Protein synthesis during a nutritional shift-up in Escherichia coli

Vol. 67, No. 1, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

PROTEIN SYNTHESIS DURING A NUTRITIONAL IN ESCHERICHIA Bruce

H. Sells,

Laboratories

Stephen

Memorial St. Received

August

and Graham

Biology,

University

John's,

COLI

M. Boyle,

of Molecular

SHIFT-UP

Faculty

Carpenter*

of Medicine

of Newfoundland

Newfoundland,

Canada

AlC 557

28,1975 SUMMARY

The rate of synthesis of ribosomal protein increased immediately following a nutritional shift-up in Escherichia &; while the rate of synthesis of elongation factors did not increase until 5-10 minutes had elapsed. The relative rates of synthesis of EFG and EFTs were constant at all times following shift-up. This constancy was not maintained between elongation factors and ribosomal protein early during shift-up. These data suggest that ribosomal and elongation factor proteins are not co-ordinately synthesized and argue against the postulate that their genes are part of one polycistron. The studies genetic

organization

one long phage

insertion

cribed,

of

polycistron.

indicating

not

of Nomura

while

the

the

those

components

located

of

actually polar

*Present

this

effects

protein

More recently,

conclusion

caused

the

a number

into

question

gene deletions were

Address:

not

distally

however, and have

findings

that

the

system

is

using

insertion

were

to the

p phage

insertion

it system

Thomas

were

and his

indicated

when gene deletions

of Biochemistry, Tennessee 37232, 203

Vanderbilt U. S. A.

1~

transwere

was concluded

that

linearly

arranged

colleagues u phaqe were

observed.

Department Nashville,

on

was presented

u phage

insertions,

synthesizing

and that

upon

An arrangement

to the

of

suggested synthesizing

chromosome. proximal

components

protein

was based

components

in one polycistron. called

conclusion

bacterial

Following

(1) originally

of the

all

transcribed.

that

components This

into

that

and Engbaek

University,

(2) have insertion

selected

aqainsl

Vol. 67, No. 1, 1975

BIOCHEMICAL

The following co-ordinate system

synthesis

occurs

rates

of

synthesis

did

of

the

protein

shift-up

early

shift-up.

and elongation these

are

during

rates

to those

the the

were

increased

obtained

shown

without In our

factors

shift-up

early

have

(3,4).

rate

determined

studies

shift-up

following

to compare

factors

factors

of elongation

15 minutes

a

synthesizing

Previous

following

whether

The relative

and elongation

phases

until

question

the protein

RNA synthesis

was designed

and to compare

of

enzyme activity

however,

the

one polycistron.

proteins

specific

investigation

somal

indicate

nutritional

very

RESEARCH COMMUNICATIONS

to examine components

and ribosomal

during

increase,

present

various

following

reports, not

the

initiated

ribosomal

protein

delay

earlier

of

cells

ribosomal

apparent

were

in such a way as to

in bacterial that

studies

AND BIOPHYSICAL

G and T

(5).

The

of synthesis

of

phase

of nutritional

during

exponential

ribo-

growth. MATERIALS AND METHODS --E. coli or glucose the

rate

growth

plus of

amino

synthesis

conditions,

bacteria to

H128 was grown

growing

lysine

was added

mature

ribosomal

fuged

and washed

chromatography

ml).

40 min.

to allow The

buffer

of bacteria, of ribosomal

factors (8).

were Both

To compare

protein

under

different

A suspension

suspension

a shift-up

assembly

of

labelled

14 C and 3B labelled

ml)

L-

an excess

and the

cells

for

were

3

(0D575 (U- 14c of unlabelled

were

ribosomal cells

of

of cells

received

1.0 min later,

incorportation

acetate

(6).

(2 MC and 10 ug per

A second

Exactly

isotope

with

lysine

growth.

to arrest

subunits.

