors in addition
to RNA polymerase II. The basal transcription factors that are
required include: TFIIB, TBP, TFIIE, TFIIF, TFIIH and TFIIJ
(see Table 1.3). The factor TFIIA is stimulatory to accurate
initiation but not necessarily required. The direct
involvement of these factors in the formation of preinitiation
complexes has been defined using gel mobility shift assays and
accurate transcription assays in which intermediates in
complex formation can be observed using subsets of factors
(Buratowski et al. 1989; Flores et al. 1991). These studies
have helped define processes for assembly of the preinitiation
complex. Some roles of individual factors have also been
defined in these studies.

In the first step of the pre-initiation complex assembly
TBP associates with the TATA element of the promoter (see
figure 1.3, complex 1). Studies by Chambon’s lab (1983)
showed TFIID is necessary and sufficient for commitment of a

particular template to the transcriptional process.

 

 

 

 

 

TFIIE, Factor 3,
8

humans

Name, Molecular mass Properties Species Cloned
alternate names (K d)
38 kd - humans yeast, 1989
TFIID, TBP, BTFI , 27 kd - yeast Interacts With the human,1990
Factor d 22kd-Arabidopsis TATA-box Darablgopsrs,
38kd-drosophila ”Sp ”990
, + others
stabilizes TFIID/DNA
TFIIA. STF 34,19,14 kd ' interaction, removes yeast, 1993
humans repressor (Dr-2)
TFHB' a 33kd - humans agzggéistg'ftgrgA human, 1991
Factor e, FA, SUA7 3 8k d _ yeast DAB complex yeast, 1992
heterodimer,
56 and 34 kd in interacts with pol n, human,1991

stimulates CTD

kinase activity

 

TFIIF,RAP30/RAP74
Factor 5, FC, B—y,

30 and S8 kd in

heterodivmer interacts
with pol ll, recuits pol

RAP30-human,rat,
1989,1992;
RAP74-human,Droso-

 

 

 

 

 

 

 

 

II to romoter, aff ts _
BTF4, Factor 9 humans rate 0‘; elongation. ec phlla, xenopus,1992.
89,62,43,40 ATP-dependent DNA
TFIIH. 8 . and helicase, CTD kinase, 62kd subunit, 1992
Factor b- BTFZ binds to DAB pol FE
35kd
TFII J binds to DABpoIFEH
unknown complex none
Table 1. 3 Basal Transcription

Factors

10
Similarly, binding of TFIID to the TATA motif was subsequently
demonstrated by DNaseI footprinting (Parker and Topal, 1984;
Sawadogo and Roeder, 1988;) and DNA-binding experiments
(Horikoshi et al. 1989; Buratowski et al. 1989). TFIID
protects a 20 nucleotide region centered around the TATA
from -36 to -17 on the adenovirus major late promoter.

The transcription factor TFIID was first isolated and
identified from human tissue culture cells as a 0.6-1.0M KCL
eluate from a phosphocellulose column (Roeder et al., 1980).
TFIID was found to stably associate with DNA templates
containing a TATA motif (Davidson and Chambon, 1983). More
recently, it was shown that TFIID activity associated with
this fraction is made of TBP (for TATA-Binding-Protein) and a
number of TBP-associated factors (TAFs). The TAFs account both
for the large size of TFIID relative to TBP and for the
ability of TFIID to respond to activators (Dynlacht et al.
1991). The gene encoding TBP activity has been cloned from
yeast, Arabidopsis, Drosophila, and human cells (Hahn et al.
1989; Horikoshi et al. 1989; Hoey et al. 1990; Kao et al.
1990;). The human protein has been found to consist of a
single polypeptide of 38 kDa.

Several studies have suggested that TFIIA appears to
stabilize the binding of TFIID to the TATA motif, thereby
generating the TFIID-TFIIA (DA-complex; see figure 1.3) (Fire
et al. 1984; Reinberg et al. 1987). It is thought that

stabilization occurs via direct contact with the conserved

 

II

III

IIE+ "H r —?
0. xv

A... v

f IIJ
0. v.

Figure 1.3 Proposed model for the order of association of the
basal transcription factors with the adenovirus major late promoter
deduced using the mobilty shift assay.

12

domain of TFIID since both yeast and human TFIID can interact
with mammalian TFIIA protein (Cortes et al. 1992). The
association of TFIIA with TFIID can occur in solution in the
absence of DNA as well as with the TFIID bound to the TATA
motif. It has also been shown that TFIIA stimulates basal
transcription when native TFIID from Hela cells is used but is
dispensable in a system reconstituted with bacterially
produced TBP (Maldonaldo et al. 1990). It appears that TFIIA
can associate with all of the preinitiation complex
intermediates analyzed (Cortes et al. 1992). Previous studies
have demonstrated that association of TFIIA with the
preinitiation complex is dependent on the presence of complex
I (As suggested in figure 1.3). TFIIA. binds in a 'rFIID-
dependent manner upstream from the TATA region from -35 to-40
relative to the transcription start site of the adenovirus-
major late promoter (Maldonado et al. 1990). The binding of
negative cofactors is thought to compete with TFIIA to bind
the TFIID complex and repress transcription (Meisterernst et
a1. 1991). Thus, an experimental system that contains few
negative cofactors would show a lesser requirement for TFIIA.
The factor TFIIA has been purified using yeast TFIID affinity
chromatography and shown to be composed of three polypeptides
of 34, 19 and 14 kDa (Cortes et al. 1992).

TFIIB is the next factor to associate with the DA
complex, resulting in the formation of the DAB complex

(complex II), (Van Dyke et al. 1988; Buratowski et al. 1989).

13

When TFIID is bound to the TATA motif, TFIIB can stably
associate to produce the DB complex. Thus the binding of TFIIA
to the TFIID-DNA complex is not required for TFIIB binding;
rather, the presence of TFIIA increases the overall stability
of the complex. The association of TFIIB with bound TFIID is
required for the association of the other remaining general
factors and RNA polymerase II on the promoter sequences. In
a model proposed by Buratowski (1992), TFIIB forms a bridge
between TFIID and pol II. This suggests that TDS4 supplies an
analogous activity for RNA polymerase III. It is possible that
a TFIIB homolog will be found in the pol I system. Isolation
of the clone encoding human TFIIB was reported by Ha et al.
(1991). This CDNA encodes a polypeptide with a predicted
molecular mass of 33 kDa.

At this point TFIIF (RAP30/74) physically interacts with
RNA polymerase II and acts as a "chaperone", escorting the
polymerase to the DAB complex to form the DABpolF complex
(complex III). Without TFIIF the polymerase is unable to form
any of the higher ordered complexes and is thus incapable of
transcription. The human TFIIF is a heterodimer whose 74 kDa
and 30 kDa subunits have been cloned (Finkelstein et al. 1992;
Sopta et al. 1989). The small subunit of TFIIF, RAP30 has
sequence homology with and is functionally analogous to the
sigma 70 subunit of E.coli RNA polymerase II and can alone act
as a chaperone without RAP74. The 30 kDa subunit of TFIIF can

recruit pol II to the DAB complex independent of the large

14

subunit but the resulting DABpolRAP3O complex is less stable
to electrophoresis than DABpolF. Addition of RAP74 stabilizes
the complex (Flores et a1. 1990). In reconstituted
transcription systems, RAP30 alone is unable to functionally
replace TFIIF (Finkelstein et al. 1992). RAP30 is required for
initiation of transcription, but RAP74 is apparently
dispensible for all initiation processes. RAP74, however, is
essential for early elongation and appears to be required for
promoter escape by polymerase (Chang et al. 1993 paper
submitted for publication).

Once the DABpolF complex is formed, TFIIE and TFIIH
associate to form the DABpolFE and DABpolFEH complexes,
respectively (Flores et al. 1992). Studies by the Reinberg lab
(Lu et al. 1992) have indicated that although entry of TFIIH
into the transcription complex is dependent on complexes III
and IV, the amounts of TFIIE required to form the DBPolFEH
complex (complex V), in the presence of TFIIH, are
approximately S—fold lower than those required to obtain
complex IV. It appears that the association of TFIIE and TFIIH
with the preinitiation complex may be cooperative.

