Regulation of the invitro synthesis of E.coli ribosomal protein L12

BIOCHEMICAL

Vol. 91, No. 4, 1979 December

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages

28, 1979

Regulation of L Gregory

Received

of the -- In Vitro Synthesis coli Ribosomal Protein Ll2

Goldberg*, Herbert

Roche Institute November

1453-1461

Tanya Zarucki-Schulz, Weissbach and Nathan

of Molecular

Biology,

Paul Brot

Nutley,

Caldwell,

New Jersey

07110

6,1979 ABSTRACT

The --in vitro B subunit which

of RNA polymerase

contains

subunit

DNA dependent

information This

of RNA polymerase.

protein

information It

LlO.

synthesized

L12 and the

B subunit

situated

within

for

was found

efficiently

the

of ribosomal

has been investigated

the genetic

and the genetic

synthesis

that

--in vitro

for

using

ribosomal

DNA, however, the first

lacks

of RNA polymerase

DNA.

DNA from a plasmid L12 and the

the promoter acids

B subunit

this

L12 and the

protein

26 amino

L12 and the from

protein

region

of ribosomal

of RNA polymerase

These

B

results

can be synthesized

are

suggest

from

that

a promoter

LlO gene. INTRODUCTION

Although ribosomal (1),

the

the genetic

proteins

One of the

which

ribosomal

proteins. four other that

are situated

synthesis

(2).

Thus,

copies

of these

protein it

proteins

*Present

information address:

for

ribosomal

at different

aspects L12 is

of this

on the r.

is

relative

(3,4).

In addition,

but stoichiometric

the transducing

proteins

Ll,

ribosome

experiments

Xrifd18 LlO,

chromosome controlled

the mechanism

amounts

recent

phage

coli

to the other

the 70s E. coli

L12,

various

is coordinately

regulation

overproduced

protein

ribosomal

RNA and the

loci

components

has been shown that

when the DNA from

genetic

for

ribosomal

interesting

of ribosomal

ribosomal

information

(which Lll,

ribosomal contains of the have shown

contains

and L12)

by

the is

incubated

University of Wisconsin, Laboratory of Genetics, 210 Genetics Building, Madison, Wisconsin 0006-291X/79/241453-09$01.00/0 1453

Copyrighf All rights

@ I979 by Academic Press, Inc. of reproduction in anyform reserved.

Vol. 91, No. 4, 1979

in an --in vitro L12 is

BIOCHEMICAL

protein

synthesized

an excellent protein

synthesizing

than

tool

LlO

to study

(5). the

This

about

--in vitro

regulation

has been reported

that

four

to five

system,

times

therefore,

of the synthesis

the genes

and 8' subunits

of RNA polymerase

situated

the LlO gene

before

described

experiments

of RNA polymerase

between

genes

the

report

from

this

and 8' subunits

evidence

ribosomal

laboratory

more affords

of ribosomal

LlO gene.

genes

LlO,

et al.,

(9),

that

however,

only

are

that

may be situated

L12 can be synthesized

from

report

for

and that within

a previous

L12 and the a promoter

B for

the adjacent

provides

a promoter

B and

somewhere

In addition

the genes

cotranscribed

The present

B

recently

L12 and the located

Ll and LlO.

suggested

L12 and the

from one promoter

a common promoter

proteins

(5)

proteins

suggested

share

ribosomal

of these

protein that

they

for

cotranscribed Fiil

of RNA polymerase

transcription

ribosomal

for

are

(6-8).

in which

6' subunits

other

system,

RESEARCH COMMUNICATIONS

L12. It

the

AND BIOPHYSICAL

further

independent

in vitro of the

proteins. MATERIALS

AND METHODS

Materials. E. coli H105 lysogenic with xrifd18 phage was obtained Massachusetts. r. from J.B. Kirschbzum, Harvard University, Cambsge, --_-__._ coli JF943 carrying plasmids pNF1337 and 1341, were a generous gift of J. Friesen, York University, Ontario. Ribosomal protein L12 was purified as previously described (10). Ribosomal wash, washed ribosomes and the 0.25 M and 1.0 M salt eluates from a DEAE-cellulose fractionation of an S-200 extract were obtained as reported earlier (11). Antisera to L12 and RNA polymerase were raised in rabbits with the aid of Freund's adjuvant. carried

