A thermostable protein factor acting on in vitro DNA transcription

BIOCHEMICAL

Vol. 45, No. 6, 1971

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

A THERMOSTABLE PROTEIN FACTOR ACTING ON IN VITRO DNA TRANSCRIPTION M. Jacquet, Institut

Received

R. Cukier-Kahn,

.J. Pla and F. Gros

de Biologie Molhculaire, Facultg des Sciences 9, Quai Saint-Bernard, Paris - France

October

9,

1971

SUMMARY A heat-stable protein factor has been purified from E. coli supernatant fractions which has the property to greatly stimulate in vitro DNA transcription when RNA polymerase is limiting. The step in transcription which is influenced by this factor is the formation of preinitiation complexes. --INTRODUCTION It is well polymerase depends play

is

regulated

act

role

by a large

in the

of Davison

ty to increase

the rate

repression (9),

(which

protein

ses partial for

can be purified we shall

which

preinitiation

report the

stimulates

transcription

at enzyme excess.

two effects, complexes

both

for

of which

A single reflect

holo-

of E. coli

appears

action

abili-

of a new cellu-

conveniency,

protein

like

their

RNA polymerase

at

extracts

is a heat

at DNA excess some

which

factors, for

fraction

"H",

fa-

7) or of psi

identification

supernatant

as Factor

(5)

stimulates

Other

been characterized

on the

rh6

of a given

(6,

(8).

and

sequences.

which

operons genes

RJJA of which

2, 3, 4)

the transcription

by the purified

from

designate

greatly

inhibition

these

we shall

(1,

CRP) protein

sensitive

of transcription paper

which

for

have also

In the present f,actor

the specificity sigma

or termination

of ribosomal

of DNA templates.

element

ble

elements

from a variety

lar

of factors, Some like

the case of the CAP (or

et al

by the DNA dependent

of initiation

on the transcription

the M protein

stable

recognition

from catabolite

This

catalyzed

variety

controlling

: such is

similarly

enzyme

RNA synthesis

used as templates.

as positive

mily of genes RNA synthesis acts

that

upon the genes

a general

Others

known

while

it

cau-

to be responsithe

level

of

(10).

RESULTS 1. - Characterization In the course procedure factor succinate

adapted stimulating eluates

of "H"

factor

of the E . coli

from

Chamberlin

DNA transcription from

a washed

RNA polymerase and Berg could

protamine-DNA

1597

(11)

purification which

be detected complex.

is outlined in the 0.2

according in Table M -0.3

to a I, M K

a

Vol. 45, No. 6, 1971

BIOCHEMICAL

AND BIOPHYSICAL

Table Outline

Alumina

1.

of RNA polymerase

and factor

Escherichia grindigg

coli

(a)

at 105,000 g p J 0.5 % Protamine sulfate precipitation 4 Pellet washed with 0.1 M DMG (c) 4 Elutions with K-Succinate :0.2M

Heating,

; 10&Z ><5

Sephadex

purification

MKE 600

2.5 hours

1st

RESEARCH COMMUNICATIONS

2nd : 0.3 min

ribosomal (b)&

G 75 fractionation + Factor "H"

discarded

supernatant

M

3rd

: 0.4

discarded

M

;

LI(

(d)

+

pellet

Ammonium sulfate (e)

4th

: 0.5

M

fractionation

(f)

chromatography 4 RNA Polymerase

(g)

DEAE cellulose

4

(a)

Disruption of cells is made in a buffer containing 0.02 0.01 M B-mercaptoethanol, 0.001 M EDTA, 50 % glycerol.

(b)

1 vol 0.5 % protamine pH 6.0.

(c)

For all protamine sulfate pellet elutions a Potter-vessel K-dimethyl glutarate pH 7, K succinate pH 6.