TFIIH consists of five subunits. A gene encoding one
subunit of 62 kDa has been cloned (Fisher et al. 1992). A
purified fraction of TFIIH is associated with a CTD kinase
activity (Lu et a1. 1992). The cloned 62kDa subunit showed no
kinase activity, although a monoclonal antibody directed

against the 62 kDa subunit inhibited both transcription and

15

CTD kinase activity (Fisher et al. 1992). In the presence of
ATP, TFIIH binding is stimulated and not dependent on the
presence of TFIIE in the complex. Recently, an 89 kd subunit
was identified that shows an ATP-dependent DNA helicase
activity (Schaeffer et al. 1993). Microsequencing of tryptic
digests of the 89 kd subunit indicate that this polypeptide
corresponds to the ERCC-3 gene product, a presumed helicase
which has been shown to participate in the nucleotide excision
repair process in humans, drosophila and yeast (Gulyas, K. and
Donohue, T. 1992). Previously, it had been shown that an ATP-
dependent DNA helicase activity was associated with the
transcription factor TFIIF(RAP30/RAP74) and another
polypeptide of molecular mass 90 kd when eluted from RNA
polymerase-associated columns (Sopta et a1. 1989). The finding
of a TFIIH-associated helicase activity in transcription
initiation has long been predicted and sheds light on the
mechanistic events that occur during this process.

Finally, the last factor TFIIJ, a factor required for
transcription when recombinant yeast or human TBP is used
instead of endogenous TFIID, yielding complex VI. (Cortes et
al. 1992). At this time little is known about TFIIJ.

At some time between the initial promoter binding and the
formation of the first phosphodiester bond, the RNA polymerase
II is converted to a highly phosphorylated form. It is thought
that this phosphorylation event represents a critical step

during initiation. The general factors TFIIE, TFIIH and TFIIJ

16
may all be involved in this step. Phosphorylation of the CTD
by TFIIH may still provide a critical role in uncoupling the
RNA polymerase from the preinitiation complex and initiating
the elongation phase of transcription.

There are several lines of evidence that implicate the
phosphorylation of the CTD as a requirement for the transition
from transcription initiation to elongation. Photoaffinity
labeling experiments demonstrated that nascent RNA
transcripts crosslink almost exclusively to the phosphorylated
IIO form in Vivo and in vitro, suggesting that it is the IIO
polymerase that elongates RNA chains (Cadena and Dahmus,
1987). In addition Lu et al. (1991) demonstrated that the
nonphosphorylated form of RNA polymerase II (IIA),
preferentially associates with the preinitiation complex,
where it is phosphorylated by a protein kinase. More recently,
it was shown that a chemically synthesized monomer of the CTD
(heptamer repeat YSPTSPS) binds the human and yeast TBP. Also
it was found that the nonphosphorylated form of RNA polymerase
II, but not the phosphorylated form, binds to TBP (Usheva and
Aloni, 1992). This may indicate that in vivo , binding of TBP
to the CTD is one of the initial steps in the formation of a
transcription-competent complex. In a model proposed recently
by Koleske et. al. (1992) the CTD is one of the components
that interacts with the DAB complex during recruitment of the
enzyme to the promoter. This interaction is directly or

indirectly influenced by SRB2,(a dominant suppressor of CTD

17
truncation mutations in yeast) and /or other factors that are
associated with TFIID. Phosphorylation of the CTD may disrupt
its association with the DAB complex, allowing RNA polymerase

to exit the promoter complex.

1.4 RNA-Polymerase-II-Associating Proteins

To identify and purify proteins important for
transcription by RNA polymerase II (RNAP II), Greenblatt and
colleagues used calf thymus RNAP II as a ligand for protein-
affinity chromatography (Sopta et al. 1985) . When calf thymus
or HeLa cell-derived extracts were passed over these columns,
three RNA pol II-associating proteins or (RAPs) were
identified and designated RAP30, RAP38 and RAP74; the number
indicates the molecular mass in kilodaltons . RAP38 appears to
be the same factor as the elongation factor S-II (Natori et
al. 1979). Using antibodies to RAP30, it was later shown that
RAP30 and RAP74 interact with each other, and that both of
these polypeptides are required to restore the transcriptional
activity of a RAP-depleted nuclear extract (Burton et al.
1988). RAP30/RAP74 was also shown to be immunologically
identical to the isolated factor TFIIF and 37(Flores et al.
(1988) and Conaway J. and Conaway R., 1989). Initially, it was
thought that By was the rat equivalent for the human factor
TFIIE (Conaway, 1989). This was because in early transcription
systems, TFIIF was present in crude TFIIE preparations

(Sawadago and Roeder, 1985).

18

other groups had taken different approaches for the
isolation of proteins that interact and/or modify RNA
polymerase II activity. For example, previous studies by
Roeder's lab showed that the 0.5 M KCL phosphocellulose
fraction contained at least two transcription factors (IIB and
IIE) that could be separated from each other cut a DEAE-
cellulose column (Dignam et al. 1983). After purification of
TFIIE, glycerol gradient studies showed that TFIIE
specifically interacts with RNA polymerase II and TFIIB
(Reinberg and Roeder, 1987a). These results suggested a weaker
interaction between TFIIE and RNA polymerase II than between
RNA polymerase II and TFIIF. TFIIB might enhance the
interaction betwween TFIIE and RNA polymerase II. The
purification of the previously described TFIIE protein
fraction resulted in the identification of a new transcription
factor TFIIF (Reinberg and Roeder, 1987b). It was then
demonstrated that when the more purified TFIIF, TFIIE and RNA
polymerase were independently sedimented through a glycerol
gradient that both transcription factors can independently
interact with and/or modify RNA polymerase II. Western blot
analysis showed that the formation of a functional complex
containing RNA polymerase II and TFIIF resulted from a direct
physical interaction between the RAP 30 subunit of TFIIF and
the polymerase (Flores et al. 1989). The fact that TFIIE was
not found on RNA polymerase II columns was thought to be due

to TFIIE having an unstable interaction with the polymerase

19
and/or an indirect interaction dependent on other factors.
Since we now know that TFIIE enters the complex late after
TFIIF and polymerase it would seem that both explanations are
possible. In this thesis I will show evidence that TFIIE is
indeed a RAP, by Western blot analysis of RAP fractions using
anti-serum produced against recombinant TFIIE.

Using a genetic approach Young and colleagues have shown
that RNA polymerase II CTD has a functional interaction with
the TATA-binding factor. In order to understand the role of
the RNAP II large subunit carboxy-terminal domain (CTD) in
transcription initiation, they investigated the function of
the SRB2 gene, which was isolated as a dominant suppressor of
CTD truncation mutations. Template commitment assays indicate
that SRBZ becomes associated with the transcription apparatus
at the promoter, and binds specifically to TFIID as revealed
by column chromatography studies. Biochemical studies by
Usheva et al. (1992) indicate that the CTD interacts directly
with the human and yeast TATA-binding factor (TBP). The
phosphorylated form of the enzyme was unable to interact with
TBP.

Recently the Kornberg lab reported the purification and
characterization of another yeast basal initiation factor,
Factor 9, which consists of three polypeptides of 30, 54, and
105 kDa with a native molecular mass of 300 kd as judged by
gel filtration (Henry et al. 1992). Factor 9 was required for

promoter-directed transcription with purified factors a, b, e,

20
TFIID, and pol II. In addition, they showed that factor g can
stably associate with RNA polymerase II by co-sedimentation in
a glycerol gradient. Factor g is thought to be related to the
human factors TFIIE or TFIIF since both of these also
independently associate with RNA polymerase II. At this time
it remains unclear whether factor 9 is homologous to any
mammalian transcription factor. A possible experiment to test
this is to see if antibodies against the human TFIIE will
react with factor g. But due to expected sequence divergence
between the two proteins this experiment seems unlikely to
give positive results. The cloning and sequencing of the genes

for factor 9 will resolve this issue.

1.5 Energy Requirements for Accurate Initiation of
Transcription

Accurate transcription initiation by RNA polymerase II at
various promoters requires the presence of either ATP or dATP
(Bunick et al. 1982; Sawadago et al. 1984). Because
nonhydrolyzable ATP analogues will not support accurate
initiation, ATP hydrolysis of the 6-7 phosphate bonds are
utilized for energy during the initiation process prior to the
formation of the first phosphodiester bond. The purpose of ATP
hydrolysis is unknown at this time, but some possible roles
are; (1) to provide energy for the formation of the first or

first few phosphodiester bond(s) of the transcript; (2) to

21

phosphorylate one or more of the general transcription factors
so that the complex becomes activated; (3) to convert the RNA
polymerase II form (11a) to the (IIo) form; (4) to establish
the open complex, by the melting of the DNA template around
the initiation site, to expose the template strand. It is not
clear what happens inside the preinitiation complex during the
ATP-dependent transition. Once the open complex is formed, the
activated complex moves toward the elongation stage if other
nucleosides triphosphates are present.

