ERIZATION

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M

PSEUDOMONAS PUT‘IDA

SITY

»o-o-.n-

of M. S.

 

0F ITP AND CT? 8‘! RNA POLYMERASE 0F" .
Thesis for the Degree
MICHIGAN STATE UNIVER
KATHLEEN M. ROSE
19-69

UNP'RIMED INTERDEP‘ENDENT POLY

 

 

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v

 

LIBRARY

TNEJWS

Michigan State
University

 

ABSTRACT

UNPRIMED INTERDEPENDENT POLYMERIZATION OF ITP
AND CTP BY RNA POLYMERASE OF
PSEUDOMONAS PUTIDA

 

By

Kathleen M. Rose

After a lag, RNA polymerase of Pseudomonas putida
catalyzed the unprimed interdependent polymerization of
ITP and CTP to form TCA insoluble material which was pri-
marily homopolymers. After fractionation of the enzyme
into two components QC I and PC II) by chromatography on
cellulose phosphate, each component was able to catalyze
the synthesis of homopolymers from ITP and CTP. However,
the length of the lag for the reaction catalyzed by PC I
was at least 2.5 times greater than the length of the lag
for the reaction catalyzed by PC II. The steady state
rate of polymer synthesis was proportional to [E]n, where
n equaled 2 for the reaction catalyzed by PC II and n
approached 3 for the reaction catalyzed by PC I. PC I
and PC II also catalyzed the unprimed interdependent
synthesis of homopolymers from ATP and UTP. Again, the

length of the lag for the reaction catalyzed by PC I was

Kathleen M. Rose

longer (2 times) than the corresponding lag for the re~
action catalyzed by PC II.

Electrophoresis of PC I and PC II on polyacrylamide
gels (Tris-glycine, pH 9.0) each showed two major protein
bands, both of which were active in the unprimed reactions
catalyzed by RNA polymerase. Electrophoresis of the
polypeptide chains derived from PC I and PC II (SDS-
sodium phosphate, pH 7.1) indicated PC I had five major
polypeptide chains, one of which was a contaminant, and

PC II had three polypeptide chains.

UNPRIMED INTERDEPENDENT POLYMERIZATION OF ITP
AND CTP BY RNA POLYMERASE OF
PSEUDOMONAS PUTIDA

 

By

Kathleen M. Rose

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Biochemistry

1969

DEDICATED
to

Dick

11

 

ACKNOWLEDGMENTS

The author wishes to express gratitude to Dr.
J. A. Boezi for his patience and counsel. Thanks are
expressed to Drs. J. L. Fairley and F. M. Rottman for
serving as members of the guidance committee. Appreci—
ation is expressed to Mrs. Monique Debacker, Mr.
Kenneth Payne and Mr. Gary Gerard for valuable dis-
cussions and to Mr. James Johnson for preparation of
the enzyme.

This investigation was supported in part by a
National Institutes of Health Fellowship (GM—39,434)

from the National Institute of General Medical Sciences.

111

TABLE OF CONTENTS

 

Page
DEDICATION . . . . . . . . . . . . . ii
ACKNOWLEDGMENTS . . . . . . . . . . . iii
LIST OF TABLES. . . . . . . . . . . . vi
LIST OF FIGURES . . . . . . . . . . . vii
INTRODUCTION . . . . . . . . . . . . 1
MATERIALS AND METHODS . . . . . . . . . A
Purification of RNA Polymerase . . . . . 4
Radioactive Assay for the Incorporatio of
CMP and/or IMP from 3H— CTP and/or 1 c ITP
Into Polymers by RNA Polymerase . . . . 5
Radioactive Assay for Polymer Formation from
3H-ATP and UTP by RNA Polymerase. . . . 6
Nearest Neighbor Analysis . . . . 6
Disc Electrophoresis on Polyacrylamide Gels . 8
General Methods and Materials. . . . . . 11
RESULTS 0 o o o I o o o o o o o o o 13
Characteristics of the Interdependent Poly—
merization of ITP and CTP by RNA Poly-
merase in the Absence of Added Template . 13
Requirements for the Reaction. . . . 13

The Time Course for the Incorporation of

3H— CMP and luc— IMP Into Polymers by

RNA Polymerase and the Effect of KCl . 13
PC I RNA Polymerase and PC II RNA Poly-

merase; Incorporation of lL‘C-IMP and

3H—CMP Into TCA Insoluble Material . . 22
Nearest Neighbor Analysis of the Polymer

Formed by RNA Polymerase with

d-32P- CTP and ITP as Substrates . . . 25

iv

The Effect of Enzyme Concentration Upon
the Incorporation of 3H-CMP and
140- IMP Into TCA Insoluble Material;
PC I . . . . . . .
The Effect of Enzyme Concentration Upon
the Incorporation of 3H- CMP and
140- IMP Into TCA Insoluble Material,
PC II . . . . . . . . . .

Characteristics of the Interdependent Poly-
merization of ATP and UTP by PC I RNA
Polymerase and PC II RNA Polymerase. .

PC I RNA Polymerase and PC II RNA Poly—
merase; Incorporation of 3H-AMP Into
TCA Insoluble Material . . . .

Nearest Neighbor Analysis of the Polymer
Formed by PC I and PC II Utilizing
o-32P-ATP and UTP as Substrates . .

Disc Electrophoresis of RNA Polymerase on
Polyacrylamide Gels . . .

Disc Electrophoresis with Polyacrylamide
Gels Using the Sodium Phosphate-SDS

system 0 O O O O O O O O O 0
DISCUSSION 0 O O O O O O O O O O I
REFERENCES . . . . . . . . . . . .

27

33

39

39

42

All

50
56
6O

 

Table

LIST OF TABLES

Page
Requirements fog the Incorporation of
3H—CMP and l C-IMP Into TCA Insolu—
ble Material . . . . . . . . . . 14
Effect of 0.1 M KCl Upon the KiEfitics of
Incorporation of 3H—CMP and C-IMP by
RNA Polymerase . . . . . . . . . l7
Nearest Neighbor Analysis of the Polymer
Formed from o-32P-CTP and ITP . . . . 26
PC I RNA Polymerase. . . . . . . . . 30
PC II RNA Polymerase . . . . . . . . 36
Nearest Neighbor Analysis of the Polymer
Formed from d—32P-ATP and UTP . . . . 43

vi

 

LIST OF FIGURES

Figure Page
1. The Time Course of the Incorporation of
H-CMP and lAc-IMP by RNA Polymerase . . 16
2. The Effect of KCl Concentration Upon the
Incorporation of 3H-CMP . . . . . . l9
3. The Incorporation of 3H—CMP at Three Hours
As An Effect of the K01 Concentration. . 19

A Time Course of the Incorporation of

u.
3H—CMP Into TCA Insoluble Material by
PC I RNA Polymerase and PC II RNA Poly—
merase in the Presence of 0.1 M KCl . . 21

5. A Time Course for the Incorporation of
l“(z—IMP Into TCA Insoluble Material by
. . 21

PC I and PC II . . .

6. A Time Course of the Incorporation of
3H-CMP Into TCA Insoluble Material by
PC I RNA Polymerase and PC II RNA Poly-
. . 23

merase in the Absence of KCl. . .