Disruption

Elongation

(4,5-3H)

with

medium)

was employed.

or undergoing

UC and 47 ng per

an additional

isolation

L-

30°C supplemented

of cellular

procedure

exponentially

(0.1

at

(enriched

classes

of exponential

lysine

the

with

medium

and nucleosides,

a double-label

4 generations

for

acids

of different

was labelled

O-2-0.4)

in salts

incubated

protein combined,

into centri-

(7). separation subunits

separated

of

ribosomes

was performed from

soluble

EF G and EF T were

204

and soluble as previously

proteins eluted

protein,

and

described

(5).

by DEAE cellulose from

the

column

in

the

Vol. 67, No. 1, 1975

same fractions.

BIOCHEMICAL

EP G was assayed

and was shown to be sensitive EF T was assayed presence

by poly

of EF G.

acrylamide

disc

to fusidic

U directed

(10)

procedure

which

was shown to contain

produced

EF G and EF T migrate

together

under

EF T activity

detected

in our

band

was not

electrophoresis in the

gels

Antibodies rabits emulsified the

from

kg)

were

of the

ear

adding

nite

at

either

23OC and then

Each

of the

dialyzed

mined

50° C and the

by scintillation

punching

out

areas

Attempts The isotope an aliquot

the

content

plates

antibody

against

of the

of

out, in the

Background

Ouchterlony

to prepare

2 days

punched

the

total

broken

cell

having

3 weeks,

animals

were bled

allowing was then

in 0.05 in

the

stored

at

pH 7.5,

a separate

plate

by

to peripheral were

against

incubated

phosphate

incubated

overbuffered

overnite

in

1.0

EF G and EF Ts band deter was determined

by

any immunoprecipipitin

protein

was measured

prior

blood

M Tris,

unsuccessful.

preparation

white

antigen

EFTu were

cellular

205

The

in an equal

the

radioactivity not

generated

After

adjuvant.

The plates

C 14 to H3 ratio

counting. of

were

mq of

serum which

for

that

EF T.

and 50 ~1 of S-100

extensively

reported

in New Zealand

after

well.

gels

gels.

contain

was assayed

center

bands

in the

1% agarose

well

Coomasie

stained

may be due to heat

2 weeks,

to the

with

of electrophoresis,

mq of antigen

After

fraction

to a center side

0.25-5

using

S-100

(10)

of 0.5-l

by centrifuqation

poured

The immunoprecipitin at

of

was added

were

1 mM EDTA.

ml of protosol

drawing

(0.01%)

Gordon

prepared

Freund's

adjuvant.

collected

plates

4 mm from

saline.

bands.

Freund's

50 1~.1 of antiserum

wells

shots

stained on the

may also

injections

in the by poly-

band

of persulphate

of complete

booster

Merthiolate

M NaCl,

This

EF G, therefore,

volume

and serum

Ouchterlony

gels.

by intramuscular

given

incomplete

to clot. 4Oc.

l-2

protein

conditions

EF G and EF TS were

in an equal

animals

volume

0.1

against

(about

these

were

(9)

of EF G.

purified

gels

Although

or the presence containing

further

and the

EF G activity.

inhibitor

of phenylalanine

were

one main

GTP ase activity

a known

polymerization

electrophoresis

(11).

during

acid,

factors

Blue

RESEARCH COMMUNICATIONS

as ribosome-dependent

The elongation

gel

This

AND BIOPHYSICAL

to

fractionation.

by withThe

Vol. 67, No. 1, 1975

sample ml.

BIOCHEMICAL

was solubilized

of

content

of

was determined elongation it

soluble

protein

in a Beckman was calculated

yuenching

following in the

by slicing

the

10 ml.

added.

band

of

gel from

the

scintillation

the

counts

by external

The C14/H3

were

correctea

standard

ratio

for

The

band containing gel

and

fluid

The vial was tightly 14 The H3 and C content of all

counter.

