Glycerol inhibition of purified and chromatin-associated mouse liver hepatoma RNA polymerase II activity

BIOCHEMICAL

Vol. 67, No. 3, 1975

GLYCEROL INHIBITION

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF PURIFIED

AND CHROMATIN-ASSOCIATED

HEPATOMA RNA POLYMERASE II Ralph

.I. Smith

and Jacob

Department of Biology, Calgary, Alberta,

Received

September

MXJSE LIVER

ACTIVITY

D. Duerksen

University of Calgary Canada T2N IN4

22,197s

of mouse liver hepatoma RNA polymerase II is depenSUMMARY : The stability dent on the type of buffer, pH and, most importantly, the glycerol concentraGlycerol above 2% or 15% shows a tion of the incubation or storage buffer. linearly increasing inhibition of enzyme activity with increasing glycerol concentration for purified RNA polymerase II and chromatin-associated RNA polymerase II, respectively. At 25% glycerol the activity of purified enzyme on DNA template was inhibited approximately 50% whereas the chromatinassociated activity was inhibited only approximately 30%. RNA polymerase I activity was not inhibited by glycerol at the concentrations examined. Since

the initial

description

eukaryotic

DNA-dependent

nucleoside

triphosphate:

been carried

out.

In most

used as a stabilizing

mouse liver Paul

hepatoma

ification, merases

chromatin manuscripts

(Smith

and Duerksen,

severly

whether

in purified with

inhibited

this

indicates

the important

steep

Cop.vright All rights

are

in preparation),

routinely Additionally,

During hepatoma

our

pur-

RNA poly-

II

activity.

activity

that

species

DNA template

as endogenous

(16,

we discovered

or single-stranded

II

of

gradients

of the RNA polymerase

or

This

by glycerol

report and

of such an inhibition. AND METHODS

for enzyme preparation mouse TLT (Taper Liver

o 1975 by Academic Press. Inc. of reproduction in an?’ form reserved.

used.

manuscript

of RNA polymerase

The source of cells preparation was from

concentration

the mouse liver

MATERIALS atin

is

buffers.

and use of

implications

extensive

and characterization

glycerol

preparations

inhibition

glycerol

and assay

15) fractionation

on native

chromatin

describes

storage

the activity

state

EC 2.7.7.6)

studies

in preparation)

characterization

glycerol

associated

13, 14,

ribo-

3, 4, 5, 6, 7, 8, 9, 10, 11) have

(e.g.,

in the

of the various

(RNA nucleotidyltransferase;

of the reported

agent (12,

and Duerksen,

separation

RNA nucleotidyltransferase,

and characterization

continuing

2) of the

RNA polymerases

purification

in our

(1,

916

and for nuclei Tumor) hepatoma

for chrom(17) grown

BIOCHEMICAL

Vat. 67, No. 3, 1975

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in the ascites form in Swiss Webster mice. RNA polymerase activity was obtained from lysed cells as an ammonium sulfate precipitate. This activity was separated into RNA polymerase I and IIA/II by DEAE-cellulose chromatography and each of these forms was further pur!fied (specific activities in the range 120-200 unitsfmg protein) by phosphocellulose chromatography (18, 5, 6) and were kept at -80" in storage buffer containing 50% glycerol. One unit of enzyme is defined as the incorporation of one picomole of UMP per minute at 37' under the conditions described below. DNA was prepared from nuclei of TLT hepatoma cells by the method of Marmur (19) as modified by Church and McCarthy (20) or by a modification of the high-molecular-weight method of Gross-Bellard Denatured --et al. (21). DNA and sheared (15 seconds) DNA were prepared as previously detailed (13, 14). After TLT hepatoma nuclear preparation (12, 13, 14, 15) by using 0.1% Triton in the cell-homogenizing sucrose buffer and eliminating the 1.7 M sucrose centrifugation step, the chromatin was released from the nuclei by suspension in 10 mM glycine buffer (22, 13, 14, 15). Chromatin was also sheared for 15 seconds (13, 14). RNA synthesis with the purified enzymes on DNA template was carried out in at least duplicates in 250 1.11 of a standard incubation medium (1, (pH 7.9), 0.1 mM dithiothreitol (Calbiochem. Calif.), 2, 18): 80 mM Tris-HCl 0.1 mM disodium ethylene diamine tetraacetate, 3 mM MnCl, 50 mM ammonium sulfate and the three nonlabelled nucleoside triphosphates (Calbiochem., Calif.) are at 150 nM while [3H]UTP (Amersham/Searle, Ontario) is at 75 nM and 66 uCi/nmole. When the endogenous RNA polymerase activity of chromatin was measured, a 2.5 ml reaction volume containing sheared chromatin had glycine (pH 7.8) at 40 mM replacing Tris-HCl, MnCl at 1 mM, ammonium sulfate at 288 mM (Smith and Duerksen, manuscript in preparation) and the bases were at l/5 standard concentration. Glycerol concentrations were as indicated in the figure legends. After stopping the reaction with reaction-stopping mixture at 0' (1, 2), RNA synthesis was determined by measuring the incorporation of labelled nucleotide into acid-insoluble material (13, 14). N-trisChydroxymethyl)methyl-2-aminoethane-sulfonic acid (TES) was obtained from Calbiochem. (Calif.) while Tris-(hydroxymethyl)aminomethane (enzyme and buffer grade) was obtained from Schwarz/Mann (N.Y.). RESULTS AND DISCUSSION The stability presence

