The interchangeability of stimulatory factors isolated from three microbial RNA polymerases

Vol. 41, No. 3, 1970

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

THE INTERCHANGEABILITY OF STIMULATORY FACTORS ISOLATEDFROMTHREEMICROBIALRNAPOLYMERASES' H, R. Whiteley' and H. Ernest Hemphill Department of Microbiology, School of Medicine University of Washington, Seattle, Washington 98105

ReceivedSeptember 25, 1970 The DNA-dependent RNApolymerases of three bacteria were resolved by phosphocellulose chromatography into minimal RNApolymerases and stimulatory factors. The latter had no intrinsic RNApolymerase activity but promoted the synthesis of RNAby the minimal enzyme. Under someconditions, the factor from one species of bacteria was able to stimulate the minimal enzyme from a heterologous species. INTRODUCTION The DNA-dependent RNApolymerases of several bacteria characterized

with regard to their

subunit structure

enzymes can be resolved into two major functional

have now been

(3, 8, 10).

subunits:

Each of these

a high molecular

weight "core" or "minimal enzyme" which catalyzes RNA synthesis and consists of several dissimilar

polypeptides,

as the "sigma factor".

and a low molecular weight monomerreferred

The latter

stimulates and specifies

minimal enzyme by promoting initiation scription

to specific

the activity

of RNAsynthesis and restricting

Pseudomonasaeruginosa and Escherichia coli,

tran-

subtilis,

we have resolved a low molecular

from each enzyme which stimulates the homologous minimal poly-

merase. In this report,

we have examined the specificity

of these factors with minimal enzymes by testing stimulate

of the

regions of the template DNA (1, 4, 7, 12, 13).

In the course of studies on the RNApolymerases of Bacillus

weight factor

to

of the interaction

the ability

of the factors

to

RNAsynthesis by heterologous minimal polymerases. MATERIALSANDMETHODS Cultures of i.

subtilis

and 2.

grow in a glucose-minimal salts

1Supported in part by funds from the American Cancer Society (grant number E538) and from the U. S. Public Health Service (grant number AM-13111) 2 Recipient of Research Career Award GM-K6-422. 647

Vol. 41, No. 3, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

mediumsupplemented with 1% peptone (11) and E. aeruginosa grown in a citrate-minimal Phase

salts

and stored

were

purified

the method

100-200

columns

chromatography buffer

minimal ing

were

0.45

M KCl.

fold

and Berg desalted

from

(the

flow-through),

M KC1 and that

of --E. coli

The RNA polymeraee 0.05

lo-20

fraction

(10-30

was incubated

reaction collected

war terminated

noted

step

ilutfon

of the

contain-

KC1 concentraprofiles,

fractions

factor

The

by buffer

by different in elutlon

portion

in 0.5 al

the

which

was eluted

were

by 0.15

chloride,

6 moles

factor were

mixtures

con-

of 2-mercaptoethanol

preparation

added

washed,

acid. dried

and

ug protein)

the reaction

serum albumin

of trichloroacetic filters,

(2-10

as desired;

100 ug bovine

by addition

reaction

each of ATP, GTP, CTP and OTP, 25 pmoles

of HgC12,

15 rln,

aeruginosa

extracted

with

addition

of ethanol,

dialyzed

against

ther

using

by

and mixture

was added

and the

Precipitate6

were

and counted

in a liquid

counter.

DNA from 5 T4 were

0.4 jmole

on Whatman GP/C glass

scintillation

were noted

was aueayed

pg protein)

at 37 C for

or DEAE-

N KCl.

5 moles

Potarslux

(2)

of

and resolved

was eluted

the g. aeruginora

activity

bacteria

in KC1 concentration.

bacteria

In the last

A-15

to Burgess

were

these

or by a modification

Biogel

increases

factore

from

from DKAE-cellulose

through

according

by 0.25

(pH 7.9).

ug of DKA.

enzyme

by passage

pc 8-C14 -ATP,

buffer

(2)

Fractions

variations

was found

adsorbed

of Trls

(5).

each of the three

factor

at mid-log

The KNA polymeraeerr

M increeent

occaeional

by centrlfugation

Burgess method

by the

0.05

harvested

needed.

The atimulatory

Although

taining:

’ until

containing

8. subtilis

were

on phorphocellulose

polymerase

t fans .

not

at -70

of Chamberlin

sephadex

with

xedium(9),

cold

phase

E 79, 4

buffered

rediaaolved

aubtillr

(pH 7.2)

in 0.015

the same NW1 - sodium

phage

rediatllled

X NaCl - 0.0015

citrate

rolution

822 and & phenol, M #odium

calf apooled

phage by

citrate,

and used without

fur-

purification. KKSDLTS AND DISCUSSION Aa &own

in Table

1, the KNA polymeraaea

648

of 2. aubtilla,

9. e

and p.