Previously, several authors had observed a DNA-dependent-
ATPase activity associated with transcription factors TFIIE
and RAP30/RAP74 (TFIIF) in partially purified fractions
(Reinberg and Roeder, 1987; Sopta et al. 1989). These
observations at present appear not relavant to a possible role
of TFIIE or TFIIF in the hydrolysis of ATP during specific
transcription initiation. This is because RAP74 is neither
required for RNA polymerase II to initiate phosphodiester bond
formation from a promoter (Chang et al. Paper submitted) nor
for ATP hydrolysis in initiation, which precedes
phosphodiester bond formation. The recently idenitifed 89 Rd
subunit of TFIIH appears to be the ATP-dependent DNA helicase
required to melt the DNA template, to facilitate the onset of
the transcription reaction. Neither RAP74 or RAP30 could be
detected in the purest preparations of TFIIH as shown in
immunoblotting' results using' two corresponding antibodies

(Schaeffer et al. 1993).

22

Studies by Gralla et al. (1992) imply that transcription
complexes generated by binding of TFIID, IIA, IIB, pol II,
IIE/IIF, and TFIIH are all closed complexes, as they form in
the absence of ATP. The closed complex containing the factors
bound to the DNA is opened upon the addition of ATP. The
opening or melting allows the polymerase access to the bases
on the template and is thus a required event in gene
activation. Transcriptional activators facilitate the assembly
of these closed and open complexes by allowing them to form
faster or more efficiently (for reviews, see Gralla 1990 and
1991). Gralla concludes that in the pol II and sigma 54
system, transcription can be stimulated by activators bound at
close or distant positions and all promoters containing
Intiator or TATA elements require ATP hydrolysis to form open
complexes (Jiang et al. 1993). This differs from the pol III
and sigma 70 systems which cannot be activated at a distance
and do not require ATP hydrolysis to melt the DNA. This
suggests there may be two distinct classes of mechanisms for
transcription initiation present in eukaryotes and

prokaryotes.

23

1.6 structure and Properties of Transcription Factor TFIIE

In order to learn about the participation of the general
transcription factors in the regulation of transcription
initiation, the approach has been to isolate each factor in a
pure form and to elucidate its function. Currently, seven
basal transcription factors have been identified and
extensively purified; TFIIA, -IID, -IIB, -IIE, -IIF, -IIH, and
-IIJ.

As for most of these factors, the understanding of the
functional properties of TFIIE has been hampered by the slow
progress of its purification. Early studies with a partially
purified TFIIE fraction indicated that it interacts with RNA
polymerase II (Reinberg et al. 1987), that it acts at a late
stage in preinitiation-complex assembly and function (Workman
and Roeder, 1987; Buratowski et al. 1989) and that it may
possess an ATPase activity (Sawadago and Roeder, 1984;
Reinberg and Roeder, 1989). Further purification, however,
resulted in the separation of TFIIE from TFIIF(RAP30/RAP74)
(Reinberg et al. 1987; Burton et al. 1988). Homogenous TFIIE
contained polypeptides of 34 kDa and 56 kDa that copurify with
transcriptional activity (Ohkuma et al. 1990; Maldonaldo et
al. 1990). In agreement with previous studies indicating an
interaction between TFIIE and RNA polymerase II it was found
that entry of TFIIE into the transcription cycle was

subsequent to the entry of RNA polymerase II.

24

Following the purification of TFIIE to apparent
homogeneity, the Roeder and Reinberg laboratories made the
following observations: (1) TFIIE is required for accurate in
vitro transcription of several class II promoters; (2) the
native molecular mass of TFIIE as determined by gel filtration
was estimated at 200 kDa; (3) TFIIE exists in solution as a
tetramer composed of two 34 kd and two 56 Rd polypeptides; (4)
the 56 kd subunit was sufficient for low levels of
transcription activity using a reconstituted transcription
assay depleted of TFIIE, (5) the addition of 34 kd subunit
resulted in stimulation of transcription, (6) TFIIE failed to
show any DNA-dependent or independent ATPase activity,
helicase, kinase, or topoisomerase activity.

Both subunits of TFIIE have been cloned from HeLa cDNA
libraries using reverse genetics (Peterson et al. 1991; Roeder
et al. 1991). The predicted molecular mass of the two subunits
are 49.5 Rd (493 amino acids) and 33 kd (291 amino acids)
respectively. The recombinant TFIIE (rTFIIE) behaves
identically to native TFIIE in gel mobility shift assays and
reconstitution assays in basal and activated transcription. In
contrast to an earlier observation concerning the 34 kd
subunit, neither TFIIE subunit alone was sufficient to
reconstitute accurate transcriptional activity. It is likely
that the earlier experiments were performed in an assay system
contaminated with the 56 kd subunit of TFIIE.

Amino acid comparisons of both subunits of TFIIE revealed

25
no close similarities with any of the other known
transcription factors or any other proteins in GENBANK and
EMBL databases. Both subunits did, however, reveal several
interesting motifs as well as subregion similarities with
other transcription factors that may provide clues to
functional roles in transcription initiation.

TFIIE-B (33 kd) is a highly basic protein of pI 9.5 with
several basic amino-acid clusters through. which it could
complex with the TFIIE-a (49.5 kd) subunit, a very acidic
protein with a pI of 4.5, suggesting that interaction of the
two subunits may involve ionic interactions. A potential
protein interaction site in TFIIE-B is a putative leucine
repeat from residues 154 to 176 (figure 1.6A and 1.6C). other
interesting regions of TFIIE-B are the serine rich region (21—
71) and the basic region sequence (251-273) which show
similarity to part of the basic region-helix—loop-helix (BR-
HLH) domain of the c-myc-related family of enhancer binding
proteins (such as Myo-Dl and E12)(Davis and Weintraub, 1987).
This basic region may be involved in protein-protein
interactions or DNA-binding.

The large subunit TFIIE-a, also contains three potential
structural motifs (leucine repeat, zinc finger, and helix-
turn-helix) that. are characteristic of" many transcription
factors (see figure 1.6B—1.GD). There is also a highly acidic
region (378-389), a kinase consensus region and a predicted

amphipathic alpha helix (12-41). Perhaps the most interesting

26

 

 

 

 

 

 

    

 

1 29-71 154- 173 251- 273
‘-‘
\ \
B .s s
S-rlch LR Basic
1 12-41 57 129-157 132 242- 259 373-339 439
a C-C-C-C .4
LR Zinc Klnue Acidic
a- Helix ““0" HT" consensus

TFIIE -i3154 I DQHDQI GLGG iLI EDI EE-
mm -a 33 NSCVKI EDMLELI KFDRKO L ISVLNNI

D.

157
TFIIE —a TNRASFKC PV STFTDLEANQLFDPMTGTF-RCTFC
s-II TNTDLFTC GK KIYTQVQTRSAD::PT-TFWCNEC
KNC
Kruppel TGEKPFEC PE KR FTRDHHLKT 7I1E|272
TFIIIA TGEKPFPC KE%C KG FTSLHHLTR
E

Figure 1.6 Structural features of TFIIE subunits B (56kDa) and a
(34kDa). A) S-rich, region rich in serine residues; LR, Leucine repeat;
Basic, region rich in basic residues; B) a—Helix, region predicted to
form amphiphatic alpha helix; Leucine repeat region; Zinc finger,
sequence that could potentially form a zinc finger; HTH, helix-turn-
helix; Kinase consensus, region similar to proteins with known kinase
activity ; Acidic, region rich in acidic residues. C) Leucine repeat. The
putative leucine repeat of TFIIE-B is aligned with that of TFIIE-a.
Revelant residues are boxed. Oppositely charged residues at the same
position may also interact. D) Zinc finger in TFIIE-a. Similarities to
Transcription elongation factor S-ll(boxed 0202 motifs). TFIIIA, and the

Drosophila developmental protein Kruppel (boxed C2H2 motifs).