Phage and plasmid DNA. The isolation out as described elsewhere (12).

of phage

and plasmid

DNA was

The complete system for protein synthesis In vitro protein synthesis. usinga partially fractionated L coli extract was identical to that previously describg$ (5). One to two ug of either xrif 18, pNF1341 or pNF1337 DNA and 14 FM [ Slmethionine were added and the reaction mixtures incubated for 90 min at 37'. After centrifugation at 7000 xg for 10 min, an aliquot of the supernatant was assayed for incorporation of radioactivity into total protein by precipitation of the protein with hot Cl CCOOH and filtering the precipitate onto a nitrocellulose disc. The filter wa s assayed for radioactivity in a scintillation spectrometer. Slab se1 electrophoresis of the in vitro synthesized products. An aliquot of the reaction mixture was analyzed by electrophoresis on 7.5%-15% gradient polyacrylamide slab gels in the presence of 0.1% sodium dodecyl-

1454

BIOCHEMICAL

Vol. 91, No. 4, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

sulfate (NaDodSO ). The gels were treated with dimethylsulfoxid ? (22.5% w/v) for fluorographic exposed to Kodak X-Omat R film at -190".

2,5-diphenyloxazole detection (13)

in and then

Assay for LlO and L12 by imnunoprecipitation. The amounts of LlO and L12 synthesized were determined by immunoprecipitation and subsequent gel electrophoresis of the immunoprecipitated products (14). It has previously been shown (15) that ribosomal protein LlO forms a tight complex with L12 and is coprecipitated with L12 by L12 antisera.

RESULTS Ribosomal

protein

L12 and LlO synthesis

In an attempt

to gain

more

for

protein

L12 we used DNA from

ribosomal

fragments

of hrifd18

relevant

to this

and 1341 contain hrifd18 -

(9).

Since

which

is

from

its

starts

the

situated normal

at codon L12 and the

of the

6' subunit

bacterial

are

DNA which

Lll, within

genes

for

24 of ribosomal

(6,7,9),

starts

LlO,

(9).

plasmids

I digests

Ll,

B subunit

LlO and

of RNA polymerase

from

one promoter

be synthesized

the

complete

genes

and terminates

within

the

to note

that

is

important

in a direction

PNF 1341

LIO Ll

Lll

I PNF 1337

Fig.

1

Schematic genetic map of the DNA of plasmids pNF 1337 and pNF 1341. Dashed lines represent plasmid DNA and the arrows indicate the direction of transcription (9).

1455

gene the

opposite

z?qy-ly++zzz-I

106

pNF1341

is oriented

Ll2

of

DNA from

includes

It

pNF1337

at codon

proteins

Ll cannot

of RNA polymerase,

of these

Pst

The bacterial

protein

cloned

Plasmids

partial

the

promoter

DNA segments 1.

cotranscribed

plasmid.

of RNA polymerase

DNA in both

for

and Ll are of Lll

in Fig.

ribosomal

the gene coding Lll

bacterial

of pNF1337

through

of the

containing

by cloning

DNA insert

in this

B subunit

p lasmids

ly

DNA templates.

position

of the

was obtained

extends

different

the

shown schematical

to the right promoter

about

The structures

The bacterial

protein

L12 and terminates

for

study

DNA (9).

of ribosomal

DNA.

information

with

Vol. 91, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL

pNF 1337

RESEARCH COMMUNICATIONS

pNF 1341

i 6000 6ooo 4000 3ooo 2ooo looo

34

42

!

24

32

40

46

FRACTION

Fig.

of the

plasmid

of these

genes

initiating

The DNA from

genes

xrifd18

with

to ribosomal

electrophoresis. obtained

after

LlO

are

the genetic

information,

DNA are

The products

were

of the

radioactive

antiserum

to L12.

However,

for

It both

consistent

the first

precipitated

products can be seen ribosomal

when pNF1341

of LlO is

plasmid,

and incubated

to NaDodSO4 gel

used as template,

synthesized.

on this

was extracted

L12 and subjected

with

any transcription

promoter.

system.