Ten

fractionation

in toluene).

scintillation

was monitored

was then

protein

containing

24 hours.

after

1 ml of Protosol.

to toluene)

The radioactivity

vial

37O for

RESEARCH COMMUNICATIONS

containing

or ribosomal

was determined

and 3% Protosol at

vial

Gmnifluor

same manner.

in a scintillation

and incubated

sample

(0.4%

protein

Gmnifluor

measured

fluid

in the

factor

placing (0.4%

in a scintillation

scintillation

isotope

AND BIOPHYSICAL

capped, samples

was

of each

isotope

spillover.

ratios.

RESULTS AND DISCUSSION The first synthesis protein

of auring

bacterial

cells

experiment total

performed

cellular

the

early

(25 ml.

was designed

protein, phases each)

ribosomal

or nutritional prelabelled

15

protein shirt-up.

with

0

to determine

HS-lysine

the

rates

and elongation Suspensions during

growth

of factor of in

30

MINUTES AFTER SHIFT-UP

Fig.

1.

Synthesis of cellular proteig for 30 min. following shift-up. Cellslxere prelabelled with H-lysine in acetate medium and with C-lysine after transfer to enriched med$um for 1 min. as described in Methods. The 14C13~ ratios of total cell protein, CJ-Cl ; elongation factor protein,A , 30s ribosomal protein,* *, and 50s ribosomal protein, were determined as described in Methods. 0 -0'

206

Vol. 67, No. 1, 1975

acetate

medium

medium

(shift-up).

incubated

of total

factor

protein

14

protein

data,

a table

indicate

during that

after

a fifteen

factor

synthesis.

at

0

time

acid

rate

synthesis

Presenting

in amino

the

the

pool

following

size

and elongation

following

cell

in the

in this

nutritional

rate

use of these

protein

synthesis

as a function

way eliminates

cause. shift-up,

of

and elongation

Making

are expressed

might

shift-up.

protein

delay.

in the C 14 to H3

the

of ribosomal

data

were

as described

protein

total

enriched

cells

increase

minute

in which

shift-up,

30 minutes

of

containing

1, illustrate

an immediate

Synthesis

of elongation

that

the

and processed

first

is

RESEARCH COMMUNICATIONS

flasks

after

in Figure

the

there

constructed

changes

twelve

305 and 50s ribosomal

synthesis.

is

protein

influence

one minute

protein,

increased

of

times

presented

determined

rates

to each

for

The data

protein

and the

-1ysine

indicate

factor

total

C

cell

results

ribosomal

transferred

AND BIOPHYSICAL

At predetermined

section.

ratio

These

were

with

methods

BIOCHEMICAL

of

any

The data,

in Table

the

of

rates

I,

ribosomal

TABLE I Differential of Time after

Ribosomal

shift-up

Rates

Proteins C14/H3 C

14

of

and Elongation Ribosomal

/H

3

Total

.016

.045 .016

15

.074 .020

Protein

c

14

/H

3

Elongation

C14/H3

Factors

Total

Protein

-30s

=

1.07

=

2.81

=

3.70

.016 -015

.015

10

Factors

Protein

50s

0

Synthesis

.025 .016 -060 .020

30

207

=

1.07

=

1.56

=

3.00

.014 .015

&l& .016

.026 .020

=

.93

= 1.00 =

1.30

Vol. 67, No. 1, 1975

protein

synthesis

soluble

protein

30 minutes)

BIOCHEMICAL

and elongation formation

following

is

and elongation

factor

variation

from

data

ribosomal

protein

factor

factor

1.