(d)

0.2 M K succinate and 0.3 M K succinate eluates are with ammonium sulfate. The precipitate is dissolved pH 7.5 and heated 15 minutes at 100' C,

(e)

After centrifugation on G 75 Sephadex

(f)

Folymerase

(g)

Chromatography is followed by stepwise elution in Tris-HCl, buffer pH 7.5 with 0.2 M, 0.25 M, 0.3 M, 0.4 M respectively. Enzyme activity is eluted at 0.3 M Tris-HCl. Specific activity of the enzyme is usually around 1 000 units/mg. 1 unit = 1 mpM of UMP incorporated in 20 min at 37 ' C.

Further heating heated

activity

studies

sulfate

to 1 vol

of S 100 in 0.1

pH 7.5, M K succinate

is used.

is obtained

having

between

shown

that

purification

on a G 75 Sephadex

column.

is

25 and 40 % Ammonium sulfate

the

stimulating

was achieved As illustrated

1598

DMG

pooled and precipitated in 0.02 M Tris-HCl,

of heat coagulated proteins, the supernatant in 0.02 M Tris-HCl buffer, pH 7.5.

at 1OO'C subsequent extract

is added

M Tris-HCl,

capacity by absorbing on Fig.

filtered saturation.

can sustain a 15 min pre1 the heat

stable

:

Vol. 45, No. 6, 1971

Figure

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

1 - Gel filtration of partially purified H-factor 1 x 80 cm G 75 Sephadex column was used ; Buffer : dinitrophenyl-alanine.

Marker

component fraction single

resolves non excluded

band

conditions, plus

into

after

two components, from

giving

rate

(Fig.

can be assigned

two sub'fractions

fraction

II.

- Effects

of "H"

Under

conditions

stimulating

by the purified whether

using

purification various

From its weight

has not yet factor

activity

gels

will

during

gel

reflect

as a

of 1 % SDS

filtration,

10 000 daltons.

corresponding

be subsequently

towards

"H"

Whether

the

two different

subunits

to the factor

plus

already

defined,

the transcription

the enzyme preparative

concentrations

migrates

in non-denaturing

in the presence

behavior

in the

a

been established.

RNA polymerase

3, illustrates

bands

pH 7.5

residing

factor

(pH 8.5)

M Tris-HCl

on transcription

which

E. coli

purified

of around

on SDS acrylamide

techniques

Figure

This

protein

or one main polypeptide

contaminating

high

B).

activity

electrophoresis

2).

a molecular

obtained

of the same factor

(peak

gel

to two distinct

101 mM dithiothreitol

factor

the gel

polyacrylamide

the stimulating

: 0.02

a typical

of "H" factor,

of h p lac5,

(holoenzyme). procedure

reported

purified

1599

"H" exerts

in Table

(12) using

as described

were

I (11)

and by Burgess native

a

@Boih or T4 DNA

The same results

described

by Babinet experiment

factor

obtained or the

(13).

h p lac 5 DNA and in the preceding

Vol. 45, No. 6, 1971

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Figure

2 - Acrylamide gel electrophoresis of H-factor Polyacrylamide gels were prepared and electrophorezed at 8 mA per gel as -A-B described by Weber and Osborn (15) (acrylamide 5 %, SDS 0.1 %). Prior to electrophoresis the extracts (20 pg) were incubated with 10 mM dithiothreitol, 0.1 % SDS, 21 % glycerol. The front was indicated by a metallic thread introduced through the bromophenol blue spot. A : commercial bovine Ribonuclease I B : "Peak B" from G 75 Sephadex C Electrophoresis in "non denaturing buffer of "peak B" : - acrylamide 7.5 % - buffer : Tris glycin pH 8.5

Figure

3 - Stimulating

effect

of the

factor

Indicated amounts of the "G 75 Sephadex" purified factor were incubated in 0.250 ml of the following mixture (final concentrations) : 40 mM Tris-HCl, pH 8.0, 5 mM B-mercaptoethanol, 8 mM MgC12, the four triphosphonucleotides, each at 0.4 mM, 3H UTP (10 PC/PM), RNA polymerase 1 unit, and h. p lac DNA, 7.5 pg. o 2 the triphosphonucleosides. The reaction was started by the addition 3 ml of cold trichloracetic acid Each sample was incubated at 37°C for 10 minutes. (5%) was added and the precipitate was filtered on (Millipore) nitrocellulose membranes, introduced in vials containing toluene omnifluor liquid, then counted in a Packard Tricarb liquid scintillation system. 1600

Vol. 45, No. 6, 1971

section.