of eukaryotic

of glycerol

in high

5, 6, 7, 8, 9, 10, 11). II

incubated

glycerol decay Tris

rate

or TES (pH 7.5)

increases buffer

is

much below

dropped

in the buffers

at different

presented

a 24 hour

in Figure

period

for

to 17% the

7.0 or above The biphasic

decay

biphasic.

nature

917

2, 3, 4,

RNA polymerase

The monophasic

minimal. rate

II

linear

activity

When the

in concen-

of enzyme activity

Bringing

7.5 greatly

(1,

pHs and at 17 and 50%

1.

is

on the

used

RNA polymerase

50% glycerol,

and becomes

is dependent

of mouse TLT hepatoma

buffers

containing

dramatically

the enzyme activity.

The stability

is

of 35% over

of glycerol

concentrations

in various

concentrations

tration

tion

at 21'

RNA polymerases

increases

of the decay

the pH of the the decay curve,

the

incubarate

inflection

of

Vol. 67, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TIME

Figure

1.

point

Effect of glycerol, buffer and pH on the stability of mouse TLT hepatoma RNA polymerase II. RNA polymerase II was diluted from the stock enzyme preparation into the indicated buffers at 50 mM at different pHs and containing 0.1 mM EDTA, 1 mM dithiothreitol and glycerol either at 50% or 17% such that 25 1.11 of preparation contained 7.4 units of enzyme. The mixtures were incubated at 21' and 25 yl removed at the indicated times and assayed for 10 minutes in the standard reaction mix of 0.25 ml containing 25 ug of sheared, denatured TLT hepatoma DNA as template. The incubation preparations for the enzyme contained the following combinations: TRIS or TES pH 7.5 and 50% glycerol (an), TES pH 7.0 and 17% glycerol (A-A), TES pH 7.5 and 17% glycerol (O-O), TRIS pH 7.9 and 17% glycerol (O-O), TES pH 6.4 and 17% glycerol e), TES pH 8.0 and 17% glycerol (A---A), and TES pH 8.5 (0 and 17% glycerol (B-e).

varying

Gissinger

with

--et al.

the incubation (9)

activation

of calf

glycerol.

Temptingly

of two forms tion

(IIA

(Smith

effect

TLT hepatoma

for

and 11,)

cells

results

of heat II

similar

(42")

in a buffer

can be correlated

unpublished

results),

differences

(9, 10, of glycerol

evident.

918

their 11).

to that

or urea

(lM,

obtained 15')

30%

the known purified

complex

presence

prepara-

subunit

In any case the

on RNA polymerases

by

in-

containing with

of the enzyme in our partially

concentrations is

is

conditions,

RNA polymerase

these

structural

of high

the pattern

thymus

and Duerksen,

up and subunit ing

(Hours)

makestabiliz-

of mouse

BIOCHEMICAL

Vol. 67, No. 3, 1975

Consequently, various

it

concentrations

RNA polymerases.

is

AND BIOPHYSICAL

necessary

to examine

on the activity Figure

2 shows

of the

the kinetics

eo. TIME

IN MINUTES

RESEARCH COMMUNICATIONS

the effect several of

of glycerol

mouse TLT hepatoma

incorporation

10 GLYCEROL

at

20

30 CONCENTRATION

of IJMP by

40 (% V:V)

50

Figure

2.