O

7. A Time Course of the Incorporation of
l C-IMP Into TCA Insoluble Material by
. . 23

PC I and PC II . . . . .

8. The Effect of PC I Concentration on the
Incorporation of 3H-CMP Into TCA Insolu-
. 29

ble Material .
9. The Effect of PC I Concentration on the

Incorporation of C—IMP Into TCA Insolu—

ble Form . 29

10. The Effect of PC I Concentration Upon the
Rate of Incorporation of 3H-CMP Into
0 0 0 32

TCA Insoluble Material. . . .

vii

 

 

Figure Page

11. The Effect of PC I Concentration Upon the
Rate of Incorporation of ll‘C-IMP Into
TCA Insoluble Material . . . . . . . 32

12. The Effect of PC II Concentration Upon the
Incorporation of 3H—CMP Into TCA Insolu-
ble Material . . . . . . . . . . 35

13. The Effect of PC II C ncentration Upon the
Incorporation of l C-IMP Into TCA Insolu-
ble Material . . . . . . . . . . 35

lb. The Effect of PC II Upon the Rate of In—
corporation of 3H—CMP Into TCA Insoluble
Material. . . . . . . . . . . . 38

15. The Effect of PC II Upon the Rate of Incor-
poration of luC—IMP Into TCA Insoluble
Material. . . . . . . . . . . . 38

16. The3Time Course for the Incorporation of
H-AMP Into TCA Insoluble Material by
PC I and PC II in the Presence of 0.1 M
KCl . . . . . . . . . . . . . Al
17. The3Time Course for the Incorporation of
H-AMP Into TCA Insoluble Material by
PC I and PC II in the Absence of KCl . . Al

18. Polyacrylamide Gel Electrophoresis; Protein
Stain. D U C . O . O . U C O . L‘6

l9. Polyacrylamide Gel Electrophoresis; Activity
Stain. . . . . . . . . . . . . A8

20. Polyacrylamide Gel Electrophoresis in SDS—
sodium Phosphate, pH 7.1 . . . . . . 52

21. Standard Curve for Protein Migration in the
SDS-sodium Phosphate, pH 7.1, Poly—
acrylamide Gels . . . . . .

. . . 5A

viii

INTRODUCTION

RNA polymerase (nucleoside triphosphate: RNA
nucleotidyltransferase, EC 2.7.7.6) can catalyze two
types of reactions. One type is the template directed
synthesis of polyribonucleotides. RNA polymerase cata-
lyzes the synthesis of polyribonucleotides with a base
sequence complementary to the template (1-5). The other
type of reaction, which is termed unprimed, is the
synthesis of ribopolymers in the absence of added tem-
plate.

RNA polymerase catalyzes two unprimed reactions.
One is the interdependent polymerization of ATP and UTP

to form homopolymers by RNA polymerase from Escherichia

 

coli, Azotobacter vinelandii and Pseudomonas putida (6-9).

 

 

 

The other reaction involves the unprimed interdependent

polymerization of ITP and CTP to form polymers in which

 

inosine and cytidine are in alternating sequence. RNA

polymerase from E. coli and A. vinelandii has been re-

 

ported to catalyze this reaction (10).

The unprimed reactions catalyzed by RNA polymerase
have been shown to have certain requirements (6-10).
Synthesis of TCA insoluble material from ATP and UTP or

CTP and ITP occurs only

.m M“ a i am. .r“ n1 W. 9- S .1 e C e e e P ... u
- . a I C l . .. . e «I n u a V .C P. .5 w... E -5 P“ M“.

 

1. In the presence of RNA polymerase.
2. In the presence of Mn+2. Mg+2 will not
substitute as the divalent ion.
3. If both nucleoside triphosphates are present
(the exception here is a small amount of ATP
incorporation by the A, putida enzyme in the
absence of UTP).
The Watson-Crick analogue of ITP, GTP, will not substitute
for it in the reaction utilizing ITP and CTP. Synthesis
of product in both unprimed reactions occurs only after a
lag. The rate of formation of product in the reaction
involving ATP and UTP (A. putida enzyme) is related to

the square of the enzyme concentration (9).

Polyacrylamide gel electrophoresis of RNA polymerase

 

from E. coli and A, vinelandii has been performed (ll, 12).
Depending on the structural form, RNA polymerase migrates
as a single band (E, coli) or as multiple bands. The A,

vinelandii enzyme migrates as two bands, the major band

 

being the more slowly moving component. An Ag situ assay

 

for RNA polymerase activity in the unprimed reactions has IR
been developed by Krakow (12). In this assay, ribopoly-

mers synthesized by the enzyme which had migrated into the

gel during electrophoresis are stained with ethidium

bromide. In both unprimed reactions with A. vinelandii

RNA polymerase polymers were synthesized by both the

major and minor protein bands.

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Polyacrylamide gel electrophoresis of E, ggAA RNA
polymerase in 8 M urea or sodium dodecyl sulfate (SDS)
and B—mercaptoethanol has been performed (11, 13). De-
pending upon the manner of purification various patterns
are seen. In its most complete structural form (0288'0).
four polypeptides are observed in the SDS gels with
molecular weights estimated at 165,000 (8), 155,000 (8'),
95,000 (0) and u0,000 (o) (lu).

RNA polymerase from E. putida has been fractionated
into two components by chromatography on cellulose phos-
phate (J. C. Johnson, private communication). This report
describes (l) the unprimed interdependent polymerization
of ITP and CTP by each of these components, (2) the un-
primed interdependent polymerization of ATP and UTP, (3)
polyacrylamide gel electrophoresis (Tris-glycine, pH 9.0)
with analysis of enzymatic activity using the AA ElEE
assay, and (A) SDS—polyacrylamide gel electrophoresis of
the polypeptide chains derived from E. putida RNA

polymerase.

MATERIALS AND METHODS

Purification of RNA Polymerase

 

RNA polymerase was purified by the method of J. C.
Johnson and J. A. Boezi (private communication). The
purified enzyme was eluted from a cellulose phosphate
column as a single peak or as two peaks depending upon
the elution procedure. When the material on the column
was eluted with a linear gradient of two column volumes
from 0.0 to 0.6 M KCl, RNA polymerase was eluted in a
single peak, referred to in RESULTS simply as RNA poly-
merase. However, when a linear gradient of six column
volumes from 0.0 to 0.4 M KCl was used, the enzyme was
eluted from the column in two peaks, designated PC I
(phosphocellulose I) RNA polymerase and PC II RNA poly-
merase. The enzyme isolated as a single peak was used
in only two experiments, that testing the effect of KCl
concentration and in the nearest neighbor analysis.

PC II RNA polymerase contained twice as much protein as

PC I RNA polymerase. PC II RNA polymerase was isolated

as an essentially pure protein. PC I RNA polymerase was
estimated to be 50-75% pure, containing, in addition to

the enzyme, a single protein component (presumably a

contaminant) which was inactive in RNA synthesis.

