Azotobacter vinelandii RNA polymerase VI. An in situ assay for polymerase after acrylamide gel electrophoresis

Azotobacter vinelandii RNA polymerase VI. An in situ assay for polymerase after acrylamide gel electrophoresis

Vol. 32, No. 1, 1968 BIOCHEMICAL AZOTOBACTER ASSAY FOR AND VINELANDII POLYMERASE J. S. BIOPHYSICAL RESEARCH RNA POLYMERASE AFTER Krako...

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Vol.

32,

No.

1, 1968

BIOCHEMICAL

AZOTOBACTER ASSAY

FOR

AND

VINELANDII

POLYMERASE

J.

S.

BIOPHYSICAL

RESEARCH

RNA POLYMERASE

AFTER

Krakow,

ACRYLAMI

K.

VI.

DE GEL

Oaley

and

AN

IN SITU

ELECTROPHORES

E.

University of California Space Sciences Laboratory Berkeley, California

COMMUNlCATlONS

IS. *

Fronk

94720

Received June 10, 1968

Analysis gel

more

one

into

activity

consistently

This

(Smith

et

for

assaying

assay

utilizes

Karstadt, visualize

Krakow,

1967)

followed

the

of

the

A.

published

gels

recovery

of

enzyme

bands

could

The

present

paper

describes

polymerase

in

1968)

or

the

synthesis the

staining

not

acrylamide of

poly

A-poly

rlC

copolymer

(Krakow

with

ethidium

bromide

U

polymers.

EX PER I MENTAL

The

RNA

protein

unprimed

by

from

However,

separately. RNA

associated

initially

elution

spaced

acrylamide

w;n

bands.

after

closely

by

activity

protein

1866).

for

polyrnerase

enzyme

determined

1967;

the

that resolved

and

assayed

al,

RNA

(Chrambach,

variable

a procedure

vinelandii

the

was

discs

was

gel.

of

activity

sliced

to

A.

indicated

than

polymerase

and

the

electrophoresis

with

be

of

vinelandii

RNA

procedure

* This work was supported from the National Institutes California, Berkeley.

PROCEDURES

polymerase

(Krakan

by

and

was Ochoa,

grants from NASA of Health to the

98

prepared 1963)

NsG 479 University

by with

and

a modification gel

filtration

GM of

12326

Vol. 32, No. 1, ‘I 968

on

BIOCHEMICAL

agarose

final

beads

(Bio-Gel

hydroxylppatite

had

a specific

AMP

incorporated

A step.

activity

I .5 The

of

into

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

m,

BioRad

RNA

I50

polymerase

units/mg

RNA per

min

Laboratory)

(I

at

37O

used

in

=

I wmole

unit

with

replacing

calf

this

the paper

of

thymus

DNA

C

14

as

-

the

template). The gels

using

manua

I.

the

enzyme the

The

upper

acid

was

resolved

Tris-Glycine gels

were

system

polymerized buffer

Chemical

Co.)

liter

artifacts

(Brewer,

1967).

phoresis

apparatus

was

used

with

was

applied

5’.

The

enzyme

sucrose

and

electrophoresis

70

min.

Protein

phoretical

ID0

were rm~)

pH 7.8; 0.6

situ

0.02!

with

were

The

incubated

0.05

E NaCl

poly rni

of

85%

thioglycolic

persulfate electro-

through

.02

at

aniline

i

the

Tris,

2.5

and

jacket

pH 7.8-10%

mamp

black

polymerase:

glass

of

the

I TP

columns

-

(100

24

unbound

0.6

0.001

following

of dye

rinsed

the

- ethldium

TNE Is

rni

per

tube

destained

for electro-

5 ml

and

buffer)

removed

gels

HnS04;

In

bromlde

50

of

complex

background.

99

for

the

TNE

stains

tubes

copolymer

ATP

and

UTP.

(18

hrs.)

The

at

!J! Tris,

37O. pH 7.8

ethidium After

photographing

bright

The red

Tris,

and

rlC

buffer.