Effect of glycerol concentration on rate of [3H]UTP incorporation by RNA polymerase II. Firstly, in a series of reaction volumes of 0.26 ml 36 pg of sheared, native TLT hepatoma DNA served as template for 2 units of TLT hepatoma RNA polymerase IIA in the presence of 4% glycerol (0 -cl (37O) 1. At the incubation times indicated reactions were terminated by the addition of reaction-stopping mixture at 0' and the samples analyzed. Secondly, large reaction mixtures containing, respectively, 28% glycerol l ), 48% glycerol (O-O) and 50% glycerol (O-a), had Cm 46 ng of sheared, native TLT hepatoma DNA serving as template for 4.5 units of a mixture of TLT hepatoma RNA polymerases IIA/IIB per a 0.26 ml volume. At the indicated incubation (37') times 0.2 ml aliquots were removed from each reaction mixture and added to reaction-stopping mixtures at Oo and analyzed.

Figure

3.

Effect of glycerol concentration on the activity of RNA polymerase II. Reaction mixtures of a volume of 0.26 ml contained 46 pg sheared, native TLT hepatoma DNA serving as template for 4.5 units of a mixture of TLT hepatoma RNA polymerases IIA/IIB (O-O). Glycerol concentrations as indicated were present in their corresponding reaction mixtures. The reaction was initiated by the addition of enzyme and terminated at 30 minutes incubation (37O) by the addition of reaction-stopping mixture at 00 and the samples analyzed. l and 0 are the 30 minute values for 28% and 48% glycerol taken from Figure 1.

919

Vol. 67, No. 3, 1975

BIOCHEMICAL

RNA polymerase

IIA

RNA polymerase

IIA/IID

rate

II

activity

by glycerol

3), is

at 25% glycerol

shown by the results

not

fashion

0

4.

the

incorporation are varied of RNA

proportional

to the glycerol

concentra-

is

approximately

inhibited

4, RNA polymerase

the range is also

These

results

inhibits

I 5 GLYCEROL

decreases

concentrations

that

by

the inhibition

I activity shown).

concentrations

evident

in Figure

within

concentration'dependent

Figure

is

directly

presented

concentrations

(Data

it

of incorporation

Glycerol

When glycerol

the activity

of RNA polymerase

as template

to the rate

48 and 50% glycerol.

85 minutes.

4 and 50% (Figure

polymerase

compared

and at the higher

at approximately

between

bition

in 28,

of UMP incorporation

ceases

tion;

in 4% glycerol

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

examined. evident

This

50%. I is

unaffected

lack

of inhi-

on double-stranded

further

show that

RNA polymerase

II

I I 10 15 CONCENTRATION

As

DNA

glycerol

whether

in a

the

I 20 (% VV 1

Effect of glycerol concentration on the activity of RNA polymerase I and II on single-stranded, sheared DNA. Reaction mixtures of a volume of 0.26 ml contained 23 pg of sheared, denatured TLT hepatoma DNA serving as template for 2.4 units of TLT hepatoma or 3.6 units of a mixture of TLT RNA polymerase I (0 -0) hepatoma RNA polymerases IIA/IID (O-O). Glycerol concentrations as indicated were present in the corresponding reaction mixtures. The reactions were initiated by the addition of enzyme and terminated at 20 minutes by the addition of reaction-stopping mixture at 0' and the samples analyzed.

920

Vol. 67, No. 3, 1975

template

is

BIOCHEMICAL

single-or

double-stranded

inhibition

of RNA polymerase

associated

with

(Figure

at relatively Duerksen,

chromatin

The cryptic

4).

high

a-amanitin-sensitive

bitory

of inhibition

obtained portion

results)

is

that

at

this

RNA polymerase

is

demonstrable

for

lysed

of this

enzyme activity

and the majority II

form

concentration

(23,

(Smith

Above this

is

is

24, Smith

only

and is due to unpublished

15% had no inhi-

concentration

approximately

nuclei

expressed

and Duerksen,

to the glycerol there

the enzyme

of the activity

of approximately

proportional

glycerol

mouse TLT hepatoma

concentrations

enzyme activity.

directly

35% glycerol

5.

this

from

the

concentration 50% inhibition

degree such of

activity.