27

finding is a sequence similar to the zinc-finger
motif containing a cluster of cysteine residues arranged in
the Cޤ(two cysteine pair, figure 1.6D) class. The structure
and function of TFIIE-a putative zinc-finger is analogous to
that of the single zinc-finger found in the transcription
elongation factor TFIIS (Agrawal et al. 1991), which is
thought to be involved in DNA-strand opening or protein-
protein interactions. This region also shows sequence homology
with the zinc finger motifs present in the UvrA and UvrB
proteins found in E.coli (Husain et al. 1986).

Recently, work done in Phillip Sharp’s lab (Parvin et
al. 1992) questions the paradigm that all basal factors (in
this particular case TFIIE) are essential for all polymerase
II promoters. They suggest that not all basal factors are in
fact general. A general factor is defined as a factor required
by all polymerase II promoters, whereas, basal factors are
required for unregulated transcription. Studies of basal
factor requirements for transcription from the immunoglobulin
heavy chain gene (IgH) showed no apparent requirement for
TFIIE, whereas basal transcription from the adenovirus major
late promoter is highly dependent upon TFIIE, in addition to
the other basal factors. In these studies, reactions were
reconstituted using partially purified basal factors from both
HeLa cells and the human B cell line BJA-B.

The strongest evidence for the TFIIE requirement and

possible role in transcription initiation was done in work

28
done in Reinberg’s lab (Lu et al. 1992). In that study they
tested whether TFIIH could phosphorylate RNA polymerase II in
the absence of the other basal factors. In these experiments
the addition of TFIIE to the assay resulted in stimulation of
phosphorylation of RNA polymerase II. This suggested that
TFIIE may stimulate the processivity of TFIIH-kinase activity.
Because TFIIE is directly responsible for introducing TFIIH to
the preinitiation complex, and also to RNA polymerase II, it
is not surprising that it stimulates TFIIH activity. The
availability of large quantities of purified recombinant TFIIE
will facilitate studies of this protein’s function in

transcription and complex assembly.

1.7 Production of Human Protein in E.coli

The advanced knowledge of the genetics and physiology of
E.coli has made this bacterium ideally suited as a host for
protein overproduction. Additional characteristics that make
it advantageous include rapid generation of biomass due to
high rates of cell growth and availability of low—cost media.
The disadvantages of this system are its limited capacity to
secrete proteins and its inability to exert certain
posttranslational modifications of proteins (such as disulfide
bond formation, glycosylation and acetylation), which can
affect the proper folding or function of the protein. other

problems more associated with human proteins include: the

29

high-level expression of many proteins can lead to the
formation of "inclusion bodies", very dense aggregates of
insoluble protein, which must be solubilized by denaturing
reagents followed by careful renaturation. A problem with
denaturation/renaturation is the yield of properly folded
protein is variable and sometimes quite low; some proteins,
especially large ones, cannot be properly refolded at all.

Translational efficiency problems are often encountered
when foreign proteins, such as human proteins, are produced in
E.coli. Translation initiation is mediated by the 5'
untranslated mRNA region. There is now compelling evidence
that not only the ribosome binding site (RBS) and the AUG
determine the efficiency of the process, but also both the
structure and sequence of this region affect the ribosome
accessibility to the transcript. Therefore, this region may be
manipulated to maximize translation initiation. Another
potential problem is that of pre-mature translation
termination due to E.coli codon preference. For example, the
gene encoding human RAP74 appears to be translated
inefficiently in E.coli. Interspersed throughout the central
1/3 of the gene are a number of rare E.coli codons, including
two consecutive Arg codons (AGG) which have been reported to
result in poor expression (Wang et al. 1993). This problem can
be resolved by recoding the protein using PCR.

The last important parameter pertaining to protein yield

is protein degradation. Proteolysis is a very selective and

30

carefully regulated process that influences the degree of
protein accumulation in E.coli. Protein degradation has
important practical consequences since many cloned proteins
are recognized as abnormal by proteolytic systems of the cell,
and are therefore rapidly hydrolyzed (Boyer et al. 1977).
Despite these potential disadvantages, E.coli is a useful host
for protein production.

The basic approach used to express foreign genes in
E.coli starts with insertion of the gene into an expression
vector. The minimal elements that an expression plasmid vector
should supply are a well characterized origin of replication
and a selection marker for plasmid propagation and
maintenance, a strong regulatable promoter, a ribosome binding
site, and appropriate cloning sites to clone the gene in the
correct orientation within the vector.

In these studies the pET16b vector (see figure 1.7) was
used to overproduce TFIIE-a and TFIIE-B. This plasmid carries
the colicin E1 replication origin (ori) allowing maintenance
of multiple copies in E.coli. The selectable marker is the
gene encoding lactamase, which confers ampicillin resistance
(amp). The strong inducible promoter is the bacteriophage T7
promoter. A very active enzyme, T7 RNA polymerase elongates
chains about five times faster than does E.coli RNA
polymerase. Background expression is minimal in the absence of
T7 polymerase because the host enzyme does not initiate from

T7 promoters. For gene expression from this promoter a

31

pET1 6b
(5711bp)

 

Figure 1.7 Schematic of Bacterial Expression Vector
pET16b. The minimal features of the plasmid include:
ori, orgin of replication; T7, regulatable promoter; amp,
ampicillin resistance selective marker; mcs, multiple
cloning site; lac, lac repressor gene.

32

specialized E.coli strain, BL21(DE3) is required that
can supply T7 RNA polymerase from the defective DE3 lysogen.
T7 pol is controlled by the lacUVS promoter and lac operator
in DE3. Addition of IPTG (isopropyl-Beta-D-
thiogalactopyranoside) tcia growing culture induces production
of T7 RNA polymerase, which in turn induces production of the
cloned gene encoded on the pET vector. Even in the absence of
IPTG there is some expression of T7 RNA polymerase from the
lacUVS promoter in ADE3 lysogens. This may present a problem
if the protein produced is toxic to .E.coli. Studier and
colleagues resolved this problem by designing vectors that
contained a T7 lac promoter (Studier et al. 1990). When this
type of vector is used for expression (pET16b and pET23a-d),
the lac repressor acts both at the lac UV5 promoter in the
host chromosome to repress transcription of the T7 RNA
polymerase gene by the host polymerase and at the T7 lac
promoter in the vector to block transcription by any T7 RNA
polymerase that is made.

Purification of recombinant proteins from host organisms
can be time-consuming. For many proteins removal of
contaminants is critical to the characterization of a
recombinant protein. One solution to this problem is to fuse
the overproduced protein to a peptide for which an affinity
purification is available. The newest pET vectors have the

advantage of carrying a stretch of either 6 or 10 consecutive

33
histidine residues 0% or Hm-tag) that can be expressed at the
N-terminal or C—terminal end of the protein. Some of these
vectors allow removal of the His-TagTM with proteases
thrombin, enterokinase or Factor X“. These modified vectors
allow purification of proteins under gentle, physiological
conditions if proteins are soluble. The His-Tagn“(available
from Novagen), sequence binds to divalent cations (Niz+) and
allows expressed proteins to be purified by metal chelation
chromatography (Hochuli et al. 1987 and Smith et al. 1988).
After washing of unbound proteins, the recombinant protein is
recovered by elution with imidazole. The procedure is rapid
and efficient, allowing' purification to greater than 90%

homogeneity in one step.

2.0 MATERIALS AND METHODS

2.1 Construction of gecombinant clones pET16szPIIE-a and
pET16blTFIIB-d

Isolation of cDNAs encoding TFIIE has been described
(Peterson et al. 1991). The plasmids pAR34E and pAR56E
containing the clones were the kind gift of Dr. Danny
Reinberg. The cDNAs for both subunits were subcloned as an
Eco RV to Nde I fragment between the NdeI and BamHI (the BamHI
site was converted to a blunt end with Klenow DNA polymerase
I) sites of pET16b by Janek Werel in our lab (see figure 2.1).
When fused. with. the TFIIE cDNAs in frame this construct
produces the 36 kd and 58 kd proteins with a short peptide
extension at the NHz-terminus: MGI-Iiflflml-im-nfliSSGHIEGRim (H10-
TFIIE-B, Hm-TFIIE-a). This sequence contains a stretch of 10

histidines and a factor Xucleavage sequence (IEGR/).