The absence

L12 is synthesized.

minimize

plasmids

2 shows a display

and pNF1337 L12

the

protein

immunoprecipitation

and

should

a plasmid

synthesizing

Fig.

when hrifd18

proteins

this

NUMBER

which

and from

protein

antiserum

(91,

from

in an --in vitro

only

324046666

Disc gel electrophoresis of the irrmunoprecipitate of ribosomal proteins LlO and L12 synthesized in a DNA-directed in vitro system. An aliquot of the reaction mixture was im%oprecppitated with L12 antisera and the precipitate was washed and solubilized. The solubilized proteins were electrophoresed on NaDodSO polyacrylamide gels, sliced and assayed for radioactivity. Further detiils are described elsewhere (14) and in the text.

2

to that

that

i

DNA is used,

with

the

26 amino

lack

acids

of of

protein. Location

of the

can be synthesized

L12 promoter.

from

pNF1341

Although

the above

DNA, an attempt

14.56

data

show that

was made to obtain

L12

more

BIOCHEMICAL

Vol. 91, No. 4, 1979

information

about

bacterial

the

promoter

amount

were

of product

pNF1337

is

from

(especially

when the

It

was found

would

pNF1341

Although

pNF1341

were

being

DNA, it used.

the amount

the

synthesis

described

above,

this

DNA is

contains

the Ll

cotranscribed

Lll)

which

is missing

were

responsible

active

for

the

little

synthesis

the

synthesis

results

DNA was initiated

PPGPP.

A bacterial

this

nucleotide

DNA.

(140

about

Synthesis have been

the

two fold

site

from

lower

somewhat promoter

that (see

in this

if

it

also

Fig.

a plasmid

of insertion

1).

Lll

before promoter

should

be

and the orientation

3 shows that

DNA whereas

As

(located

same promoter

Fig.

pNF1337

is

promoters

promoter

this

identical.

did

not

from

PM) did synthesis

support

a bacterial

might

there

not

inhibit

that

promoter

came from

there

is

no

is excellent

which

of RNA polymerase. have presented

studies

not

with

oNF1341

DNA was deshown).

Recently,

evidence

from

by ppGpp but

of L12 from

1337 and hrifd18 -

of ppGpp (data

1457

L12 synthesis

to be affected

the synthesis

of L12 from

in the presence

of reports

the view

be expected

of B and g' subunits

a number

the

DNA,

a plasmid

of Lll

cpm

and pNF1341

DNA was studied.

Thus,

of L12,

lo6

DNA.

promoter

In contrast

creased

of Ll

that

pNF1341

35 codons

DNA (9).

be expected. per

DNA except

bacterial

since

are

Xrifd18

to pNF1341

a single

of Ll,

to the direction

pNF1337

pNF1337

if

promoter

synthesized

of plasmid

DNA, the

seen with

of L12 would

if

normal

However,

a plasmid

expect

the role

last

pNF1337

plasmids

of Ll from

The only

the

synthesis

synthesis

of the DNA in both or very

from

(9).

opposite

from

one would

similar

from

for

protein

of Ll from

gene and about

and Ll are

from

of L12 were

explore

system,

to that

of the L12 synthesized

is more than

--in vitro

pNF1341

low synthesis

into

To further

of L12 from

is oriented

pmoles

incorporated

respectively. with

segment

and 9.3

If the

to be close

initiated

very

synthesis.

the L12 promoter

DNA were

bacterial

17.2

of 35[S]methionine

synthesis

be expected

transcription) that

the

in its

known to contain

transcription

plasmid

involved

used for

formed

DNA which

of the

promoter

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

there

suggesting

that

Vol. 91, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL

pNFl337

Fig.

the

3

6 and 6' subunits LlO and/or

whether

pNF1341

subunit

of be

the

other

L12 (6-9).

DNA, which

from

the

about

same

the genes

in

the

shown).

No synthesis

as noted

above,

either

amounts

DNA

from

DNA contains

with

for

the

synthesis 3 that

that

xrifd18 -

or

seen with

a truncated

to ascertain

B but not of the

DNA

the

the

6'

6 subunit.

pNF1341

from

of the subunits

showed

ribosomal

of interest

of RNA polymerase.

of B and 8' is

this

the Fig.

6 subunit

inmunoprecipitation, when

therefore,

direct

could

of the

are cotranscribed

was,

contains

in which

by the

It

radioautogram

synthesis

experiments

quantitated

of RNA polymerase

RNA polymerase,

seen

direct

pNfl341

Slab gel tlectrophoresis of the in vitro radioactive products from xrif 18, pNF 1337 and pNF 1341 DNA directed incubations. The incubations were carried out as described in the text. Aliquots were removed, subjected to slab gel electrophoresis and then to fluorography. Details of the electrophoresis and fluorography are noted in the text.

proteins

can

RESEARCH COMMUNICATION

It

can

In addition, synthesized

ratio

of

were

LlZ/S(S')

pNF1341

was

used

pNF1337

DNA (Fig.

was

(data

3),

not

since

B gene.