Vol. 41, No. 3, 1970

Table I RNAPOLYMERASE ACTIVITY OF FRACTIONSFROMPHOSPBOCELLDLOSE Source of Polymerase

Phosphocellulose Fraction

~ CPMcl4 AMP Incor xated T4 DNA

(a) Minimal enzyme (b) Factor Combineda&b B. subtilis

E79 DNA

2,627

(a) Minimal enzyme (b) Factor Combineda&b

E. aeruginosa

B22 DNA

308 38

(a) Minimal enzyme (b) Factor Combineda&b

501

79

20 1,478

20 1,950

220 40

180 40

1,550

1,690

aeruginosa were dissociated by phosphocellulose chromatography into two fracone fraction

tions:

("minimal enzyme") had low activity

templates, while the second fraction activity fold.

(“factor")

on native phage DNA

had no intrinsic

polymerase

but when combined with the minimal enzyme stimulated the latter It should be noted that similar

several

results were obtained with the B. subtilis

and r. aeruginosa polymerases whether T4 DNAwas used as the template or whether a correlated phage E79.

DNAwas used such as that of B. subtilis

The data presented in Table I (and also in Tables II-IV)

obtained by addition lations

phage 822 or P. aeruginosa

of Z-10 ug of factor

protein;

significantly

were

higher stimu-

were observed with larger amounts of factor.

The interchange of the factor

preparations was complicated by differences

in the salt requirements of the three RNApolymerases. (13) and Fuchs --et al.

As shown by So -et al.

(6), the RNApolymerase of E. coli

by monovalent cations , particularly

by potassium.

is greatly

This effect

stimulated

is shown in

Figure 1 which also demonstrates that the LP aeruginosa enzyme was similarly stimulated by Xl. subtilis at all

As noted earlier

enzyme was inhibited

(11) and as shown in Figure 1, the g.

by monovalent cations.

stages of purification. 649

This inhibition

was observed

Vol. 41, No. 3, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

M KCL

Fig.

1

The effect of KC1 on the three RNA polymcrase.

Each reaction mixture from a UEAE-cellulose

synthesis

of RKA by

contained 30 y of protein column fraction.

Table II

Effect

of Adding Heterologous Factors to Minimal Polymerase

Factor Added

I None

E. coli -v ;. subtilis E. aeruginosa

P. aeruginosa

M KC1

CPMcl4 AMP Incorporated

0.05

407

0.05

635

0.05

1,461

0.05

a17

0.2

785

0.2

3,780

0.2

8,634

0.2

4,019

650

BIOCHEMICAL

Vol. 41, No. 3, 1970

In view of these KC1 effects,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the three factors were tested with homologous

and heterologous minimal enzymes at low and high concentrations iments without KC1 were not tried, this salt. slightly

since the phosphocellulose fractions

contained

Table II demonstrates that the E. coli minimal enzyme was only stimulated by the addition of heterologous and homologous factors

low concentrations three factor indicate

Kxper-

of KCl.

of

KCl,

preparations

that the factors

whereas significant

stimulation

in the presence of 0.2 M KCl.

at

was observed by all These latter

results

from the three bacteria were interchangeable when

minimal enzyme. Qualitatively

similar

results were obtained with the p. aeruginosa minimal

enzyme as shown in Table III.

enzyme, the minimal polymerase

was stimulated by heterologous and homologous factor stimulation

was maximized in the presence of 0.2 M KCl.

The g. subtilis First,

preparations and this

minimal polymerases (Table IV) revealed a different

the stimulatory

effect

of the homologous factor was greatest at low

Table III

Effect

of Adding Iieterologous Factors to z. aeruginosa Minimal Polymerase

Factor Added None II. aeruginosa

pattern.

M KC1 0.05 il.05 0.05

g. subtilis

0.05

None p. aeruginosa g, coli

0.2

& subtilis

CPMcl4 AMP Incorporated 576

1,792 805 1,392 537

0.2

2,793

0.2

1,300

0.2

1,688

651

Vol. 41, No. 3,197O

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table IV

Effect

of Adding Heterologous Factors to B. subtilis Minimal Polymerase

CPMcl4 AMPI

Factor Added

M KC1

None 2. subtilis

0.05 0.05 0.05

g. aeruginosa

0.05

None ;. subtilis

0.1

E, aeruginosa None

g. aeruginosa concentrations

of the heterologous factors had no effect.