(I3

0.05B

ATP

temperature.

desired

ml

rn!

with

room

test

the

0.01

stained

at (If

of

the

mixture: 0.6

overnight

(TNE)

in

reaction

replaced

with

electrophoresis,

placed

For

CTP

incubate

g EDTA

ug/ml

placing

to

were

and

U synthesis.

and

allowed

After

2 ml

Aapoly

gels

bromide

by

RNA

the

5 ml

for

reactions

a clear

performed

mercaptoethylamine,

0.6

hrs.

from

containing

reaction,

ribopolymer

for

removed

nJ1 UTP

gels)

assay

ml

and

polyanalyst

circulating

.05

Canalco

ly. a-In

gels

stained

of

possible

Buchler

water

was

was

The

in

ml

avoid

the

persulfate

0.1

to

5% acrylamide

in

ammonium

contained

per

on

described

with

produced

at

electrophoresis

buffer

electrophoresis

(Pierce

by

agalnst

-

x

Vol.

32,

No.

1, 1968

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

RESULTS

The

unprimed

by

RNA polymerase

to

those

1867;

of

ITP

The Mn

+ CTP or

synthesis

synthesis

of

1.

carried

1868).

polymer

poly

of

acrylamide

reactions

required

(i.e.,

Figure

in

Krakow,

material

synthesis

4-b ATP

poly

Aepoly

U or

gels

showed

requirements

out

formation

and

the

+ UTP)

(Figure

1).

Aapoly

U or

in

solution

of

be

pairs present

copolymer

similar and

bromide

to

Characteristic rlC

copolymer

(Krakow

ethidium

complementary must

rlC

of in

Karstadt,

staining

of

substrates

support

ribo-

the

unprimed

solution

is

the

Assay for RNA polymerase in acrylamide gels. 10 ug of & vineiandli RNA polymerase were applied to the gels and eiectrophoresed at 2.5 mamp per gel for 70 minutes. Gel 1 was stained for protein. Gel 2 was assayed for poly Aapoly U synthesis; Gel fras assay mix; gel 4, ATP and UTP 3, Hn++ omitted The gels were incubated omitted fran assay mix. for I6 hours at 37O and stained with ethidium bromide as indicated in the text.

100

Vol.

32, No.

1, 1968

onset in

BIOCHEMICAL

of the

with

than synthesis

Figure

in in

minutes

at

diffusion

2.

of

regions

of in

detectable

Aspoiy

observed

When high

the

RESEARCH

COMMUNICATIONS

poiymerase

enzyme

region

was

activity

which

assayed also

subsequently

stained

bromide.

onset poiy

BIOPHYSICAL

reaction.

precipitate

ethidium

polymer)

the

gels,

a white

The

the

in

acrylamide

showed red

turbidity

AND

the

37’.

This of

U synthesis

soluble gel

the

(i.e.,

ethidium in

systems. is

about is

The

acrylamide lag

30 minutes,

presumably

components

the

due of

the

bromide

to

gel

phase in the

reaction

stained

for

solution time into

is poiy

later Aapoiy

about

15

required the

for gel

as

Effect of incubation time on poiymerase activity in gels. IO ug of RNA poiymerase were resolved as in Figure 1. Gel I was stained for protein. Gels 2 and 3 were assayed for poiy A-poly U synthesis; gel 2 was incubated for 1 hour at 37O, gel 3 for 5 hours 5 and 6 were assayed for riC copolymer at 37O. Gels synthesis; gel 4 incubated for 1 hour at 370; gel 2-E were stained with 5 for 5 hours at 37O. Gels ethidium bromide.

101

U

Vol. 32, No. 1, 1968

well the

as

BIOCHEMICAL

the

optimum

for

shows

the

minor

regions

1 ug

results

of

RNA

obtained

at

pattern

and of

protein

3.

be the

of

seen

applied

from

to

which

The

major

protein

(Figure

assay

and

activity.

the after

5 hr or

the

in -m

situ

assay

is

acrylamide

gel

(Figure

with

excellent

3).

of

the

is

a major,

This

band

best

the

there

and

above

up

of

a comparison

2)

is

show

limit

protein

polymerase enzyme

buffer

activity lower

of

gel

polymer

polymerase

5 ug

the

reaction.