2.2 Growth and Lysis of cells

BL21(DE3) cells were transformed by each plasmid and the
cells were grown at 37° C in 10 liters of LB medium containing
100 ug/ml ampicillin until Aubreached 0.6, at which time IPTG
was added to a final concentration of 0.4 mM to induce
synthesis of T7 RNA polymerase. After 3 hr induction, cells

were harvested by centrifugation at 4,000 rpm for 15 min. in

34

35

A.
pET16b/TFIIE—B

Factor Xa

Nco l
mmmmssmmcnn

Ndel

T7 Eco RV/Bam Hi
5’ _ i—n—H - a l
AGGA

B.
pET16b/TFIIE- a

 

 

 

NCO I Factlor Xa
scar?
1.7 N iel Eco RV/Bam Hi
5*- m— . l |
AGGA

Figure 2.1 Schematic of TFIIE-or and TFIIE-[3
Overproduction Clones. A) pET 16b/TFllE-B; this clone
encodes H1o-TFllE-B (34kDa subunit). B) pET16b/TFllE-a;
this clone encodes H1o-TFllE-a (56kDa subunit). NH2-
terminal extensions are indicated by standard one letter
aminoacid code. AGGA= ribosome attachment sequence.
The dark line indicates human DNA.

36
a Sorvall RC3B centrifuge at 4° C. All subsequent steps were
done at 4°C except where noted. The methods for preparing the
cell extract and for purification on the Ni2+ column were
adapted from information provided by the manufacturer
(Novagen, Madison, WI). Cells were resuspended using a dounce
homogenizer in 100 ml of lysis buffer (20mM tris-HCL (pH7.9),
5 mM imidazole, and 500 mM NaCl containing 1 mg/ml lysozyme).
Cells are then lysed by sonication (Ultrasonic Model 220-F, 5x
for 30 seconds, with a 30 second rest in between at a setting

of 5 using a microtip).

2.3 Purification of TFIIE-a (58 Rd subunit)

Cells containing TFIIE-a were centrifuged at 5,000 rpm
for 10 min. in a Sorvall SS-34 rotor to remove debris. The
supernatant fraction containing soluble Hlo-TFIIE-a was applied
to a column made up with 10 ml of His-bind““resin. The column
was washed with 150 ml of 1x wash buffer (500 mM NaCl, 20 mM
tris-HCL (pH 7.9) and 60 mM imidazole). The protein was then
eluted into 60 ml of 1x elution buffer (1M imidazole, 500 mM
NaCl and 10 mM tris-HCL,(pH 7.9). Prolonged incubation of

TFIIE-a at 0.1 M KCL results in precipitation.

37
2.4 Purification of TFIIE-fl (36 Rd subunit)

Cells containing TFIIE-B were centrifuged at 5,000 rpm
for 10 min. to remove debris. The cell pellet was then
solubized by resuspending in 1x binding buffer (5 mM
imidazole, 500 mM NaCl , 20 mM Tris-HCl, pH 7.9) containing 6M
urea. For subsequent steps of washing and elution 6M urea was
also used (1x wash and 1x elution buffer). The supernantant
fraction containing denatured TFIIE-B was applied to the Niz+
column containing 10ml of resin followed by, 150 ml wash with
1x wash buffer and subsequent elution with 100 ml of 1x
elution buffer. Prolonged incubation of TFIIE-B in low salt

conditions (0.1M KCl) also results in precipitation.

2.5 Production of Anti-TFIIB-a and Anti-TFIIE-fl Anti-
serum and Western Blot Analysis

Polyclonal anti-serum was raised in rabbits against
bacterially produced TFIIE-a and TFIIE-B proteins . Preimmune
serum was taken from four New Zealand white female rabbits.
Two were immunized with 150 pg of filo-TFIIE-a and the other two
with the same amount ofIHw-TFIIE-B. Protein was emulsified in
Hunter's Titre MaxmifHR-l research adjuvant using 250 pl of
adjuvant and aqueous solution containing protein. Rabbits were
boosted on.days 25 and.39 with 100 pg of protein. Rabbits were

bled on days 46 and 53.

38

Western blots were performed essentially as in Harlow
and Lane (1988) . Proteins were separated by SDS-PAGE and
transferred electrophoretically to nitrocellulose membranes
(Bio-Rad). Nonspecific interaction of the antibody was
prevented.by incubating'in.blocking solution for 30 minutes in
50 mM tris-HCL (pH 8.0), 150 mM NaCl and (1X TBS), plus 3%
gelatin followed by washing 2x for 5 min in 1X TBS . The
immunoreaction was performed using a 1:500 dilution of
antiserum. The membrane was washed 2X for 5 minutes in 1X
TTBS, before incubating with the second antibody 1:3000
diluted goat anti-rabbit IgG (Bio-Rad). The immunoreactive
bands‘were‘visualized.with,201ml of color development solution
(20 ml of 1X carbonate buffer (100 mM Na H003,1 mM MgC12) ,6 mg
of NET (p-nitro blue tetrazolium chloride) and 3 mg of BCIP
(5-bromo-4-chloro-3-indoyl.phosphatejp-toluidine.salt), (Bio-
Rad). The reaction was stopped by rinsing with TBS. Pre-
stained protein low and high molecular weight markers (Bio-
Rad) were used to monitor electroblotting and as a size

marker.

2.6 Preparation of protein-affinity fractions for
Western analysis

Eluates from an RNA polymerase II affinity column and
anti-CTD affinity column were collected from 250 m1 of whole

cell nuclear extract in 100 mM KCl, followed by wash in 100 mM

39
KCl and eluted at 500 mM KCl. The control column was Affi-gel
(Bio-Rad) containing no protein. Eluates were tested for the
presence of TFIIE. Proteins that bind to the RAP column and
not the control column are considered RNA polymerase II-
associating-proteins or RAPs. The protein fraction that binds

to the anti-CTD column should also contain RAPs.

2.7 In vitro transcription Assay

The template ‘used for' runoff transcription ‘was the
adenovirus major late promoter (Ad-MLP) subcloned as an Xho I
and Hind III fragment (coordinates -256 to +196) relative to
the Ad-MLP cap site) between the XhoI and Hind III sites of
the vector pBluescript II KS (+)(Stratagene). The DNA has a
single Sma I site located at +217 relative to the AdMLP cap
site. This template after digestion with Sma I was used to
produce a runoff transcript of 217 nucleotides (see figure
2.6). The :methods for in ‘vitro ‘transcription. have ,been
previously described (Burton et al. 1986) . Reactions were done
at 30°C. Whole nuclear extract and antibodies were combined
with AdMLP DNA (60 pg/ml) and preincubated for 60 min. The
preincubation reaction volume was 20 p1; reactions at 30°C for
30 min. Transcription buffer contained 12 mM Hepes pH 7.9, 12%
glycerol, 60 mM KCL, 8 mM MgC12, 3.12 mM EGTA, 0.12 mM EDTA
and 1.2 mM DTT. 600 pM ATP, CTP, UTP and 25 pM GTP(5 pCi/

reaction -732P GTP) were added in 5 pl aliquots. Reactions were

40

 

 

 

 

 

ATP
. . . . CTP
Preinitiation UTp Elongation
, o GTP*
I 50 mm, 30 C . I 30 min, 30°C
I l I
Nuclear extract Stop
DNA. template , EtOH ppt
_ . . PAGE
+l Antibodies Autoradiography
Quantitation
pBluescript ks +
217 nt RNA
AdMLP
ITATAAAI r> 1
+1
Sma
Figure 2.6 Schematic of in vitro transcription assay

used in antibody inhibition studies. The top figure describes
the protocol for a typical reaction. The bottom figure depicts
the DNA template used (adenovirus major late promoter) and
the RNA transcript produced during accurate transcription
when the template is digested with Sma l .

41
stopped, phenol — chloroform extracted, ethanol precipitated,
resolved on 6% PAGE containing 50% (w/v) urea and visualized
by autoradiography. Quantitation of accurate transcription was

done using a Molecular Dynamics Phosphorimager.