Although the overall specific activity of PC I in the
gh-l DNA-directed synthesis of RNA was the same as that
of PC II, the specific activity of the enzymatically
active component in PC I was twice that of PC II.
Radioactive Assay for the Incorporation
of CMP and/or_IMP from HS—CTP and/or

l‘*C-ITP Into Polymers by
RNA Polymerase

 

 

 

 

This assay measured the incorporation of 3H-CTP
and/or l)‘lC-ITP into material which was insoluble in
trichloroacetic acid (TCA). The standard reaction mix-
ture contained 0.02 M Tris-HOAc, pH 8.1, 2.0 mM Mn(OAc)2,
0.1 M KCl, 0.uu mM 3H-CTP, 0.uu mM ITP (nonradioactive or
lLAC-labeled) and 0-250 ug RNA polymerase/ml. Any enzyme
dilutions which were necessary were made in buffer con-
taining 0.05 M Tris—HOAc, pH 8.1, 1 mM Mn(OAc)2 and 0.01
M dithiothreitol. The reaction was started by the addition
of RNA polymerase and incubated for up to five hours at
30°C. During the incubation samples were withdrawn and
five ml of cold 10% TCA were added to each sample to stOp
the synthesis of polymer. Approximately 200 ug of carrier
DNA (salmon sperm) were then added. The sample was
allowed to remain at 0—A°C for 15 minutes and then the
precipitate was collected by filtering on a nitrocellulose
membrane. Each sample tube and filter was rinsed with

three 5 ml portions of 10% TCA. The filter was allowed

to air dry several minutes, then placed in a scintillation

 

vial and dried at 90°C. Five ml of a solution containing
4 g BBOT per liter of toluene were added to each sample.
The sample was then counted in a Packard TriCarb liquid
scintillation spectrometer with gain and window settings

as indicated: 3H alone, gain 65%, window discriminator

14

50-1000; 3H and C in same sample, 3H channel, gain 65%,

lb

window 50-300, C channel, gain 15%, window discrimi-

nator 250-1000. The overlap of lLAC into the 3H channel

3 14

was 17%. No overlap of H into the C channel was ob—

served.

Radioactive Assay for Polymer Formation
from JH-ATP and UTP by RNA Polymera§g

 

 

The standard reaction mixture for polymer formation
from ATP and UTP was the same as that for polymerization
of CTP and ITP except that o.uu mM 3H-ATP and 0.AA mM UTP
were used as the nucleoside triphosphates. Incubation

and analysis were carried out as described previously.

Nearest Neighbor Analysis

 

The reaction mixture for the nearest neighbor
analysis contained 0.02 M Tris-HOAc, pH 8.1, 2 mM Mn(0Ac)2,
0.1 M KCl, 0.4A mM CTP, 0.4“ mM ITP, 5 HS 0-32P-CTP/ml
and 100 ug RNA polymerase/ml. Nearest neighbor analyses
were also performed for polymers substituting 0.AA mM
ATP and 0.4M mM UTP and using c-32P-ATP in the above re-
action mixture. The reaction mixtures were incubated six

hours at 30°C. The turbid solutions were precipitated

with five ml of 10% TCA and filtered through Whatman GF/C
glass fiber filters using a Millipore filtering apparatus.
The tube and filter were then washed with an additional

15 ml of 10% TCA. After several minutes of air drying,
the filters were placed into vials. Three ml of 0.3

N KOH were added to the vials containing the filters.

The hydrolysis mixtures were incubated 15 hours at 30°C.
After removing the filters, the solutions were centrifuged
10 minutes at 10,000 x g. The supernatant solutions were
decanted and the pH of these solutions adjusted to 7 with
Dowex 50 in the H+ form. The Dowex was filtered off and
the solutions were frozen and lyophilized to dryness.

The residue was dissolved in 0.5 ml of water.

The samples were analyzed via descending paper
chromatography on Whatman, No. A1, paper using isobutyric
acid: ammonium hydroxide: water, 66:1:33, v/v/v, as the
solvent. Ten pg samples of the standards, 5' IMP and
2'(3') CMP or 2'(3') AMP and 2'(3') UMP were chromato-
graphed separately to identify the products. Fifteen ul
of the hydrolysates were also placed on the spots as
Inarkers which, after development of the chromatogram,
could be located with the use of an ultraviolet lamp.
.After preliminary location of the radioactivity with a
Packard radiochromatogram scanner, the paper strips were

out into 1.0 x 1.3 cm rectangles and placed in vials.

 

Five ml of BBOT—toluene (A g/l) were added as fluor. The
samples were counted in a Packard TriCarb liquid scintil-

lation spectrometer (gain 4%, window 50—1000).

Disc Electrophoresis on Polyacrylamide Gels
Two types of electrophoresis buffers were employed
using the polyacrylamide gel system. The first utilized
Tris—glycine buffer, pH 9.0. The second involved the use
of the sodium dodecyl sulfate (SDS)-sodium phosphate
buffer, pH 7.1, system of Shapiro (15). In both cases
the acrylamide gels were 5% cross-linked.
The solutions used for the Tris—glycine system as
adapted from B. J. Davis (16) were
A. 1 N H01 A8 m1
Tris 36.3 g
Tetramethylethylenediamine 0.23 ml
H20 to 100 ml. The pH of the solution was
adjusted to 9.0 with 1 N H01.
C. Acrylamide 20 g
Bis-acrylamide 0.735 g
H20 to 100 ml.
G. Ammonium persulfate 0.1“ g
H20 to 100 ml.
The solutions were combined, 1A:2C:lH20:AG, and

immediately 1.2 ml were poured into A mm x 70 mm glass

tubes. Care was taken to avoid air bubbles. The tubes

 

were then filled with water and allowed to polymerize 40
minutes at 26°C. After polymerization the gels were
allowed to equilibrate at O—A°C for 30 minutes. All
subsequent procedures were performed at 0—A°C. A
standard analytical disc electrophoresis apparatus such
as described by Davis (16) was employed. The upper and
lower buffers were 0.025 M Tris-H01, 0.2 M glycine, pH
9.0. The upper buffer contained 5 x 10-5% bromophenol
blue as the tracking dye. Protein was applied to each
tube by use of a syringe; 20 pg samples of protein, con-
taining 5-10% glycerol, were layered on the gels. The
running time for the gels was 110 minutes at 2.5 mamps/
gel. Protein migrated toward the cathode.

Two staining techniques were applied; one, utiliz-
ing Coomaasie Brilliant Blue, to stain for protein, the
other utilizing ethidium bromide to stain for poly—
nucleotides formed by RNA polymerase, AE_§A§AJ after
the appropriate reaction. I

For the protein stain, the gels, after removal from I
'the electrophoresis tubes, were fixed for 20 minutes in
7.5% HOAc made 5% in methanol (MeOH) and then stained for
Eit least two hours in MeOHzHOAczHBO, 5/1/5, v/v/v, made
0.25% in Coomaasie Brilliant Blue. Gels were either
ciestained electrophoretically with 7.5% HOAc, 5% in MeOH,
or by equilibration for several days with 5—10 volume

changes of the same solution.