These

synthesis

essentially

shift-up,

ribosomal

AND BIOPHYSICAL

however, protein reveal

experiments directly

repeated

to cells

growing

following

growing cells

more precisely

were

intervals

exponentially reached

At later

the

relative

rates

total

protein

versus that

the

rates

of

extent

initial

and in this

times

than

of

(15 and

of synthesis

of

show a marked

synthesis the

the

mid log added

supplements

was exactly in Figure

medium

Equal

of

rates

flask.

the

of

elongation

isotope

was terminated

of unlabelled

lysine

and the

cells

minutes,

to allow

ribosome

particles.

volume added

of cells to each

centrifuged protein

the

labelled

cell

A change by six

protein

in

minutes

30s and 505 ribosomal

during shift-up,

the

first the

rates

of

synthesis

rate

protein of

synthesis

synthesis

of

ice

growth

cell

remains

minutes

the

crushed in

shift-up.

-1ysine

added.

by the

acetate

addition forty-five

cells

elongation

increased By ten

was were factor

2 show that

during

protein

had increased

into

medium

in Figure

constant

factor

times

and an equal

protein,

the

synthesis by 30%.

first

ten

was

The rates

10% and 30% respectively minutes

30s and 505 ribosomal

208

14

labelled

The results

of elongation

and by ten

two minutes

total

At the

to be assembled

The double

determined.

protein the

over

during

of

of nutritional

an additional

synthesized

shorter

of these

one minute for

added

When the

mixturr:

and C

cells.

C 14 to H3 ratios

and ribosomal

minutes.

HJ-lysine

flasks.

was selected

were poured

with

in five

medium.

incubated

were

of g. &

concentration

after

the

made at

enriched

protein

of C 14 -1abelled

sample

of total

were

ribosomal

The cells

and the

rate

observed

all

placed

the

flasks

of the

supplements

a concentrated

for

Incorporation

to shift-up,

a suspension

The final

as described the

of

were

of growth,

to each

response

and measurements

portions

medium

phase

2, one of

cellular case nutritional

in acetate

in acetate

were

indicated

the

shift-up.

supplements

of

as a function

synthesis. To record

the

expressed

equivalent.

to a much greater

change

RESEARCH COMMUNICATIONS

following

protein

had at

the

Vol. 67, No. 1, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

.30-

.25 l ‘-a

l .20 -

I 0

s

.15/ 105 ::-:/::.:: B===szsL .05 0, 0

Fig.

least

Synthesis

doubled.

growth,

in a co-ordinate

the

not

Cells

previously

methods

separate

cl4 -1ysine

uc/ml,

protein in the

double

and elongation

experiments during

factor

in equimolar

suggest

a shift-up.

that

they

The data

to

isolate

in presence

elongation

proteins

are

amounts are

not

presented

Methods

during synthesized

in Table

1

analysis and the

C

14

were

from

The S-100

fractions

on Ouchterlony

plates.

209

pulse-labelled

intervals

separated

to H3 ratio

shown in Figure

to EF G and EF Ts were prepared.

at various

fraction

section.

factors

of H3-lysine

470 rig/ml) (S-100)

diffusion excised

used

EF G and EF T, antibodies grown

(1.0

soluble

were

I

10

contention.

2 did

bands

/

and are present

these manner

this

Since

using

ribosomes

interrelated

exponential

described

,

of

Although

physiologically

the

I

2 4 6 8 Time After Shift-up I hllnutes 1

cellular protein durin the first 10 min. of a 11s were labelled with 5 H-lysine in acetate ;yu;,, Ee C-lysine after transfer to enriched medium as described in text. The l4 C/ 3 H ratios of total cell protein, 0, elongation factor protein, 4 ., 30s ribosomal aprotein,0 0, and 50s ribosomal protein, e--------o' were described in Methods.

2.

supports

I

in the

following the

disrupted were

then

with shift-up bacteria analyzed

The immunoprecipitin

EF G and EF Ts bands

and as

Vol. 67, No. 1, 1975

determined.