3.0 RESULTS

3.1 Production and Purification of TFIIE-a (58 kDa subunit)

TFIIE-a (58 kDa subunit) was isolated from E.coli
BL21(DE3) containing the translation vector pET16b/TFIIE—a.
pET16b/TFIIE-a produces Hw-TFIIE-a with the added sequence
MGHHHHHHHHHHSSGHIEGRHMLD at the NHfiterminal end of the
protein. The whole cell protein is visualized by 10% SDS-PAGE
and Coomassie blue stain. Production of TFIIE-a before and
after induction with 0.4 mM IPTG is shown in Figure 3.1A.
TFIIE-a produces a notable protein band on SDS PAGE of cells
after IPTG induction although production is not as efficient
as TFIIE-B (compare lanes 2,4 and 6 of figure 3.1A to lanes 3
and 4 of figure 3.1B). TFIIE-a produced in E.coli is soluble.
TFIIE-a was purified to near homogeneity under native
conditions using Ni2+ affinity chromatography (see figure 3.2,
lanes 3 and 4). Lanes 3 and 4 contain approximately 2 ug and
5 ug of purified protein. The yield obtained of TFIIE-a from
a liter of bacterial culture is approximately 6 mg based on
compared observations of protein on Coomassie stained SDS-PAGE

to known low protein marker amounts.

42

43

 

97 — i
66— "i ""
H1o m %
TFHE-u
5 _ 5 if? . 7:: Liié ‘EL: :7 7
h Rea-3 it; 7 g 7
31— is. ,

 

 

 

Figue3.1A Ovetpodlctionof fielagesuburit of H1o-TFllE-a 'n
bacteriaLsing the expressimplwrid pET16b/TFllE—a. The gelshown
isa 10% poyacybrnBeSDS-PAGEgelsta’nethhCoomassiebbedye
Theeseparateoobriesmegownandamlyzed foreffidencyofpdection
(odony1,|anesland2:colony2 lares3and4zoola1y3,la'es53r16).
LamlfiardS-lOulofbacteridectmctbeforehdJcfionw: ' .
LameSZA and6 -10ulofbacterialexuact 3m:rsafter'r1dicdon(1)
vvithOAliPl'G. Tneappalmtn'loleuiarweig'rtofthereoombimnt
protein '5 58kd(56+2).

44

 

97—
66— ‘6

u
45—6 1.... -, .1 r l.
' . “in ’ - - —“1o -

 

 

 

f“ i" *' TFIIE-f5
31 — - r '
22—_ .
M 1 2 3 4

Figure3.18 OverpdectionofthesmalsnfltofTFllE(H1o-TFIE-B)
hbactaiawhgflmeemrefionplasniddfile/TFIE—fimefguresims a

10% Slfi-Polyagylamkiegelstainedwitl'lCoormSebluedye. Twoseparate
oobriesweamlyzedforgrowth.00lmy1,Laneslard3-20ulofu1ind1wd
bacterialextract(U)and3l'rsafterrmctionwim0.4mMIPTG. Odony2,LanesZ
and 4 - ZOuI ofurinduoedand bacterialextract Stoursafter'rmwmfl) with
0.4 mM PTG. Theappaent mdewlaweightofthereoombirantprotehis36 kd
(34 +2).

45

45

31

22

 

Figure 3.1C Purified and Partially purified
TFIIE-[3. The gel shown is a 10% polyacryiamide
SDS-PAGE gel stained with coomassie blue dye.

Lanes 1 and 2 contain approximately Sug of purified
H10-TFllE-B, lanes 3 and 4 contain approximately
15 ug of H10-TFllE-B with smaller and larger poly-

peptides of various molecular weights.

 

46

 

 

 

 

TFIIE - a
mo-
TFllE - B
M 1 2 3 4
Figure3.2 One step purification of reoomb'nmtpmteins H10 -TFllE-[3

ande -TFllE-B ushg a N2+ aff'nity ooumn(fromNovagen). Thefigure
showsa 10%pdwcrylamide SDS-PAGEgelstalnedwlthCoomassieblue dye.
M)lo~ molecular weigit marker (BioRad)Lanel-5ug of H10 -TFllE-(3;
LaneZ-lSug of H1o-TFllE-B;Lane3-2ug of H1o-TFllE-a; Lane4-

Sug ofH-lo-TFllE-a; Forpun'fication ofH1o-1HlE-B,1x elution buffer
oontainingGM ureamsusedtosohlbfizetheprote’n H1o-TFllE-a vmspurif-
iedundernativeoondtions.

47

3.2 Production and Purification of TFIIE-fl (36 kDa subunit)

TFIIE-B was purified from E.coli BL21(DE3) cells
containing the translation vector pET16b/TFIIE-B. This vector
producesZHw-TFIIE-B with the sequence MGHwSSGHIEGRHLMD fused
to the NH2-terminal end of the protein. TFIIE-B accumulated
within inclusion. bodies as an insoluble aggregate.
Purification was performed under denaturing conditions (6M
urea ) according to the pET—His protocols of Novagen. Figure
3.13 shows results of the whole cell extract before and after
induction ‘with IPTG as ‘visualized on 10% SDS-PAGE ‘with
Coomassie blue stain. The small subunit migrated at a rate
consistent with its molecular weight of 36 kd and appears to
be the most abundant protein present in the extract. Figure
3.1C shows purified and partially purified TFIIE-6. Under
native conditions only partial purification was possible and
as a result smaller and larger polypeptides were also found
after'Ni¢+affinity'purification(see lanes 3 and 4). Figure 3.2
shows results of purification using Ni2+ affinity
chromatography using 6M urea for purification of TFIIE-6 (see
lanes 1 and 2). Approximately 5 and 15 ug of protein is shown
in lanes 1 and 2. The yield of Hm-TFIIE-B from a liter of
bacterial culture is approximately 15 mg. Based on compared
observations of known protein amounts on Coomassie stained

SDS-PAGE.

 

48

3.3 Western blot Analysis of recombinant TFIIE

Western blot analysis was performed using anti-serum
against the bacterially produced proteins in E.coli. Each
corresponding subunit of recombinant TFIIE (58 kDa and 36 kDa
subunits) was detected. Experiments were performed to
determine the sensitivity of detection of the antiserum
generated or titre. Figure 3.3A shows an immunoblot containing
the following amounts of Hm-TFIIE-a; 500 ng, 250 ng, 125 ng,
50 ng, 25 ng, 10 ng and 5 ng. Our antibody can be used to
detect Hw-TFIIE-a to the low ng range, which is consistent
with results obtained using RAP fractions (figure 3.3C, lane
R2). The control lane (lane C) contained 125 ng of Hw-TFIIE-B.
Figure 3.38 shows an immunoblot of Hm-TFIIE—B with decreasing
amounts of protein; 500 ng, 250 ng, 125 ng, and 25 ng. The
control lane contained 125 ng of Hw-TFIIE-a. A 1:500 dilution
was used for the primary antibody for each blot. Immunoblots
of RAP fractions (Figure 3.3C Lanes R1 and R2) provides
immunological evidence that the recombinant proteins are
derived from expression of cDNAs for each subunit of TFIIE.
Results reveal that both subunits of TFIIE are present in RAP
fractions in ng quantities (positive controls - 25 ng). Eluate
from the anti-CTD affinity column gave a weak reaction for the
TFIIE-a,(Lane C2), but TFIIE-B was below the level of

detection in this experiment (Lane C1).

49

 

 

106—‘

80—‘

 

“10' —‘
TFIIE -a
49

 

 

32

 

27

 

Figure 3.3A Western blot analysis for detection of the large
subunit of TFIIE ( TFIIE - a ). Lanes 1 -7, H10 - TFIIE - a
protein : 5, 10, 25, 50, 125, 250, and SOOng respectively.
Lane C, ”10' TFIIE - )3, 125 ng (negative control). ‘

 

50

 

106—
80—
49—
- —”10'
32— . - - TFIIE-B

 

 

 

Figure 3.33 Western blot analysis for detection of small
subunit of TFIIE (TFIIE - B ). Lanes 1-4, H10 - TFIIE - [3
protein : 500ng, 250ng, 125ng and 25ng respectively.
Lane C, H10 - TFIIE - a, 125ng (negative control).

51

anti-TFIlE-B anti—TFIlE-a

 

80—
66—

49—

32—

 

 

 

C1 R1 NC PC1 M C2 R2 NC PCZ

Figure 3.3C Western blot analysis of small and large
subunit of TFIIE in RAP fractions from RNA polymerase ll affinity
column and anti-CTD affinty column. Lane C1 and C2, anti-CTD
affinity column eluate, lane R1 and R2, RAP column eluate, lane
NC, negative control column eluate, lane PC1, positive control,
25ng recombinant H10 -TFllE- B, lane M, prestain molecular

weight markers, lane PCZ, positive control, 25ng recombinant
H10- TFIIE - (1.