10

To test for RNA polymerase activity in the gels,
after completion of electrOphoresis and removal from the
glass tubes, the gels were rinsed for ten minutes in
0.01 M Tris-H01, pH 7.8, 0.05 M NaCl, 0.001 M EDTA (TNE)
and then immersed in 5 m1 reaction mixtures containing
2 mM ITP (or ATP), 2 mM CTP (or UTP), 2 mM Mn(OAc)2,
0.04 M Tris—HOAc, pH 8.1, and 0.015 M B-mercaptoethanol.
Reactions were allowed to incubate at 30°C for 20-24
hours. The gels were rinsed again in TNE and then
stained for at least six hours in ethidium bromide,

100 ug/ml in TNE. Excess dye was removed by equili-
bration in TNE overnight. The presence of fluorescent
orange bands indicated the formation of ribopolymer-
ethidium bromide complexes.

In the SDS-sodium phosphate, pH 7.1, gel system,
solution A was replaced by A-SDS which contained 0.8 M
sodium phosphate, pH 7.1, 0.8% SDS, 0.23 ml tetramethy—,
ethylenediamine and H20 to a final volume of 100 m1.
Gels were poured as before, except that all procedures
for the SDS-sodium phosphate, pH 7.1, gels were performed
at room temperature. The electrophoresis buffer con-
tained 0.1 M sodium phosphate, pH 7.1, made 0.1% in SDS.
Before layering standard proteins on the gels, the pro—
tein solutions were denatured for three hours at 37°C
in 0.1 M sodium phosphate, pH 7.1, 1% SDS and 1% B-

mercaptoethanol and then dialyzed overnight against

 

11

0.01 M sodium phosphate, pH 7.1, containing 0.1% SDS and
0.1% b-mercaptoethancl. Protein solutions were then made
2-4% in sucrose to facilitate layering on the gel. Since
RNA polymerase had been dialyzed as the last step in the
purification procedure, dialysis treatment was not per-
formed, but protein was applied directly to the gels
after the denaturing treatment. In all cases 20 ug of
protein was applied to the gel. Electrophoresis was
carried out at 7.5 mamp/gel for two hours. Protein
migrated toward the cathode. Fixing and staining the
gels were conducted as described previously.

In both electrophoresis systems, pre-electrOphoresis
of the gels for 30 minutes at 10 mamps/tube to remove the

ammonium persulfate did not alter the results.

General Methods and Materials

 

Salmon sperm DNA, type III, used as carrier in the
radioactive assay was purchased from Sigma Chemical Com-

pany. Bact—T—Flex nitrocellulose membrane filters, type

B-6, were obtained from Carl Schleicher and Schuell Com-
pany. The unlabeled nucleoside triphosphates were from
P. L. Laboratories, Inc.

All radioactively labeled nucleoside triphosphates
were purchased from Schwarz Bioresearch, Inc. 3H-CTP
had a specific activity of 1.3 c/mmole, 500 uc/ml.
1[AC-ITP had a specific activity of 24.4 mc/mmole, 10

uc/ml. Before use the labeled triphosphates were

l2

lyophilized to dryness in order to remove all EtOH and

H20 was added back to restore the original volume. Radio-
active mixes of lLAC—ITP or 3H—CTP were made by addition of
the appropriate unlabeled nucleoside triphosphate so the
final specific activity of the solution was approximately
1000 opm/nmole. Appropriate volumes of the mixes were
then added to the reaction mixture to give the desired

32

molarity. d-32P-CTP and d— P-ATP were used in the nearest
neighbor analyses as water solutions after lyophilization
but without dilution with the unlabeled nucleoside tri-
phosphates.

The acrylamide and bis-acrylamide used in gel
electrophoresis were obtained from Canalco and were re-
crystallized in acetone as described by U. E. Loening (l7).
Coomaasie Brilliant Blue was purchased from Colab Labor-
atories, Inc. Bovine hemoglobin, rabbit muscle aldolase
and yeast pyruvate kinase were gifts of Dr. Clarence
Suelter. Bovine serum albumin was purchased from Armour

Pharmaceutical Company. Egg white lysozyme was obtained

from Sigma Chemical Company.

 

RESULTS

Characteristics of the Interdependent
Polymerization of ITP and CTP by
RNA Polymerase in the Absence
of Added Template

 

 

 

 

Requirements for
the Reaction

 

 

RNA polymerase catalyzed the interdependent polymeri—
zation of ITP and CTP into TCA insoluble material in the
absence of added template. In addition to the enzyme,
polymerization required the presence of both nucleotide
triphosphates and Mn+2. Mg+2 did not substitute as the
divalent ion. GTP would not substitute for its analogue,
ITP. These results are summarized in Table 1 for PC I
RNA polymerase and for PC II RNA polymerase.

The Time Course for the
Incorporation of 3H-CMP
and qu—IMP Into Polymers

by RNA Polymerase and the
Effect of KCl

 

 

 

 

 

The kinetics of synthesis of polymers from CTP and
ITP by RNA polymerase are presented in Figure 1. De—
tectable synthesis of polymer occurred after a lag and
then proceeded at a steady state rate. The difference
between the upper and lower sets of curves reflects an

effect of the K01 concentration. KCl shortened the lag

l3

14

 

.ooom pm compmozocfi go mason conch nouns HE\ompmthmoo

Icfi opmzamongoqoe mUHmooHoss mmHoE: mm ommwmhaxm ohm mpasmom .28 m mm: mfio¢ovwz .25

32.0 mo Goapmspcoocoo Hmsfim m on poops mm: mam .HE\wn ooa was coapmspsmocoo oezuco
one .mmomemz 92¢ mq<Hmme¢z ca omnfimomoo mm who: moLSQxHE coapommh osmocmpm one

 

 

 

 

Ho.o Ho.o Ho.o Ho.o . cocoa onaovmz

ooocHEo Aoaovcz

Ho.o Ho.o Ho.o Ho.o ooccaso «Acaovcs

I Ho.o I Ho.o oooos mew .ocbcHEo meH

I Ho.o I Ho.o oocpHEo meH

No.0 I Ho.o I omccHEo ago

Ho.o Ho.o Ho.o Ho.o occbaso onenesssoo 42m

em AA Hm om scones ocoHoEoo
He\mzHIoeH Hs\mzoImm Hs\mzHIo:H Ha\m20Imm
moHoEQ moHoEQ mmHoEC moHoEc

cameosaaom azm HH om cmmnosaaom azm H om
.Hmfisopme

oscsaomca «me once mzHIoes can azoImm no soacmnoobooca one soc meccsonflsoomII.H mamas

15

Figure 1. ——The time course of the incorporation of

3H-CMP and 14C— IMP by RNA polymerase. The reaction mixtures
were as described in MATERIALS AND METHODS. The enzyme con—
centration in the reaction mixtures was 100 ug/ml.
0——o 3H—CMP incorporation with the standard reaction mix-
ture (0.1 M in KCl); A-—-A 14C-IMP incorporation a so in
the presence of 0.1 M KCl; o——o 3H—CMP and A—--A 1 C-IMP
incorporation when KCl was omitted from the reaction mix-
ture.