As can be seen,

concentration

until

experiment, plate,

no enhancement

initial

of transcription

then

clearly

has already stimulation some light

transcription value

stimulation.

how this

remaining

constant.

decreases

system

in

factor

effect

by varying increases

with

on transcrip-

the DNA concen4, the initial DNA concentration

at DNA excess.

been noticed

A similar DNA concentration response workers (Zillig, personal communication).

by other

reaching here about but definite inhibitory

this

DNA was used as tem-

As shown on Fig.

first

factor

be observed.

of the

was influenced

in the control

with

corresponding,

denatured could

features

effect

increases

is reached, rate

the general

RESEARCH COMMUNICATIONS

rate

When heat

of the transcription

to explore

enzyme concentration

rate

AND BIOPHYSICAL

plateau

fold

we have examined

tration,

the

a certain

to a four

In order tion,

BIOCHEMICAL

tenfold ; in contrast, effect at limiting

factor addition DNA concentrations,

curve Maximal

causes

$---I* ‘. \

I I

/’

\ ‘Y

I ,

\

\

\

I I

I

I

\

\

\

\ \

I I

\

I

I

Figure

4 - Effect

as described factor.

Indicated under

From these lated

either

experiment effect

of H-factor

results,

symmetrical

was analyzed

\ Y

at different

DNA concentrations

the differential

effect

DNA concentration

to the one just under

conditions

\ \

d’

amounts of @g,-, i h DNA were used with RNA polymerase (1 unit) Fig.3,

to the absolute

\

1601

of factor

"H" could

or to the DNA/enzyme

described where

in 250 ~1 of reaction and 18 ~g of partially

was realized

enzyme concentration

be corre-

ratio.

in which increases

mixture purified

the

An factor in the

BIOCHEMICAL

Vol. 45, No. 6, 1971

Figure

5 - Effect

for

observed a high

at different

RNA polymerase

a fixed depends

amount

of DNA. It

is

ratio

at a low DNA/enzyme

of factor

"HI' itself,

of transcription

In order the factor,

a series

RNA polymerase nucleotide

to specify

the step

of experiments

was incubated

substrates,

affected

factor

(at

mixture

that

the kind

a stimulation

being

obtained

these

data

ratio.

also

is present in larger ved : when the factor observed even at a high DNA/enzyme ratio. - Stage

(Fig.51

clear

on the DNA to polymerase

and an inhibition

the concentration

III.

concentrations

Indicated amounts of RNA polymerase were added to the reaction nnder Fig. 1) containing 5 pg of @SO i h DNA and : no factor 12 ug H-factor 30 pg H-factor 60 pg H-factor

(described -.-0 -a-A-

system

of H-factor

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Moreover,

influences amounts

the type

an inhibitory

of effect

of effect effect

at

show that obser-

can be

by "H" factor in RNA transcription were

37'C)

"H" being

carried

out in

in the presence present

1602

which

is

influenced

the presence of native

or not.After

by

of rifampicin

QSOih DNA and

one minute,

enough

Vol. 45, No. 6, 1971

BIOCHEMICAL

AND BIOPHYSICAL

1603

RESEARCH COMMUNICATIONS

ON PREINITIATION

3

1 1 1 1 1 1 1 1 1 1 1 1

0 30 0 30 0 30 0 30 0 30 0 30

250 ul of reaction mixture (as defined started after 5 minutes of preincubation plus 4 ug/ml rifampicin.

