An Escherichia coli K12 mutant carrying altered ribosomal protein (S10)

Vol. 75, No. 1, 1977

BIOCHEMICAL

AND BIOPHYSICA’. RESEARCH COMMUNICATIONS

AN ESCHERICHIACm Kl2 MUTANTCARRYING ALTERED RIBOSOMALPROTEIN(SlO)

Michihiko Kuwano, *Hatsumi Taniguchi, Hideya Endo and **Yoshinari Ohnishi

Mayumi Ono

Cancer Research Institute and *Department of Bacteriology, Faculty of Medicine, Kyushu University, Fukuoka 812 and **Department of Bacteriology, School of Medicine, Tokushima University, Tokushima 770, Japan.

Received

January

24,1977

SUMMARY : An Escherichia coli mutant (JE14373) carrying decreased stability of stable RNAspecies was found to have altered electrophoretic mobility of a 30s ribosomal protein (SlO). Recombinants covering str gene (76 min on E. M linkage map by Bachmann, Low and Taylor, 1976 (ref. 1)) obtained from a cross of CSH64x JE14373, restored normal SlO protein. The size analysis of RNAslabeled for 15 min with [3H]uridine showed 50 to 60 % decrease of 16s RNAin this mutant strain, but almost no decrease of 23s RNAat 10 or 40 min after addition of rifampicin. On the other hand, no change was observed in the stability of both rRNA pieces in its parental PA3092 strain even at 40 min after addition of rifampicin. Remarkable progress has been made in the elucidation interaction

within

of protein

- RNA

the Escherichia coli ribosome by biochemical study on

nuclease digests of the complexes formed between ri.bosomal RNA (rRNA) and proteins

(r-proteins)

(2, 3, 4).

To clarify

rRNA, genetic mutants of ribosome (see ref. tool.

interaction

of r-protein

5) could be utilized

On the other hand, stable RNAincluding

However, regulatory

mechanismsto stabilize

to exist in E. coli

(10).

as powerful

rRNA and transfer

to turn over slowly during steady state growth of llscherichia rRNA or transfer

with

coli

RNAis known (6, 7, 8, 9).

RNAwere

suggested

Two genes (srnA and -srnB) were responsible for pro-

moted turnover of stable RNA at 42'C when RNAsynthesis was stopped (ll), also that these genes seemnot or rRNA. turnover stability

to link to the structural

and

genes for r-proteins

Thus, we decided to isolate E. -coli mutants with increased rate of of stable RNA.

In this report,

we will

describe correlation

of r-RNA

with an altered 30s r-protein.

Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

156 ISSN

0006-291

X

Vol. 75, No. 1, 1977

BIOCHEMICAL

AND BIOPHYSICAL

MATERIALS

RESEARCH COMMUNICATIONS

AND METHODS

Bacterial strains Screening of mutants carrying altered turnover of RNA among about 1,200 temperature sensitive g. -coli K12 mutants will be described elsewhere (Kuwano, Ono, Endo, Hori, Hirota, Nakamura and Ohnishi, manuscript in preparation). The parental strain is PA3092 (F-, z, &, lacy, 9, his, a, strA, malA, x&, argH, supE), from which a mutant strain JE14373 was derived. ?&the cross experiment, a nalidixic acid resistant strain was isolated spontaneously from JE14373 strain and CSH64 (Hfr, G, with injection order from M, strA) (12) was used as donor strain. Assay for RNA degradation and analysis of RNA size by sucrose density gradient centrifugation Exponentially growing cells (2 to 4 x 10' cells/ml) in minimal medium were (27 Cijmmole, New England labeled for 2 or 15 min with 1 pCi/ml [3H] uridine Nuclear, Boston) at 3O"C, and then transferred to 37'C immediately after RNA synthesis was stopped by addition of 500 pg/ml rifampicin and 200 w//ml uridine. After addition of rifampicin, aliquots (0.2 ml) withdrawn from the cultures at the indicated times were added to 2 ml of ice-cold 10 % trichloroacetic acid. The radioactivities collected on glass fibre filter were counted by Beckman Liquid Scintillation Counter. The highest value of i3H]To prepare labeled RNA remaining on the filter was expressed as 100 W. radioactive RNA, the cells (30 ml culture) were labeled at 30°C for 15 min with 1 pCi/ml [3H]uridine and then RNA synthesis was stopped by rifampicin (500 pg/ml) to follow fate of size distribution of [3H]labeled RNA at 37'C. addition of rifam[3H]RNA extracted from the cells at 0, 10 and 40 min after picin was placed on a 10 to 30 % sucrose density gradient containing 0.1 M sodium acetate (pH 5.1)-l mM EDTA-0.01 % sodium dodecyl sarcosinate as described previously (13) and was spun for 4hr at 39000 rpm with a Spinco L-2 centrifuge. Preparation of r-proteins and gel electrophoresis Ribosomal proteins of 70s ribosome from 2 to 4g cells were extracted with acetic acid after the method of Waller and Harris (ref. 14). Some 400 to 500 pg r-proteins were applied to two-dimensional polyacrylamide gel electrophoresis developed by Kaltschmidt and Wittmann (ref. 15) and staining was done with coomassie brilliant blue. RESULTS The mutant

strain

isolated

by screening

guanidine

mutagenized

followed

the fate

JEl4373

which

of stability E. coli

of labeled

of pulse-labeled

K12 strain RNA after

of rifampicin.