'4C-IMP/ml

nmoles 3H-CMP or

16

 

 

 

 

 

Time (hr)

Figure l

17

and increased the steady state rate. (The rate was calcu—
lated from the straight line portion of the ascending
curve and expressed as nmoles nucleoside monophosphate
(NMP)/m1/hr. The length of the lag was estimated by
extrapolating the rate curve to the abscissa.) The
kinetics of incorporation of 3H—CMP relative to lLAC—IMP

in each reaction were similar. The rates and lengths of
lags calculated from Figure l are presented in Table 2.

TABLE 2.-—Effect of 0.1 M KCl upon the kinetics of the
incorporation of 3H-CMP and l4C-IMP by RNA polymerase.

 

KCl Omitted 0.1 M KCl

 

Rate (nmoles 3H—CMP/ml/hr) 9 25
Rate (nmoles luC-IMP/ml/hr) 10 26
Lag (min) 90 45

 

The effect of KCl concentration upon 3H-CMP incor-
poration into TCA insoluble material is shown in Figures
2 and 3. In Figure 2 the effect of KCl upon the time
3

course of H-CMP incorporation is presented for various
K01 concentrations. For all concentrations tested, KCl
increased the rate of the reaction. In Figure 3 the
amount of 3H—CMP incorporated after three hours is plotted

versus the KCl concentration. The maximum incorporation

occurred when the reaction mixture was 0.1 M in KCl.

 

18

Figure 2.-—The effect of the K01 concentration upon
the incorporation of 3H—CMP. The enzyme concentration in
the reaction mixture was 100 ug/ml and the reaction mixture
as described in MATERIALS AND METHODS with the K01 concen-
tration as indicated. O-—O no KCl present; A——A 0.05 M
K01; A——A 0.1 M K01; I——O 0.2 M K01.

Figure 3.—-The incorporation of 3H—CMP at three hours
as an effect of the K01 concentration. The nmoles of 3H-CMP
incorporated after three hours (data taken from Figure 2) are
shown versus the K01 concentration.

19

Figure 2

 

 

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nu MW nu nu
an an 9‘

IOOP

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w EEEUIIM moose

20

Onu

 

 

 

 

2
o 10.
\U \)
Zlhx M
e I I
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TI III/II
C
e . :/_OVLO
m m m a mo 2 o
.E m «o _E\n=20.I II» .336:

Figure 3

20

Figure 4.--A time course of the incorporation of 3H-CMP
into TCA insoluble material by PC I RNA polymerase and by
PC II RNA polymerase in the presence of 0.1 M K01. The re-
action mixtures were as described in MATERIALS AND METHODS.

The enzyme concentration was 100 ug/ml in both cases.
O——O PC I. A——A PC II.

Figure 5.--A time course of the incorporation ofluC-IMP

into TCA insoluble material by PC I and P0 II. The reaction
mixtures and enzyme concentrations were as described for

Figure 4. 0—0 PC I. A-i—A PC II.

21

 

 

 

 

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R\\ \,   '"
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3
Time (hr)

2
Figure 5

C3

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2

 

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<I\<l

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OD

IUJ/dW 3- H; SGIOWU

(3
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CU

Time . (hr)

Figure 4

22

PC I RNA Polymerase and PC II
RNA Polymerase; Incorporation
of 140-IMP and 3H—CMP Into
TCA Insoluble Material

 

 

 

 

Since RNA polymerase could be fractionated into two
components by chromatography on cellulose phosphate, the
capacity of these components to catalyze the unprimed
interdependent polymerization of CTP and ITP was investi-
gated. As shown in Figures 4 and 5, the kinetics of
incorporation of luC—IMP relative to 3H—CMP for the re—
action catalyzed by PC I RNA polymerase were similar.
Likewise, the kinetics of incorporation of lLAC-IMP rela—
tive to 3H-CMP for the reaction catalyzed by PC II RNA
polymerase were similar. By comparison of the reactions
catalyzed by the two components of RNA polymerase in 0.1
M K01, the length of the lag for the reaction catalyzed
by PC I was 90 minutes, whereas the length of the lag for
the reaction catalyzed by P0 II was 25 minutes (averaging
the data for CMP and IMP incorporation). The rate of the
reaction catalyzed by P0 II was greater than that cata-
lyzed by PC I (34 nmoles NMP/ml/hr versus 18 nmoles NMP/
ml/hr, again averaging the data for both substrates).
Similar kinetics of the reactions catalyzed by PC I and
PC II were observed when K01 was omitted from the reaction
mixture. The results are presented in Figures 6 and 7.
The lag for the reaction catalyzed by PC II in this case

was 50 minutes and for PC I, 140 minutes. The rate of

incorporation of the nucleoside monophosphates in the

 

23

Figure 6.--A time course of the incorporation of 3H—CMP
into TCA insoluble material by PC I RNA polymerase and by PC II
RNA polymerase in the absence of K01. The reaction mixtures
were as described in MATERIALS AND METHODS, omitting K01. The
enzyme concentration was 100 ug/ml in both cases. O——O PC I
RNA polymerase. A——A PC II RNA polymerase.

Figure 7.-—A time course of the incorporation of lLAC-IMP

into TCA insoluble material by PC I and PC II. The reaction
mixtures and enzyme concentrations were as for Figure 6.

O-—O PC I. A-—A PC II.

 

 

 

24

 

 

 

 

 

 

 

J I I I l ‘ l o
s s s s - s 2 o
Iw/dWI-Ow‘ SG|OUJU
f I U
\ \ «I
<1 0
an
G
<1\ N
G
' \
I J I l I J o
s s 2 s s 2 ° ‘

IUJ/dWO-Hg sanwu

Time (hr)

Figure 7

Time (hr) .

Figure 6

 

25

 

absence of K01 was 17 nmoles NMP/ml/hr for the reaction

catalyzed by PC II and 5 nmoles NMP/ml/hr for PC I.

Nearest Neighbor Analysis of
the Polymer Formed by RNA
Polymerase with a—JZP-CTP
and ITP as Substrates

 

 

Nearest neighbor analyses were conducted using RNA
polymerase, PC I RNA polymerase and PC II RNA polymerase

2

with 0-3 P-CTP and ITP as substrates. The results, which

are presented in Table 3, indicate at least 95% (average

= 98%) of the 32

P cochromatographed with the 2'(3') CMP
standard. Thus the sequence CpC occurred an average of
98% of the time, while the sequence IpC occurred less than
2% of the time. The polymer product must then contain
long stretches of cytidine. Since the ratio of incor-
poration of inosine to cytidine was close to one for all
three forms of the enzyme (see Figures 1, 4, 5, 6, 7) the
polymer product must also contain long stretches of ino-
sine. These results are consistent with the product be-
ing mostly homOpolymers of cytidine and of inosine or
being blocks of inosine connected to blocks of cytidine.
Nearest neighbor analyses were carried out both in 0.1

M K01 and in the absence of K01 for PC I and PC II. No

significant differences in the products were detectable.