DISCUSSION The

resulting proteins present

synthesis

of

E.

in a ribosome of both as four

coli

containing

subunits copies

ribosomal

except (3).

Many

proteins

is

stoichiometric for of

a coordinate

amounts

ribosomal

protein

the

for

14.58

genes

these

of all

L12(L7) proteins

event

of the which have

is been

Vol.91,

No.4,

found

to exist

units

(1).

this

protein

in clusters

would

be the increased

separate

promoters

for

are

report DNA from

further

plasmid

acids

subunit

a promoter

pNF7341,

of LlO,

relative

to L12.

subunits

are

present that

which

cotranscribed

a plasmid

in these

(9)

contains

an intact

little

if

little

readthrough

note,

any Ll

however,

pNF1341

DNA.

possible

that

DNA.

is

This

lack

an intact

evidence

that

one must for

the promoter

results

indicate

DNA from

of inhibition

protein

and the

not

is required

L12 synthesis. 1459

L12

of both indicates

Ll2

that

and the promoter

that

a plasmid

B (and

B and 8' which

there

is

is no evidence

6 subunit

synthesis

is to be noted in the

that plasmid,

Ll and synthesizes there

is

very

promoter.

been explained, to elicit

6')

efficiencies

as pNF1341 for

24

L12 and the B

equal

it

the

first

by ppGpp of L12 synthesis

has still

LlO gene

point,

site

in the present

site

L12 and the

this

this

has been shown that

be cautious,

and direction

lacks

bacterial

it

of

the structural

ribosomal

DNA with

a comnon bacterial

These

are two

cotranscribed

that

The data

promoter

suggest

same site

observation

for

Thus

the

responsible

3).

of the the

coding

are

within

the synthesis

would

Ll gene but

there

for

has been presented

was suggested

idea.

To support

in the

(Fig.

evidence

DNA and hrifd18 -

Although

studies.

cloned

and it

lacks

from

promoter

--in vitro

pNF1337

data

responsible

perhaps

may be present

direct

pNF1341

is

L12 and B and 6' subunits

Preliminary

These

on pNFl341

L12 that

Alternatively

the genes

can still

from

for

of L12

from which

to this

of RNA polymerase.

synthesized

the overproduction

of RNA polymerase.

support

to the

LlO and Ll2

(5),

for

relative

since

from which

site

LlO

transcriptional

problem

for

Recently,

binding

B and 6' subunits lend

amino

promoter

protein

represent

One,

cotranscribed.

gene of ribosomal

are

L12.

a separate

and --in vitro

promoter

of L12 mRNA.

an RNA polymerase

and the

--in vivo

of a unique

more active

RNA polymerase

might

both

of polycistronic

poses

One explanation

synthesis

and a second

that

however,

proteins. presence

to be part

of L12,

is overproduced

ribosomal

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and appear

The synthesis

other

the

BIOCHEMICAL

1979

We do directed

but

it

a ppGpp effect

by

is on

Vol. 91, No. 4, 1979

BIOCHEMICAL

Based on the present promoter

in pNF1341

to other

studies

from

a phage only

complete

genetic

(6-8)

part

appeared

are

the

of LlO.

located not

which

proteins would

three Ll,

a promoter

it

of the

Hind

with

l-10,

placed

the

within

of these

proteins

with

is

L12,

Thus, is

of RNA polymerase also

is possible

are

experiments

in

L12 or B (from

proteins genes

the

a promoter

have recently

these

initiated

site

to the

between that

right

the --in vivo

the expression

from

a normally

weak promoter

and others

(6-g),

conditions. based

on this

work

units

for

synthesis

and the

the

af3' subunits

LlO would

the structural in much lower include

1460

of the

be transcribed would

gene for amounts

a mechanism

and

be cotranscribed

from

L12, for

Ll and

individually,

LlO.

than

that

ribosomal

of RNA polymerase.

of RNA polymerase

L 12 must

restriction

synthesize

of the

It

were

LlO gene.