7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 2.5 2.5 1.25 1.25

in 10 min

used. The reaction was nucleoside triphosphates,

482 1668 534 1848 480 1788 822 2250 1000 600 960 220

(uumoles/ml)

UMP incorporated

COMPLEX FORMATION

in the legend of Fig. 3) were at 37'C by adding the requisite

0 0 ATP ATP UTP UTP 4 NTP 4 NTP 0 0 0 0

Components present during preincubation (5 min, 37'C.J _-____----__--------------------------------------------------Polymerase H-factor Triphospho-nucleosides DNA (pg) (units) 0.1 uM for each (!a)

EFFECT OF "H-FACTOR"

Table

BIOCHEMICAL

Vol. 45, No. 6, 1971

rifampicin

was added

activity

incorporated

was added

only

factor

present

is

is

is noticeable.

step

(13),

site

to the factor of factor

preincubated strates,

with it

with

ratio additions

experiment

initiation

DNA and RNA polymerase, the simultaneous

during

the preincubation

factor

"II"

at some late

the

not

change

RNA synthesis

in RNA synthesis. is

with

a prerequi3, a marked

the factor

of nucleotide

was sub-

and triphosphonu-

was increased of these

concerns

the factor

of the initia-

of Table

The presence

the extent

primarily

6 - Effect of H-factor on the Nitrocellulose h DNA in the presence of varying amounts

Indicated amounts of 1 pg of 3H Ic DNA (10 pC/mg) in and 2 mM S-mercaptoethanol with Each sample was c-----o -). 20 ml of 10 mM Tris-HCl, pH 8.0, dried and counted.

presence

prein-

no effect

step stage

of rifampicin factor.

DNA

that

be shown when

complexes

interaction of the polymerase step, namely specific rise to rifampicin resistant complexes (10).

Figure

their

could

additions resistant

did

the propagation

even in the absence

by adding

period

on overall

simultaneous

"H" 2, when

or inhibit

in the 1 minute

are

In the experiment rate

factor

one to conclude

whether

on transcription.

at enzyme excess effect

to test

of rifampicin

settled

involved

radio-

As shown in Table stimulate

leads

and not with are

experiments

rifampicin.

the DNA/enzyme This

effect

In parallel

can either

"H" on the transcription

The number

and decreased

with

and TCA precipitable

and rifampicin

was of interest

5 min before

cleosides.

reinitiation

it

triphosphonucleosides

tion effect

upon

When factor

related

Since

together

to rifampicin,

depending

of the factor action

1 minute

prior

system.

any further

in 10 min was measured.

after

transcription cubation

to block

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

at DNA excess

of ATP or UTP effects.

Hence,

the preinitiation

promoter

sites,

giving

filter retention of radioactive of RNA polymerase.

RNA polymerase were incubated 10 minutes, at 4"C, with : 10 mM Tris-HCl, pH 8.0 250 pl of buffer containing ( -----x----) or without 8 pg of purified H-factor filtered on nitrocellulose membrane and washed with 50 mM NaCl buffer with gentle suction. Filters were 1605

Vol. 45, No. 6, 1971

BIOCHEMICAL

Results using

obtained

radioactive amounts

tration tions

this

this

kind

it

even

of RNA polymerase of enzyme is

where

Although

by the nitrocellulose

h DNA indicate

in the absence ting

(data

not

technique bind

However

retain

preliminary

by limiat a concen-

DNA by itself

(Fig.

to various

formation

(14) DNA even

DNA retention of factor

and subject

"HI' can influence

of nucleotide

retention

"H" can weakly

in the presence

practically

is

factor

factor shown).

enhanced

of analysis

in the absence

purified

does not

that

membrane

that

greatly

latter

confirms

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

6).

interpreta-

of enzyme DNA complexes

substrates.

DISCUSSION "H" factor stimulate

appears

of the initiation Factor

to share

DNA transcription process.