26

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He\wn 00H mm: soapmmpcmonoo mazucm one

Hox ms» Spas mQOmBmS 02¢ ma<HmMB<S CH Umnfihommn mm mhmz mmMSQNHE QOHpommh one

.mmmmnpconmg cfi oopmofiocfi mm coauMMPGmonoo

 

 

 

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mm mms om Aoochso Homv H om
mm omm.m How Aaox z H.ov HH om
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Ugo mocozwmm QQH mocosvom

one a d a

EU I II

A V mzo mmm As ov mzH mmm

 

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27

The Effect of Enzyme Concen—
tration Upon the Incprpor—i

ation of JH—CMP and J-‘*CI--IMP

Into TCA Insoluble Material;
PC I

 

The kinetics of the incorporation of 3H—CMP and
luCuIMP into TCA insoluble material at various PC I
concentrations are presented in Figures 8 and 9. By
comparison of Figure 8 with Figure 9, the kinetics of
3H—CMP incorporation for any enzyme concentration were
similar to the kinetics of incorporation of l[AC-IMP.

The steady state rate and the lengths of the lags cal-
culated from the data presented in Figures 8 and 9 are
shown in Table 4. As the enzyme concentration increased
the steady state rate increased. At high enzyme concen—
trations the length of the lag was 80 minutes. The
effects of enzyme concentration upon the rates of in-
corporation of 0MP and IMP are presented in Figures 10
and 11. The dependence of rate upon enzyme concentration
was not a linear function but was exponential in form.
Plotting the log of the rate versus the log of the enzyme
concentration gives a line whose slope is representative
of the power of the reaction with regard to enzyme con-
centration. The slope of the line thus plotted (not

Ishown) approached 3, indicating the rate may be a function

(Df the [enzyme]3.

28

Figure 8.--The effect of PC I concentration on the
incorporation of 3H—CMP into TCA insoluble material. Standard
reaction mixtures, which contained 0.1 M K01 were as described
in MATERIALS AND METHODS. PC I concentration as indicated.
0——o 42 ug/ml; A-—A 84 ug/ml; o——o 97 ug/ml; A——A 126 ug/ml;

H 168 ug/ml; H 210 LIE/ml.

Figure 9.--T e effect of PC I concentration on the
incorporation of 1 C-IMP into TCA insoluble form. Reaction
mixtures and enzyme concentrations were as for Figure 8.

29

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00
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CON

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3O

 

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ow mOH ms mmH
as Hm es emH
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om HH m :w
omH m m N:
Es as Assam: ices cisafimw Ices eoamfimwé

 

.cnmnossHod azm H omII.s mamas

31

Figure lO.--The effect of PC I concentration upon the
rate of incorporation of 3H-0MP into TCA insoluble material.
Data is taken from Table 4.

Figure 11.--The effect of PC I concentration upon the
rate of incorporation of 140-IMP into TCA insoluble material.
Data is taken from Table 4.

32

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_E\c_2o..n_ on

9.0. mm. ww.

cm
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33

The Effect of Enzyme Concen-
tration Upon the Incorpor-
ation of 5H—CMP and i40-IMP
Into TCA Insoluble Material;
P0 II

 

 

 

 

 

The kinetics of the incorporation of 3H-CMP and
luC—IMP into TCA insoluble material at various PC II
concentrations are presented in Figures 12 and 13. As
for PC I, luC-—IMP incorporation in the reaction catalyzed
by PC II was similar to the 3H-CMP incorporation at any
enzyme concentration (comparing Figure 12 with Figure 13).
Table 5 presents the steady state rates and lengths of the
lags determined from the data presented in Figures 12 and
13. As the enzyme concentration increased, the steady
state rate of the reaction increased. The length of the
lag was 25 to 40 minutes. The steady state rates are
plotted versus the protein concentration in Figures 14
and 15. As for PC I, the rate of the reaction catalyzed
by P0 II was not a linear function of enzyme concentration,

but the two parameters were exponentially related. Plotting

the log of the rate versus the log of the enzyme concen~

 

tration (not shown) yielded a straight line with a slope
of 2, indicating the rate of the reaction varied with the
square of the enzyme concentration.

By comparison of the reactions catalyzed by PC I
and PC II (Tables 4 and 5) the length of the lag for the
reaction catalyzed by PC I was 50—80 minutes longer than

the corresponding lag for the reaction catalyzed by PC II.

34

Figure l2.--The effect of PC II concentration upon
the incorporation of 3H-CMP into TCA insoluble material.
Reaction mixtures were as described in MATERIALS AND
METHODS. P0 II concentration as indicated. 0——O 40 ug/ml;
A—A 80 LIB/ml; O——O 98 ug/ml; A—A 120 ug/ml;l-—.l60 ug/ml;
n——u 200 ug/ml.

Figure 13.—-The effect of PC II concentration upon
the incorporation of lliC-IMP into TCA insoluble material.
Reaction mixtures and enzyme concentrations were as for
Figure 12.

 

 

 

 

 

 

 

I
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3

 

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67 /Z-;
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240 -

lUJ/dINO-Hg sanwu

Time (hr)

re 13

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°\'\:\‘.\°\ _.
\
3: 3: 3. A A o°
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Time (hr)

36

mpmum museum

 

4

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.mQOmBmS Qz¢ mA¢HmMB<S CH vmnfihommv mm mpmz mmhdprE COHpommm

 

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om mo mm omH
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.cnsnoeHHoo «2m HH omII.m mmmae

 

37

Figure l4.--The effect of PC II upon the rate of in-
corporation of 3H—CMP into TCA insoluble material. Data is
taken from Table 5.

Figure 15.—-The effect of PC II upon the rate of in-
corporation of l4c-IMP into TCA insoluble material. Data is
taken from Table 5.

38

 

 

/
/

 

5!?
0 4O 80

I20 t-
IOO _
O
O
O
O
O

 

 

I60 200

O
I
I20

 

M?
0 4O 80

 

J I
<3 8 o o o 8 o

JLl/IUJ/dWO-Hg seIowu

ug Protein/ml

Figure 15

ug Protein/ml

Figure 14

39

Although the rates for the reaction catalyzed by PC II
were greater than those for the reaction catalyzed by
PC I at low protein concentrations, the rates appear to

converge at higher protein concentrations.

Characteristics oflthe Interdependent
Polymerization of ATP and UTP by 1
PC I RNA Polymerase and PC II
TRNA Polymerase

PC I RNA Polymerase and PC II
RNA Polymerase; Incorporation
of 3H—AMP Into TCA Insoluble
Material ‘3

 

The catalysis of the interdependent polymerization
of ATP and UTP into homopolymers by RNA polymerase (iso-
lated as a single peak by chromatography on phosphocellu-
lose) in the absence of added template has been previously
described (9). Since that study, RNA polymerase has been
isolated as two fractions, PC I and P0 II, both of which
were active in the DNA directed synthesis of RNA and in
the unprimed interdependent polymerization of ITP and CTP.
The capacity of the two components of RNA polymerase to
catalyze the unprimed interdependent polymerization of
ATP and UTP was investigated. The time course of the in-
corporation of 3H-AMP into a TCA insoluble form by PC I
and P0 II is shown in Figure 16. As for the reaction
involving ITP and CTP, the length of the lag for the
reaction catalyzed by PC II was shorter than that for

PC I (20 minutes versus 40 minutes). The steady state

40

Figure l6.—-The time course for the incorporation of 3H-AMP
into TCA insoluble material by PC I and P0 II in the presence of
0.1 M K01. The standard reaction mixtures were as described in
MATERIALS AND METHODS. The enzyme concentration in both cases
was 100 ug/ml. O——O PC I. A——A PC II.