The

protein

can

pH of

appearance

of

As

the

unprimed

incubation.

about

Figure

in

the

earliest

overnight

band

difference

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is

protein

stained slow

contiguous

moving, with

a

Sensitivity of the --in situ polymerase assay. Gels l-3 contain 5 pg and gels 4-6, ! pg of RNA polynmrase. The gels were run as given in Figure I. Gels I and 4 were stained for protein. Gels 2 and 5 were assayed for poly A*poly U synthesis and gels 3 and 6 were assayed for rlC copolymer synthesis. The assays were incubated for 16 hours at 370 and stained with ethidium bromide.

102

Vol.

32,

No.

diffuse of

8lOCHEMlCAL

1, 1968

area

protein

and

probably in

of

ionic

forms

of

refiects

polymerase

the

to

enzyme

and

a continued with

resulted

in

protein

moved

consonant

treatment

of

caused

sedimenting

the

4.

RNA polymerase

The

minor

the

diffuse

bands

of

the

region

results 24s

4)

g.

the of

of

the

split

the

correspond

to

in

the

to

major

band

minor

Walter

et --

RNA

polymerase

gel

dimer.

prior

form

found

monomeric

probably

I ncubat

ion

of

electrophoresis and

bands. al

band

band

dimer,

the

transformation

COMMUNICATIONS

a minor

major

area of

RESEARCH

by

The

(Figure

a quantitative around

bounded

the

a disappearance

with

heparin

is

dissociation

heparin

into

BIOPHYSICAL

activity.

strength.

polymerase

Figure

which

corresponds

low

that

activity

AND

(1967)

al I of This who

was showed

particles into

the

with material

15s.

Dissociation of RNA polymerase dimer form by heparin. Gel I contained 15 ug of RNA polymerase, gel 2 contained I5 ug of RNA polymerase plus 5 ug of The gels were electrophoresed as in heparin. Figure I and stained for protein.

103

BIOCHEMICAL

Vol. 32, No. 1, 1968

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DISCUSSION

The and

assay

described

preserves

the

electrophoresis. by

the

--In

situ.

in

the

patterns

the

of

ease

of

operation

obtained

after

acrylamide

The

stained

protein

patterns

are

the

polymers

synthesized

was

no difference

after

staining

interesting

to

note

that

there

pattern

noted

for

poly

A.poly

acrylamide

demonstrating

that responsible

there

is

for

the

disc closely

U or

no

apparent

two

types

matched

rlC

copolymer

difference of

in

unprimed

i ons . The

the

only,

small

protein

is

advantage

detail

RNA polymerase

react

the

is

the

fine

obtained

It

synthesis,

has

and

amount

of

the

absence

in ethidium

difficult

minor,

problem

ethidium

bromide,

of

polymer

bromide-protein to

mistake

encountered a cationic synthesis

complex for

the

with

bright

the dye,

(Figure

stains red

a dull dye-ribopolymer

assay which

1). buff

is binds

to

However, red

and complex.

REFERENCES

I.

:: 4.

Brewer, Chrambach, Krakm, Krakow,

i:

Krakow, Smith,

7.

Walter,

H. Science, 156, 256 (1967). A., Analyt. Biochem., l& 3 (1966). Biochim. Biophys. Acta, in press. J. S., J. S. and Karstadt, H., Proc. Natl. Acad. Sci., 58, 2094 (1967). J. S. and Ochoa, S., Biochem. Z., 338, 796 (1963). Wil I iams, D. L. and Martinez, D. A., Ratliff, R. L., J. Biol. Chem., 242, 590 (1967). G., Zillig, W., Palm, P. and Fuchs, E., Eur. J. Biochem., 2, 194 (1967). J.

104

A.

M.