GLYCEROL

Fi .gure

In addition,

up to a concentration

on this

RESEARCH COMMUNICATIONS

DNA.

activity

RNA polymerase

Glycerol effect

II

ammonium sulfate

unpublished

results).

AND BIOPHYSICAL

CONCENTRATION

(% V:V)

Effect of glycerol concentration on the activity of chromatin endogenous RNA polymerase activity. Reaction mixtures of a volume of 2.5 ml contained the described proportions of ingredients (see materials and methods) but replaced Tris with glycine at 40 mM concentration. Ammonium sulphate at a final concentration of 288 mM for endogenous RNA polymerase activity, was present in each reaction mixture as well as the indicated glycerol concentrations. The reactions were initiated by adding 0.2 ml (200 ug chromatin DNA) of a sheared TLT hepatoma chromatin preparation. After 60 minutes incubation (37O) reaction-stopping mixture at 0' was added and each sample analyzed.

921

Vol. 67, No. 3, 1975

BIOCHEMICAL

In conclusion, TLT hepatoma with

the

strable

whether

single-or

results.

whether

from

of glycerol

(data

inhibition

It would

now be important

inhibitory

cells.

If

so,

effects the

present,

great.

of the RNA polymerases

in estimating lished

template

DNA is

not shown).

where

cannot

capacities

level

the

from

TLT hepatoma fractions,

these

glycerol

from other

amounts

glycerol

of transcrip-

or not

evaluation

demonThe

be determined

obtained

of chromatin

associated is

has

eukaryotic

of RNA-

of glycerol

of the purification

of mouse liver

still

out

whether

varying

The details

diluting At what

to determine

on mouse

used as template.

since

to the quantitative

measurements,

rather

inhibition

This

on RNA polymerases

polymerase-activity are

nuclei.

is manifested

implications

effect form or

is reversible

glycerol

similar

tion

lysed

inhibitory

in a purified

double-stranded

the enzyme activity

this

has a dramatic II

obtained

effect

restores tion

RNA polymerase

chromatin

inhibitory

glycerol

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

are

and characterizacells will

and their

use

be pub-

elsewhere. ACKNOWLEDGEMENTS

This

We wish to thank Dr. Izhak J. Paul for his valuable investigation was supported by the National Research

suggestions. Council of Canada.

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

8. 9. 10. 11. 12. 13. 14. 15. 16.

Roeder, R.G., and Rutter, W.J. (1969) Nature (Land.), 224, 234-237. 2543-2553. Roeder, R.G., and Rutter, W.J. (1970) Biochemistry, 2, Chesterton, C.J., and Butterworth, P.H.W. (1971) Eur. J. Biochem., l.9, 232-241. (1971) Proc. Nat. Acad. Weaver, R.F., Blatti, S.P., and Rutter, W.J. Sci. U.S.A., 68, 2994-2999. (1972) Eur. J. Biochem., 28, 277-282. Gissinger, F., and Chambon, P. (1972) Eur. J. Biochem., 8, 283-290. Kedinger, C., and Chambon, P. Mondal, H., Mandal, R.K., and Biswas, B.B. (1972) Eur. J. Biochem., 463-470. 5, (1973) Eur. J. Biochem., 36, 286-293. Cacace, M.G., and Nucci, R. Gissinger, F., Kedinger, C., and Chambon, P. (1974) Biochimie, 56, 319-333. Schwartz, L.B., and Roeder, R.G. (1974) J. Biol. Chem., 249, 5898-5906. Schwartz, L.G., and Roeder, R.G. (1975) J. Biol. Chem., 250, 3221-3228. Duerksen, S.D., and McCarthy, B.J. (1971) Biochemistry, lo, 1471-1478. (1974) Molec. Cell. Biochem., 4, 197-203. Duerksen, J.D. Duerksen, J.D., and Smith, R.J. (1974) Int. J. Biochem., 2, 827-844. (1975) Molec. Cell. Biochem., In press. Paul, I.J., and Duerksen, J.D. Murphy, E.C., Hall, S.H., Shepherd, J.H., and Weiser, R.S. (1973) Biochemistry, 12, 3843-3853.

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Vol. 67, No. 3, 1975

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