Figure l7.——The time course for the incorporation of 3H-AMP
into TCA insoluble material by PC I and P0 II in the absence of
K01. Reaction mixtures and enzyme concentrations were as de-
scribed for Figure 16, omitting K01. 0-—O PC I. A——A PC II.

 

41

HH.oH:mHm

as as:

 

 

c n I N . o
LIIdIIITIdIGIaVGIjo
4

,.\

l

 

 

ON

0?

00

00

00.

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ma ondwfim

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e. m
—

 

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.u 10¢

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m

109.0

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M
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42

rate for the synthesis of polymers from ATP and UTP by
P0 II was also greater than for PC I; 89 nmoles 3H-AMP/
ml/hr for P0 II as compared to 18 nmoles 3H—AMP/ml/hr
for PC I. As shown in Figure 17, the same type of in-
corporation profiles were seen in the absence of K01,
the length of the lag for the reaction catalyzed by
PC II being 60 minutes and by PC I, 100 minutes. The
rate for the reaction catalyzed by P0 II was 22 nmoles
3H-AMP/ml/hr and for the reaction catalyzed by PC I,
2 nmoles 3H—AMP/ml/hr.
Nearest Neighbor Analysis of
the Polymer Formed by PC I
and P0 II Utilizing c-JZP—ATP
and UTP as Substrates

Nearest neighbor analyses were performed using
d—32P-ATP and UTP to determine if the products of the
reactions catalyzed by PC I and PC II were homopolymers.
The results are presented in Table 6. The ApA sequence,
but virtually none of the UpA sequence was detected,
indicating the AMP containing product was r(A)n. The
UMP containing product must then by r(U)n. Analyses
were performed in 0.1 M K01 and in the absence of K01.

No significant differences in the product sequences

were noted.

12-‘

43

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mm mom.m
OO AOH.N
OOH OOH.OH
OOH HmO.e

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mm
ma
mm
o

I,

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Ascov mapImmm

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Awmpquo Hoxv H om
Adom S H.0v HH om

AHOM z H.0v H om

 

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HmEmHog can go mHmmHmcm Honszmc pmommmZIn.m mqm<e

44

Disc Electrophoresis of RNA Polymerase
on Polyacrylamide Gels

 

 

Electrophoresis of PC I RNA polymerase and PC II
RNA polymerase was performed as described in MATERIALS
AND METHODS for the Tris—glycine system. After destain—
ing, several protein bands were evident for both PC I
and PC 11 (Figure 18). In both cases the largest per-
centage of protein was in a slowly moving band labeled
1. For PC I, 1 was split into two closely migrating
bands, 1 and 1', 1' moving slightly faster. For PC II,
1' was absent. However, a band moving more slowly than
1 was observed and is labeled 1". PC I and PC II also
had a fast moving component which seemed to be split
into two or more bands. These bands are labeled 2.

PC I had an additional slowly moving component which
migrated only a short distance into the gel.

To test the enzymatic activity of the various
bands, electrophoresis of 20 ug samples of PC I and P0 II
was performed. The resulting gels were incubated in
reaction mixtures containing ITP and CTP or ATP and UTP
as described in MATERIALS AND METHODS. Duplicate PC I
gels were incubated in reaction mixtures from which Mn+2
had been omitted. The resulting ribOpolymers were
stained l£.§lEE With ethidium bromide. The gels were

photographed under ultraviolet light and are pictured

in Figure 19.

f

45

Figure 18.-~Po1yacrylamide gel electrophoresis; protein

stain. Electrophoresis was performed as described in MATERIALS
The resulting gels

AND METHODS for the Tris—glycine system.
were stained with Coomaasie Brilliant Blue. 20 ug of protein
On the left is PC I RNA polymerase.

was applied to each gel.
P0 II is to the right.

 

 

 

1!

 

1”

Figure 18

 

PCII

47

Figure 19.——Polyacrylamide gel electrophoresis; activity
stain. Electrophoresis was carried out as for Figure 18, apply-
ing 20 ug of protein to each gel. The resulting gels were in-
cubated in reaction mixtures and stained with ethidium bromide
as described in MATERIALS AND METHODS. Photographs were taken
utilizing ultraviolet light. The set of three gel on the left
represent gels which were incubated in reaction mixtures with
ATP and UTP as substrates. From the left, PC I, PC II, PC I
with Mn(OAc)2 omitted from the reaction mixture. The set of
gels on the right were incubated in reaction mixtures contain-
ing ITP and CTP. From left to right, PC I, PC II, PC I with
Mn(OAc)2 omitted from the reaction mixture.

 

 

48

2 can:

 

49

For the synthesis of polymers containing AMP and
UMP (the set of gels pictured on the left), bands 1, l'
and at least part of 2 were active for the reaction
catalyzed by PC I and l and 1' were active in the re-
action catalyzed by PC II. Although not evident from
the photograph, PC II band 2 synthesized a small amount
of polymer from ATP and UTP.

For the synthesis of polymers containing IMP and
CMP (the set of gels pictured on the right in Figure 19),
PC I, band 1 and part of band 2, were active. In the
gel to which P0 II had been applied, only band 2 appeared
active in the synthesis of IMP, CMP containing polymers
(application of more protein, however, resulted in some
synthesis in band 1). The results obtained above were
reproduced for another preparation of PC I and P0 II.

Thus at least part of all bands present in the
polyacrylamide gels were capable of catalyzing reactions
characteristic of RNA polymerase. The only exception was

the very slowly moving, sharply banding protein appearing

 

near the top of the PC I gel. Whether this is a contami-
nant of the preparation or an inactive high molecular

'weight aggregate of RNA polymerase is as yet undetermined.

50
Disc Electrophoresis with Polyacrylamide Gels
Using the Sodium Phosphate-SDS System

Proteins with a known subunit molecular weight and
PC I RNA polymerase and P0 II RNA polymerase were de—
natured with SDS and B—mercaptoethanol in order to
separate the polypeptide chains of the proteins.
Electrophoresis was subsequently performed as de—
scribed in MATERIALS AND METHODS. The resulting gels
were stained for protein and photographed. The results
are presented in Figure 20. The distance traveled by
any subunit is logarithmically related to its molecular
weight (15). Thus in Figure 21, the molecular weight of
the subunits of the standard proteins are plotted on a
logarithmic scale along the ordinate and the distance
traveled by that subunit is plotted on the linear abscissa.
(The distance was measured in cm from the top of the gel.)
As shown in Figure 20, some reaggregation of subunits

occurred during electrophoresis to give dimers or trimers F

mt. . -

of these polypeptide chains. The straight line shown in

 

Figure 21 was used as a standard curve to estimate the i
molecular weights of the polypeptide chains obtained
from similar electrophoresis of PC I and P0 II.
PC I had five major polypeptide chains, labeled
from the top of the gel down (see Figure 20), A, B, C,
D, E with their estimated molecular weights as follows:
A. 150,000-160,000

B. 80,000

51

Figure 20.-—Polyacrylamide gel electrophoresis in SDS-
sodium phosphate, pH 7.1. Electrophoresis was performed using
the SDS—sodium phosphate system described in MATERIALS AND METHODS.
Protein was denatured as described in the text and 20 ug were
applied to each gel. From the left, bovine serum albumin, yeast
pyruvate kinase, rabbit muscle aldolase, egg white lysozyme, bovine
hemoglobin, PC I RNA polymerase and P0 II RNA polymerase.