M was assigned

whereas

factor

1OO'C for

"H",

likely

for

the following

fraction

size

than

namely

factor

at high,

sensitivity

;

like-sigma factor

"H" appears

that

the two effects

and an inhibition entities

at

at low DNA/ does not

appear

electrophoretically

effects.

and inhibition

effects

namely

stage

can be heated

to a single,

c)

for

: both

:

activities

To account

M factor at an early

protein,

protein

and inhibitory

in DNA transcription,

heat

Furthermore

b) the stimulatory the stimulation

act

M. The possibility

corresponding

both

on their

of activity.

to two distinct

preparation

exhibits

lies

; both

pronase-sensitive,

a stimulation

reasons

(9'

a thermolabile

clearly loss

be attributed

a) a factor defined

is

apparent

of smaller could

of being

it

in common with

ratio

difference

the property

by the factor,

enzyme ratios

The major

although

15 min without

to be a protein exerted

some properties

at low enzyme/DNA

the formation

the factor

are both both

relate

of preinitiation

mode of action

thermostable. with

the same stage

complexes.

different

explanations

can be

advanced. l.kind

of negative

enzyme-DNA tion

The primary

complexes

sites.

sized

control

being

activity,

able 2.-

volvement

could

model initiation

are not

however

difficult

for

binding

in

with

terms

of some

the formation

to non specific

part

of the specific number

sites

of polymerase

being

of

initiasyntherate unsaturated,

molecules

sites. factor

"H" effect would postulate its ine.g. by favouring local DNA unwinding. the factor would stimulate transcription by

process,

saturated energy

be explained interfer

a large portion of the chains factor "H" would reduce the overall

by a greater

these

An alternative

the activation is

with

would

excess,

sites,

be explained

to interact

sites

in

in excess,

in the normal

When promoter diminishing

at unproper

it

inhibiting

is

When DNA is

"H" could

whereby

by preferentially

initiated

the stimulation

a model

of factor

When RNA polymerase

of RNA synthesis. becoming

effect

required

to reconcile

for with

1606

interaction the

inhibitory

with

these effect

sites

; such

observed

at

Vol. 45, No. 6, 1971

enzyme excess

unless

"H" mode of action In particular, this

factor

BIOCHEMICAL

some additional and of its

the nature is under

AND BIOPHYSICAL

hypothesis

physiological

of the

transcription

being

relevance product

RESEARCH COMMUNICATIONS

made. Elucidation of factor will require further work. made in the presence

of

investigation.

ACKNOWLEDGEMENTS This work was supported by grants from the Fonds de Developpement de la Recherche Scientifique et Technique, the Centre National de la Recherche Scientifique, the Commissariat a 1'Energie Atomique, the Ligue Nationale FranGaise contre le Cancer, and the Fondation pour la Recherche Medicale FranGaise. REFERENCES 2. 3. 4. 5. 6. 7.

8. 9. 10. 11. 12. 13. 14. 15.

Burgess, R.R., A.A. Travers, J.J. Dunn and E.K.F. Bautz, Nature, 221, 43 (1969) Sugiura,M., T. Okamoto and M. Takanami, Nature, 225, 598 (1970). Travers, A.A., Nature, 225, 1009 (1970). Travers, A.A., and R.R. Burgess, Nature, 222, 537 (1970). Roberts, J.W., Nature, 224, 1168 (1969). Eron, L., R. Arditti, G. Zubay, S. Connaway, J. R. Beckwith, Proc. Nat. Acad. Sci., 68, 215 (1971). Emmer, M., de Combrugghe, B., I. Pastan and R. Perlman, Proc. Nat. Acad. Sci., 66, 180 (1970). Travers, A.A., R. Kamen, M. Cashel, Cold Spring Harbor Symp. Quant. Biol., 35, 415, (1970). Davison,J., K. Brookman, L. Pilarski, H. Echols, Cold Spring Harbor Symp. Quant Biol., 35, 95 (1970). Bautz, E.K.F. and F.A. Bautz, Nature, 226, 1219 (1970). Chamberlin, M. and P. Berg, Proc. Nat. Acad. Sci., 48, 81, (1962) Babinetn C., Biochem. Biophys. Res. Comm., 2, 639 (1967). Burgess, R.R., Ann. Rev. Biochem., vol 40, 711, (1971). Jones, O.W., and P. Berg, J. Mol. Biol., 22, 199 (1966). Weber, K., and M. Osborn, J. Biol. Chem., 244, 4406 (1969).

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