A highly

ted at an intermediate stimulation

by the e,

with the E. aeruginosa factor. reported a stimulation factor.

260

0.2

133 252

0.2

565 stimulation

sigma factor gave similar

I

was obtained with one and the g. e

preparation

results.

factor

(kindly

Experiments conduc-

(0.1 M KCl) also failed

and again yielded only slight

to show

stimulation

On the other hand, hosick and Sonnenshein (10)

of the & subtilis

minimal enzyme by the E. calf

sigma

It is not known at the present time whether this descrepancy with

respect to the effect of enzyme isolation, differences strains.

0.2 0.2

cation concentration factor

I

237

674

g,

provided by G. Hager and Dr. B. Ball)

469

1,257 288

(the E. aeruginosa factor),

purified

295 2,068 290

0.1 0.1 0.1

Secondly, only slight

of KCl.

I

Incorporated

of the --E. coli properties

factor

is due to differences

of the minimal enzyme and/or factors

in the cation requirements of enzymes isolated

stimulated only by the homologous factor 652

or to

from different

The present experiments showing that the 2. subtilis

can be significantly

in methods

minimal polymerase preparation

BIOCHEMICAL

Vol. 41, No. 3, 1970

while the g. subtilis in the interaction

factor

can stimulate heterologous enzyme suggests that

of minimal polymerasee and factors,

imposed by the specificity The factor

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

more restrictions

are

of the polymerase than by the factors.

preparations

obtained from the three species of bacteria

in this investigation

have the following

100-200 fold purified

polymerases by chromatography on phosphocellulose,

although they are eluted by different the preparations

in glycerol

weights of approximately

concentrations

they are resolved from

of KCl.

Sedimentation of

or sucrose gradients yielded estimated molecular

90,000 for the -E. coli and p. aeruginosa factors

60,000 for the 2. subtilis activity

similarities:

factor.

and

The preparations appear to be free of DNAase

as judged by the fact that incubation of the factor

radioactive

studied

DNAfor 18 hours at 37 C failed

to solubilize

preparations with

any TCA-precipitable

material. In agreement with the findings of Bautz -et al. by the minimal enzyme of E. &

of similar

The three factors

was

isolated in our

minimal polymerase, promoted the synthesis

product RNAs. These RNAswere asymmetric, had high molecular weights

and similar

profiles

gave identical

results

on sucrose gradients;

will

moreover, the three --in vitro

in RNA-DNAcompetition-hybridization

the competitor RNAwas T4 "early" (details

the product synthesized

in the absence of added sigma factor

symmetrical and had a low molecular weight. experiments, when added to E.

(l),

RNAisolated

RNAs

expetfments where

5 minutes after

phage infection

be presented elsewhere).

REFERENCES 1. 2. 3. 4. 5. 6. 7.

Bautz, E, K. F., P. A. Bautz and J. J. Dunn, Nature 223, 1022, 1969. Burgess, R. R., J. Biol. Chem.224, 6160, 1969. Burgess, R. R., J. Biol. Chem. 244, 6168, 1969. Burgess, R. R., A. A. Travers, J. J. Dunn and E. K. F. Bautz, Nature 221, 43, 1969. Chamberlin, M. and P. Berg, Proc. Nat. Acad. Sci. U.S. 48, 81, 1962. W. Zillig and G. Walter, European J. Biochem. Fuchs, E., R. L. Millette, 3, 183, 1967. Goff, C. G. and E. G. Minkley, Leptit Colloquia on Biology and Medicine: RNA Polymerase and Transcription (p. 124), American Elsevier Publishing Co., Inc., N. Y., 1970. 653

Vol. 41, No. 3, 1970

8. 9. 10. 11. 12. 13. 14.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Johnson, J. C. and J. A. Boezi, Fed. Proc. 29, 891 Abs) 1970. Lessie, T. G. and H. R. Whiteley, J. Bacterial. 100, 878, 1970. Losick, R. and A. L. Sonenshein, Nature 224, 35, 1969. Mizuno, S. and H. R. Whiteley, J. Bacterial. 95, 1221, 1968. Schmidt, D. A., A. V. Mazaitis, T. Kasai, E. K. F. Bautz, Nature 225, 1012, 1970. So, A. G., E. W. Davie, R. Epstein and A. Tissieres, Proc. Nat. Acad. Sci. u. s, 58, 1739, 1967. Sugiura, M., T. Okamoto, and M. Takanami, Nature 225, 598, 1970.

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