 

 

 

4
A'.
a

 

Figure 20

53

Figure 21.-—Standard curve for protein migration in the
SDS—sodium phosphate, pH 7.1, polyacrylamide gels. Distances
were determined from the gels pictured in Figure 20. The dis-
tance migrated is plotted versus the subunit molecular weight
of the protein (on log scale). BSA = bovine serum albumin.

Hb = hemoglobin. The symbol (x2) or (x3) indicates the sub—
units were in the form of a dimer or trimer.

 

54

 

 

 

 

20 \
o BSAIXZ)

IO -
'I" 9
9 a -
x 7 - 0 BSA
4— 6 -
.c:
O
.5 5 .. lysozyme
3 pyruvate O 0 (x3)

4 .. “"080 o aldolase
L.
2 HbixZ)
:, 3 _ Iysozymeoo
0 (x2)
0
'6
2

2 _ Hb

lysozyme \
| l l l
O I 2 3 4

Distance (cm)

Figure 21

 

55

0. 50,000
D. 40,000
E. 32,000

PC II contained three major polypeptide chains,
A, D and E. Because of the relatively large amounts of
D and E present, it is difficult to distinguish them in
Figure 20. PC II was missing polypeptide 0 (50,000)
completely and only a trace amount of B (80,000) was
detected. Polypeptide C is derived from a protein in-
active in RNA synthesis and presumably is a contaminant

of the preparation (J. C. Johnson, private communcation).

 

DISCUSSION

After a lag RNA polymerase from E. putida catalyzes
the unprimed interdependent polymerization of ITP and CTP
into TCA insoluble material which is primarily homopoly-
mers of inosine and cytidine. In contrast, RNA polymerase

from E. coli and from A. vinelandii catalyzes the unprimed

 

synthesis of copolymers from ITP and CTP in which inosine
and cytidine are in alternating sequence (10). After the
E. putida enzyme is fractionated into two components (PC I
and P0 II) by chromatography on cellulose phosphate, each
component is able to catalyze the unprimed synthesis of
homopolymers from CTP and ITP. Although the products of
the reactions catalyzed by the two components of RNA
polymerase are the same, the length of the lag for the
reaction catalyzed by PC I is at least 2.5 times greater
than the corresponding lag for the reaction catalyzed by
PC II. A possible mechanism for the unprimed interde—
pendent polymerization of ITP and CTP might involve the
slow synthesis of either r(I)n or r(0)n. This polymer
might then serve as template for the synthesis of the
complementary polymer, which, in turn, could serve as

template for the synthesis of its complement. Thus,

56

 

57

with polymers of inosine and cytidine available as tem-
plates, rapid synthesis of the homopolymers ensues. RNA
polymerase from E. putida catalyzes the unprimed inter-
dependent synthesis of r(A)n and r(U)n (9). RNA poly-

merase from E. coli and from A. vinelandii catalyzes the

 

same reaction, also synthesizing homopolymers from ATP
and UTP (6-8). As for the unprimed synthesis of polymers
from ITP and CTP, the products of the reactions catalyzed
by PC I and PC II from ATP and UTP are homOpolymers.
Again, however, the length of the lag for the reactions
catalyzed by the two components of RNA polymerase is
different, with the length of the lag for the reaction
catalyzed by PC I being approximately two times longer
than the corresponding lag for the reaction catalyzed by
PC II.

The rate of the unprimed interdependent synthesis
of polymers from ITP and CTP by P0 II RNA polymerase is
proportional to the square of the enzyme concentration.
The rate of synthesis of TCA insoluble material from ITP
and CTP by PC I is also related exponentially to the
enzyme concentration, with the rate of the reaction
approaching proportionality to [enzyme]3. The rate of
synthesis of homopolymers from ATP and UTP by E. putida
RNA polymerase is related to the square of the enzyme
concentration (9). A mechanism similar to that dis-

cussed by Toyama et a1. (9) for the unprimed synthesis

 

58

of r(A)n and r(U)n by E. putida RNA polymerase may also
apply to the unprimed polymerization of ITP and CTP.

The hypothesis involves a monomer—dimer conversion of
the enzyme during the unprimed reaction, with the enzyme
existing in the inactive monomeric form at the beginning
of the reaction. As the reaction proceeds the gener-
ation of polynucleotides facilitates the conversion of
monomer to dimer, increasing the concentration of active
enzyme.

The protein pattern appearing after electrophoresis
of P0 1 and P0 II on polyacrylamide gels (Tris—glycine,
pH 9.0, system) shows a major slow moving component and
a minor, faster, moving component. The structure of the
bands relative to forms of the enzyme is as yet undeter-
mined. Although both bands appear to be composed of
several components, at least part of both bands is active
in the unprimed reactions catalyzed by RNA polymerase.
Relative to the amount of protein which appears to be
present in the bands, the faster moving band appears more
active in the synthesis of polymers from ITP and CTP than
the slower moving band. The synthesis of homopolymers
of adenosine and uridine does not show this differential
effect. RNA polymerase from A. vinelandii also has two
protein bands after electrophoresis on polyacrylamide
gels. Both bands of the A. vinelandii enzyme appear

active in the unprimed reactions catalyzed by RNA

 

 

59

polymerase (12). The relationship of these bands to
those observed with E. putida RNA polymerase is unknown.
After electrophoresis in the SDS-sodium phosphate,
pH 7.1, system, the patterns on the polyacrylamide gels
indicate PC I has five major polypeptide chains and P0 II
has three chains. Preliminary data (J. C. Johnson, pri-
vate communication) indicates one PC I chain (0. M. w.
50,000) is a contaminant. Polypeptide chain B (M. W.
80,000) is then the primary difference between PC I and
PC II on the SDS polyacrylamide gels. Depending upon the
manner of its purification, E. 22;; RNA polymerase also
gives different polypeptide patterns upon electrophoresis
in the SDS polyacrylamide gel system (11). After the
E. ggAA_enzyme (0288'0) is passed through a cellulose
phosphate column, it loses the ability to synthesize RNA
using T4 DNA as template. However, it can still synthesize
RNA using E. ggAA_DNA as template. This difference in
catalytic activity is attributed to the loss of the poly-
peptide chain o(M. W. 95,000) by chromatography on cellu—
lose phosphate. The similarity of the estimated molecular
weights of the polypeptide chains of the enzymes from
E. ggAA and E. putida (B = 165,000, 8' = 155,000, A =
150,000-160,000; a = 40,000, D = 40,000, E = 32,000;
o = 95,000, B = 80,000) suggests that, by analogy, RNA
polymerase from E. putida may exist as A2DEB (PC I) and

as A2DE (PC II).

 

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