The results

demonstrate total

the

change

protein.

increases

Both

occur

in Table

the

ratios

of

reported

rate

this

experiment

in the

rate

of synthesis

rate

increase

shift-up;

the

increase

are

synthesis. in the rate

ribosomal

any of

These

data

of

synthesis

increase

in

these

studies

and suggests

that

lag

2 and

before

of the

(see Figure

factor

for

ribosomal

is

much slower 14

2 have synthesized

proteins

result

elongation

The C

Figure

protein

the

is

1).

to significant

substantiate

of EF synthesis

described

elongation individual

shown as in Table

a 5 minute

protein

the

RESEARCH COMMUNICATIONS

of EF G or EF Ts relative

rate

proteins

and show that than

of

in ribosomal

individual

rate

of

the

elsewhere

much lower shift-up

of

the

from

in their

following

AND BIOPHYSICAL

EF G and EF Ts demonstrate

1 regarding

factors than

BIOCHEMICAL

during

to

H3

been at a

nutritional

(12). The data

his

colleagues

not

part

of

presented

one polycistron.

support

ribosomal

It

is

proteins

interesting

the

conclusion

of Thomas and

and elongation to note

following

factors shift-up

are that

TABLE 2 Differential Time after

shift-up

0

Bates

of Synthesis

of EF G and EF Ts

C14/H3 in EF G 14 3 C /H in total protein

0.045 0.047

0.96

C

14

C14/H3 in EF TS 3 /H in total protein

k0 085 = 1.81 0.047

-

=

5

-0.043 0.046

= 0.94

0.088 0.046

=

10

-0.051 0.045

= 1.13

0.098 0.045

= 2.18

15

-0.058 0.046

= 1.26

0.11 0.046

= 2.39

20

-0.073 0.049

= 1.49

0.134 0.049

= 2.74

30

-0.088 0.060

= 1.46

0.153 0.060

= 2.55

210

1.91

BIOCHEMICAL

Vol. 67, No. 1, 1975

the

relative

rates

of

synthesis

The same constancy

is

not

ribosomal

through

proteins

of

AND BIOPHYSICAL

the

two elongation

maintained, the

however,

early

RESEARCH COMMUNICATIONS

periods

factors

between of

are

constant.

elongation

factors

and

shift-up.

ACKNOWLEDGEMENTS We thank preparations the

Dr.

Brot

of

of elongation

Damon Runyon

National

Nat.

Roche

Institute

factors.

Memorial

Fund,

Foundation-March

of

These

the

of Molecular studies

Medical

were

Research

Biology

supported

Council

for

purified

by grants

of Canada

from

and the

Dimes.

REFERENCES

1.

Nomura, M. and Engbaek, Gen. Genet.

2.

Cabezon, T., Faelen, Molec. Gcn. &net.

3.

Schlief,

R.

4.

Harvey,

R. J.

5.

Carpenter,

6.

Boyle,

7.

Carpenter,

8.

Nathans,

9.

Nishizuka, Enzymology

(1967)

F.

J. Mol

(1970)

J.

S. M. and Sells,

D. and Lipmann, Lipmann, B, 713.

(1970)

Biol.

Natl.

Acad.

M.,

Bollen,

A.,

B. H.

B. H. F.

Gordon,

11.

Maizel, J. V. (1971) in Virology, 180.

12.

Carpenter,

101, (1973) (1974)

FEBS Letters

Proc.

R.,

1526. (1975)

Nat.

Res.

Biophys. Acad. J.

31.

Acta

Sci.

(1968)

Comm. 51, 287,

U.S.A.

23.

322. 47,

497.

Methods

in

H. eds.

Methods

912.

in Maramorosch, B. H.

35,

Biophys.

Biochem.

F. and Lucas-Lenard,

G. and Sells,

Thomas,

69,

574.

Biochem.

(1972)

2,

SC. U.S.

41.

(1961)

Biochemistry

10.

27,

Bacterial.

B. H.

G. and Sells,

J.

Proc.

M., De Wilde, 137, 1 25.

G. and Sells,

Y., XII

(1972)

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K. and Kaprowski, Eur.

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123.