 

52

3.4 Inhibition of Accurate Transcription by.Anti-TFIIB serum

Anti-serum against TFIIE subunits 36 kd and 58 kd were
tested for ‘their ability to inhibit accurate
transcription from the.AdMLP. The results are shown in figure
3.4. Both anti-TFIIE-a and anti-TFIIE-B show inhibition when
added to in vitro transcription reactions of Hela nuclear
extracts. This result supports previous evidence that.TFIIE is
required for basal transcription from.the AdMLP. Anti-TFIIE-B
inhibits transcription by more than 90%, while anti-TFIIE-a
inhibits about 70%. Partial inhibition islalso seen when anti-
RAP30 serum is added to the reaction, inhibiting around 95%.
In addition, anti-RAP74 serum is less effective, inhibiting

about 50% (3.0. Wang, unpublished results).

(%)

transcription activity

Relative

53

 

 

 

 

120
—El— anti-34kd
—o— anti-56kd

100-! .
—I—' Preimmune
—°— anti-RAP30

80-

60-

4o-

20-

0 - I . I ‘ I '

0 1 2 3 4
volume of Abs (pl)
Figure 3.4 Inhibition of Accurate Transcription by

Antiserum Raised Against Basal Subunits. Graph shows
the effect of various antibodies raised against TFIIE
subunits 34kd and 56 kd, RAP30, and preimmune serum.
Total reaction volume is 20 pl.

 

4.0 DISCUSSION

An efficient method for the production and purification
of human TFIIE subunits a and B in bacteria has been
described. The method of Ni?+- affinity chromatography
provides a versatile, economic and convenient means of
purification either under nondenaturing or denaturing
conditions for a wide variety of proteins. Studies are now
under way to test the activity of recombinant TFIIE in in
vitro reconstitution assays.

TFIIE has been proposed to be a heterotetramer (azfiz)
structure during its formation of a preinitiation complex on
a promoter (see figure 4.1A). This is based on gel filtration
chromatography that suggests that the 36 Rd and possibly
the 58 kd subunit may form dimers in solution. When the
recombinant proteins were mixed and subjected to gel
filtration, they behaved as a complex of approximately 230 kd,
which is similar in size to native TFIIE (Peterson et al.
1992). TFIIE, like many of the other basal factors contains
many highly charged acidic, basic and hydrophobic regions that
may, be involved in protein-protein interactions. When
individual subunits are isolated in the absense of their

normal partners, binding domains may cause aggregation.

54

 

a

A a m... ‘13; 55

55

 

 

\\\\\\\\\\ \\\\\\\

L ; c4
' «5'9.»
to,

 

Figure 4.1A Current Model of the Preinitiation Complex.
Factors D (TBP), A, and B associate first to form the DAB
complex on the TATA box region of the promoter. Then RNA
polymerase II (RNAP II) and TFIIF. Followed by factors E,
H, and J which enter the complex late. Both TFIIE and TFIIF
are depicted as heterodimers. TFIIH is a large complex with
at least 5 subunits.

 

56

Multimerization of individual subunits may contribute to
another problem that occurs during isolation of the proteins.
That is, the separation of intact protein from protein
fragments. Intact TFIIE-B copurifies with fragments of TFIIE-
B, which may result from inefficient mRNA translation or
proteolysis (see figure 3.1B, lanes 3 and 4). Dissociation of
TFIIE-B from fragments is observed in 6 M urea.

Antiserumlgenerated,against.recombinant subunits appears
to be useful for detection of TFIIE subunits in RAP fractions.
Detection of the large subunit of TFIIE was much stronger
(around 25 ng when compared to positive control) then the
detection of the small subunit which was barely visible. One
would expect both TFIIE subunits to be present in equal
molar amounts. One explanation may be that the large subunit
binds to the RNA polymerase column more efficiently than the
small subunit. Another is that the small subunit disassociates
more readily during purification. This would be consistent
with previous data by Inostroza et al. 1991 which shows the
large subunit.tolbe sufficient for low levels of transcription
activity, while the small subunit is stimulatory when added.
Perhaps the interaction with RNA polymerase is important for
its activity and [or function.

Antiserum raised against TFIIE subunits shows specific
inhibition of in vitro transcription assays. The antiserum may

be a useful reagent in future studies in producing a TFIIE

57

depleted extract, immunoprecipitation studies of TFIIE, and
for testing the quality of RAP fractions eluted from RNA
polymerase II-affinity columns or anti-CTD affinity columns.
The results provide additional support for the requirement of
TFIIE in basal transcription from the AdMLP. Preimmune serum
also gave moderate inhibition (25%), this may be due to the
presence of RNases in serum. Purification of the antiserum on
an affinity column prior to the in vitro transcription
reaction may resolve this problem.

Although structural motifs and sequence similarities give
clues to possible functions of TFIIE, little is known about
its role in transcription initiation. Previous studies showed
that the purified HeLa cell 56 kd subunit of TFIIE alone has
some TFIIE activity, whereas the 34 kd subunit is stimulatory
(Inostroza et al. 1991) . Reconstituted basal transcription
from the AdMLP is dependent on both subunits to reconstitute
transcriptional activity (Peterson et al. 1991) . Various
studies with a partially purified TFIIE have indicated that it
interacts with RNA polymerase II (Reinberg et al 1987b; Flores
et al 1989) , that it assocites with the transcription complex
at a late stage during complex assembly after pol II and TFIIF
(Buratowski et al. 1989). It is still unclear whether the
interaction with RNA polymerase II is direct or indirect,
although results obtained with RNA polymerase II- affinity
studies (figure 3.3C) suggest a direct interaction. Crude

TFIIE fractions from HeLa cell nuclear extracts possess a DNA-

 

58

dependent ATPase activity, but no activity is associated with
the highly purified TFIIE suggesting possible interactions
with the 89 kd subunit of TFIIH, which was shown recently to
possess an ATP-dependent-DNA helicase activity (Ohkuma et al.
1990; Schaeffer et al. 1993). The basal factor TFIIH also
enters the preinitiation complex late after TFIIE and it may
interact with TFIIE and RNA polymerase II. There is evidence
that TFIIE may stimulate the processivity of TFIIH- kinase
activity which also suggests a possible interaction between
the two factors (Lu et al. 1992).

Other questions to be explored include: 1) do homologs
of TFIIE exist in the RNA polymerase I and polymerase III
systems?; 2) is there a TFIIE equivalent in yeast or other
systems. ? land, if so, hOW’ closely' related. are they in
structure and function? 3) does TFIIE cycle off of polymerase
during the transition from initiation to elongation? 4) what
other factors interact with TFIIE and what are their
functions? Antisera raised against TFIIE subunits may be
useful in determing TFIIE equivalents and /or homologs in
human or other systems.

As mentioned above it will be important to test whether
the recombinant TFIIE subunits will function in a highly
purified transcription system. The purification of TFIIE is an
important step toward this goal. Work is under way in our
laboratory to set up a reconstitution assay for TFIIE. Until

then we are unable to demonstrate biological activity with the

 

 

59
cloned proteins. Another important goal to the transcription
field is the development of a system of completely defined
components that will accurately transcribe from promoters.
This would include the following purified proteins reported to
make up the system: TBP, TFIIB, TFIIF (RAP30/RAP74) , TFIIE (34
and 56 kDa subunits), TFIIH (at least five subunits), TFIIJ
(undefined) and RNAgpolymerase II (10 subunits). Since not.all
components of TFIIH and TFIIJ have yet been defined, as well
as cloned and expressed, a completely defined system is not
possible at present. (Yet, the availability of large quantities
of purified recombinant TFIIE is another step closer to this

larger goal of obtaining a complete transcription system.

 

5. 0 APPENDIX

This section includes information on the aminoacid
sequence and structure of TFIIE subunits, calculated molecular
weight, isoelectric point, and extinction coefficient. Also
included is sequence data of recombinant TFIIE subunits with
the additional 22 amino acid peptide. This can be compared
with the wildtype human sequences. The 22 amino acid peptide
adds.an.additional calculated molecular'mass of 2520.7 daltons

to each subunit of TFIIE.