there

--in vivo

not for

L12 and

of RNA polymerase

on their

promoter

--in vitro

same promoter,

are synthesized

based

not clear.

suggested

Lll,

subunits

B and B' subunits

in the expression are

(9)

and the

This

studies.

that

et al.,

145),

subunits

B and B' subunits

pNF1341 did

DNA in vitro

is

Fiil

III

DNA that

between

of the

the conclusion

that

plasmid

8 and B' subunits located

acid

6~'

in these

L12 and the

transcriptional

use the

L12 and the

to the right

However,

under

In sumnary are

right

which

experiments pNF1341

L12 and the to the

L12 and the

carrying

is amplified

there

amino

located

We do note only

LlO,

have been detected

The differences

of L12 from

that

one promoter,

DNA) they

and --in vitro

Lll

suggested

LlO gene from

plasmid

from

of a promoter

are consistent

bacteria

(starting

synthesis

of xrifd18

possibility

in one operon.

which

fragment

L12

in contrast

was no L12 or ~6'

restriction

LlO gene,

the

is

the

cotranscribed.

located

III

there

conclusion

to eliminate

results

that

that

This

for

B and 8' subunits.

LlO gene would

suggested

LlO gene.

showed

a Hind

a promoter

the

location

logical

L12 and the

from

within

the most

for

cotranscribed

present

RESEARCH COMMUNICATIONS

information

authors

in the

the

which

of the

LlO and these

However,

is within

containing

included

result

results,

AND BIOPHYSICAL

Since

the

B and B'

the cotranscription a shift-down

in the

BIOCHEMICAL

Vol. 91, No. 4, 1979

synthesis It

or translation

has previously

of the messenger

been shown

autogenous

regulation

understand

the regulation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

that

(17,18),

the

RNA for

synthesis

and further

of the synthesis

the

B and B' subunits.

of these

studies

are

subunits

is

in progress

of L12 and the

under

to

B and B' subunits.

ACKNOWLEDGEMENT The authors for

supplying

would

us with

like the

to express various

their

plasmids

appreciation and for

to Dr.

many helpful

J.

Friesen

discussions.

REFERENCES ::

1: 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Nomura, M. and Murgen, E.A. (1977) Ann. Rev. Genetics 2, 297-347. in Ribosomes, eds. Nomura, Kjeldgaard, N.O. and Gausing, K. (1974) M., Tissieres, A. and Lengyel, P. (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York), pp. 369-392. Subramanian, A.R. (1975) J. Mol. Viol. 95, 1-8. Hardy, S.J.S. (1975) Mol. Gen. Genet. m, 255-274. Goldberg, G., Caldwell, P., Weissbach, H. and Brot, N. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 1716-1720. Yamamuto, M. and Nomura, M. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3891-3895. Linn, T. and Scaife, J. (1978) Nature 276, 33-37. Linn, T.G. and Hayward, R.S. (1979) Mol. Gen. Genet. Newman, A.J., 169, 195-204. Fiil, N.P., Bendiak, D., Collins, J. and Friesen, J.D. (1979) Mol. Gen. Genet. 173, 39-50. Brot, N., Marcel, E. and Weissbach, H. (1973) J. Biol. R., Yamasaki, Chem. 248, 6952-6956. Kung, H.-F., Spears, C. and Weissbach, H. (1975) J. Biol. Chem. 250, 1556-1562. Miller, J.H. (1972) Experiments in Molecular Genetics (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). Bonnet-, W.M. and Laskey, R.A. (1974) Eur. J. Biochem. 46, 83-88, Chu, F., Caldwell, P., Weissbach, H. and Brot, N. (1977r Proc. Natl. Acad. Sci. U.S.A. 74, 5387-5391. Chu, F., Caldwell, P., Samuels, M., Weissbach, H. and Brot, N. (1977) Biochem. Biophys. Res. Comnun. 76, 593-601. Post, L.E., Strycharz, G.D., Nomura, M., Lewis, H. and Dennis, P.P. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 1697-1701. Fukuda, R., Taketo, M. and Ishikame, A. (1978) J. Biol. Chem. 253, 4501-4504. Zarucki-Schulz, T., Jerez, C., Goldberg, G., Kung, H-F., Huang, K-H., Brot, N. and Weissbach, H. (1979) Proc. Natl. Acad. Sci. U.S.A. (in press).

1461