60

 

Session Name:

clvaxl

$ TYPE TFIIE34K.PEP

TRANSLATE of: tfiie34k.txt check: 2239 from:
generated symbols 1 to:

TFIIe34k

51

101

151

201

251

MDPSLLRERE

SKQNSDHSNG

EILDETQHLD

LLRLLDQHDQ

LFFNDKSCQF

KKVAPIQRRK

$ TYPE TFIIESGK.PEP

TRANSLATE of: tfiie56k.txt check:
generated symbols 1 to:

Tfiie56k

51
101
151
201
251
301
351

401

.pep Length:

MADPDVLTEV

LLKFDRKQLR

NVVKYKLDHM

TFRCTFCHTE

YEILEPEPTE

TQNVVINMDD

DMDAFQEREE

GSDSESETSE

RPFSYSEVSQ

292.

.pep Length: 292 October 8,

LFKKRALSTP
SFNLKALSGS
IGLKQKQWLM
RGLGGILLED
SVDEEFQKLW

KPASQKKRRF

440.

440
PAALKRLAKY
SVLNNLKGDK
RRRIETDERD
VEEDESAMPK
IPALKQSKDH
QEDLHRASLE
GHAGPDDNEE
SDDDSPPRPA

RPELVAQMTP

October 8,

61

1992

VVEKRSASSE

SGYKFGVLAK

TEALVNNPKI

IEEALPNSQK

RSVTVDSMDE

KTHNEHLAGV

1992

VIRGFYGIEH

FIKCRMRVET

STNRASFKCP

KDARTLLARF

AATTAGAASL

GKSAKERPIW

VMRALLIHEK

AVAVHKREED

EEKEAYIAMG

1285 from:

191 to:

13:48

SSSSSSKKKK

IVNYMKTRHQ

EVIDGKYAFK

AVKALGDQIL

EKIEEYLKRQ

LKDYSDITSS

64 to:

14:00

ALALDILIRN

AADGKTTRHN

VCSSTFTDLE

NEQIEPIYAL

AGGHHREAWA

LRESTVQGAY

KTSSAMAGSV

EEEDDEFEEV

QRMFEDLFE*

Type:

Type:

1066

TKVEHGGSSG

RGDTHPLTLD

PKYNVRDKKA

FVNRPDKKKI

GISSMQESGP

Kit

1383

SCVKEEDMLE

YYFINYRTLV

ANQLFDPMTG

LRETEDVNLA

TKGPSYEDLY

GSEDMKEGGI

GAAAPVTAAN

ADDPIVMVAG

P Check:

P Check: 2512

1543

62
Session Name: clvaxl

$ TYPE TFIIE34KH10.PEPSORT
PEPTIDESORT of: Tfiie34kh10.Pep check: 2383 from: 1 to: 313

TRANSLATE of: tfiie34k.txt check: 2239 from: 191 to: 1066
generated symbols 1 to: 292.

With Enzymes:
April 19, 1993 09:15

Summary for whole sequence:

Molecular weight = 35564.15 Residues = 312
Average Residue Weight = 113.988 Charged = 16
Isoelectric point = 10.46

Extinction coefficient = 19120
Residue Number Mole Percent
A = Ala 13 4.167
B = Asx 0 0.000
C = Cys 1 0.321
D = Asp 19 6.090
E = Glu 21 6.731
F = Phe 10 3.205
G = Gly 20 6.410
H = His 20 6.410
I = Ile 14 4.487
K = Lys 39 12.500
L = Leu 29 9.295
M = Met 6 1.923
N = Asn 11 3.526
P = Pro 10 3.205
Q = Gln 15 4.808
R = Arg 17 5.449
S = Ser 34 10.897
T = Thr 10 3.205
V = Val 15 4.808
W = Trp 2 0.641
Y = Tyr 6 1.923
Z = Glx 0 0.000
A + G 33 10.577
S + T 44 14.103
D + E 40 12.821
D + E + N + Q 66 21.154
H + K + R 76 24.359
D + E + H + K + R 116 37.179
I + L + M + v 64 20.513
F + W + Y 18 5.769

63

Session Name: clvaxl

$ TYPE TFIIE56KH10.PEPSORT
PEPTIDESORT of: Tfiie56kh10.Pep

TRANSLATE of: tfiie56k.txt check:

generated symbols 1 to: 440.
With Enzymes:
April 1

Summary for whole sequence:

Molecular weight = $1972.35
Average Residue Weight = 112.983
Isoelectric point = 4.92
Extinction coefficient = 28380
Residue Number Mo
A = Ala 45

B = Asx 0

C = Cys 6

D = Asp 35

E = Glu 54

F = Phe 15

G = Gly 24

H = His 23

I = Ile 18

K = Lys 24

L = Leu 32

M = Met 16

N = Asn 14

P = Pro 20

Q = Gln 11

R = Arg 30

S = Ser 28

T = Thr 25

V = Val 25

W = Trp 2

Y = Tyr 13

Z = Glx 0

A + G 69

S + T 53

D + E 89

D + E + N + Q 114

H + K + R 77
D+E+H+K+R166

I + L + M + V 91

F + w + Y 30

check: 5548 from: 1

1285 from: 64 to:

9, 1993 09:19

Residues = 460
Charged = ~35

le Percent

9.783

0.000

1.304

7.609
11.739
.261
.217
.000
.913
.217
.957
.478
.043
.348
.391
.522
.087
.435
.435
.435
.826
.000

(.0

ONOULflmmNi-waONUlWU'ILD

15.000
11.522
19.348
24.783
16.739
36.087
19.783

6.522

1383

to:

461

64

Session Name: clvaxl

$ type tfiie34k.pepsort
PEPTIDESORT of: Tfiie34k.Pep check: 2512 from: 1 to: 292

TRANSLATE of: tfiie34k.txt check: 2239 from: 191 to: 1066
generated symbols 1 to: 292.

With Enzymes:
October 8, 1992 13:53

Summary for whole sequence:

Molecular weight = 33043.45 Residues = 291
Average Residue Weight = 113.551 Charged = 16
Isoelectric point = 10.46

Extinction coefficient = 19120
Residue Number Mole Percent
A = Ala 13 4.467
B = Asx 0 0.000
C = Cys 1 0.344
D = Asp 19 6.529
E = Glu 20 6.873
F = Phe 10 3.436
G = Gly 17 5.842
H = His 8 2.749
I = Ile 13 4.467
K = Lys 39 13.402
L = Leu 29 9.966
M = Met 5 1.718
N = Asn 11 3.780
P = Pro 10 3.436
Q = Gln 15 5.155
R = Arg 16 5.498
S = Ser 32 10.997
T = Thr 10 3.436
V = Val 15 5.155
W = Trp 2 0.687
Y = Tyr 6 2.062
Z = Glx 0 0.000
A + G 30 10.309
8 + T 42 14.433
D + E 39 13.402
D + E + N + Q 65 22.337
H + K + R 63 21.649
D + E + H + K + R 102 35.052
I+L+M+V 62 21.306
F + w + Y 18 6.186

65
Session Name: clvaxl

$ type tfiie56k.pepsort
PEPTIDESORT of: Tfiie56k.Pep check: 1543 from: 1 to: 440

TRANSLATE of: tfiie56k.txt check: 1285 from: 64 to: 1383
generated symbols 1 to: 440.

with Enzymes:
October 8, 1992 14:01

Summary for whole sequence:

Molecular weight = 49451.64 Residues = 439
Average Residue Weight = 112.646 Charged = -35
Isoelectric point = 4.55

Extinction coefficient = 28380
Residue Number Mole Percent
A = Ala 45 10.251
B = Asx 0 0.000
C = Cys 6 1.367
D = Asp 35 7.973
E = Glu 53 12.073
F = Phe 15 3.417
G = Gly 21 4.784
H = His 11 2.506
I = Ile 17 3.872
K = Lys 24 5.467
L = Leu 32 7.289
M = Met 15 3.417
N = Asn 14 3.189
P = Pro 20 4.556
Q = Gln 11 2.506
R = Arg 29 6.606
S = Ser 26 5.923
T = Thr 25 5.695
V = Val 25 5.695
W = Trp 2 0.456
Y = Tyr 13 2.961
Z = Glx 0 0.000
A + G 66 15.034
S + T 51 11.617
D + E 88 20.046
D + E + N + Q 113 25.740
H + K + R 64 14.579
D + E + H + K + R 152 34.624
I + L + M + V 89 20.273
F + W + Y 30 6.834

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