Wild

for

50 to 55 % breakdown

2 min showed

Therefore thought

type

shows temperature

strain

to correspond

the pulse-labeled addition of .I314373

to the strain,

to stable

culture various

(Fig. Hfr

strain

la). strains

To locate were 157

was blocked

by addition at 3O'C

20 min at 37°C (Fig. steady

state

By contrast,

degraded

was

1, we

had been pulse-labeled

of RNA within

RNA fraction.

RNA of .I314373

As shown in Fig.

RNA synthesis

RNA during

growth,

RNA at 42°C from nitroso-

(PA3092).

PA3092 which

45 to 50 % of pulse-labeled

sensitive

of growth

la). was

some 70 % of

within

20 min after

rifampicin

a mutation

affecting

RNA stability

crossed

with

this

mutant.

BIOCHEMICAL

Vol. 75, No. 1, 1977

0

10

20

Time after

Fig.

It

1.

that

all

to that

of recombinants

covering

20

30 ( min

)

after

either

degraded

of bulk

degradation

However,

for

unstable

was shown in Fig.

in

within

colonies

curve

of RNA as com-

to be located

hand,

decay

However,

la.

temperature

while

the mutant

strain

close

strain

breakdown within

(Fig.

rifampicin

la and b),

RNA degraded

in the mutant

was observed

in wild

RNA (presumably

type

these

to e

and

gene

with

[3H]uridine

degraded

within

of 40 to 45 %

30 min

(Fig.

covering Thus,

addition.

lb).

under

some 15 to 20 % RNA among stable strain, strain

mRNA) degraded

158

curve

growth

20 % of the RNA in the recombinants

30 min after

all

sensitive

when RNA was labeled

of rifampicin,

15 min,

condition

the decay

10 to 15 % of RNA of PA3092

addition

Mal+

and the representative

strain,

gene was found

RNA was observed

labeling

fraction

only

among Nal-R

showed

to show still

On the other

data).

Even when labeled region

locus

found

sensitive

15 min at 3O"C,

of labeled

PA3092 str

were

temperature

30 min at 37'C

any,

10

(Str-S)

(CSH64 x JE14373)

of parental

recombinants

(unpublished

-str

12 recombinants

from a cross

parable

for

0

of ntamplcin

(A>

obtained

this

30

addition

Comparison of RNA decay curves. The cells of PA3092 (0), .I314373 (0) and a Str-S recombinant cultured to exponential phase at 30°C were labeled for 2 min (a) or for 15 min (b) with 1 )~Ci/ml [3H]uridine at 30°C and then the fate of labeled RNA was examined at 37'C after addition of rifampicin. The value of 100 % corresponds to the highest radio-activity in each experiment.

was found

Str-S

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

whereas and Str-S at similar

only

little,

recombinants. rate

in both

if

Vol. 75, No. 1, 1977

BIOCHEMICAL

0

10

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

20

10 Fraction

Fig.

parental

and the mutant

RNA stability not

were

extensive

varied

previously

(10). density

of PA3092

[3H]~A

obtained

la).

Almost

when RNA decay

type

of stable

of degradation from

for

showed

even at 40 min after

1).

of stable

identical

was analyzed

labeled

for

both

mutant

data

on the

at 30°C or 42°C

reached

the

RNA was examined

exponentially

of

growing

by cells

temperature,

the

and [3H]RNA was extracted

16s and 23s rRNA were

stable

(Fig.

to be affected

159

addition

to 80 to 100 % as described

The sucrose-density

was stopped

to show more

must be noticed

15 min at permissive

up to 37'C

(2 : 1) of 23s : 16s rRNA seemed not

it

30 min after

of ribosomal

addition.

RNA synthesis

was found

However

RNA within

After

was shifted

rifampicin that

RNA (Fig.

analysis. were

culture

this

the degradation

gradient

and .I314373

strain,

10 to 20 X, but never

Thus,

10 and 40 min after of

(see Fig.

the wild

degradation

rifampicin

temperature

strain

also

from

the extent

sucrose

20

shown).

Different

that

10

Sedimentation profiles of ribosomal RNA of PA3092 and JE14373. [3H] RNA's extracted from PA3092 treated with rifampicin for 0 min (a), 10 min (b) and 40 min (c) or from JEl4373 with rifampicin for 0 min (d), 10 min (e) and 40 min (f) were analysed by centrifugation on a sucrose density gradient containing sodium dodecyl sarcosinate. Two peaks from left to right indicate 23s and 16s rRNA, respectively.

2.

(data

20 number

gradient

at 0, patterns

in the parental

strain

Za, b and c) and the ratio during

incubation

with

BIOCHEMICAL

Vol. 75, No. 1, 1977

Fig.

3.

Separation of 70s ribosomal proteins by two-dimensional polyacrylamide gel electrophoresis (15). 70s r-proteins were derived from PA3092 (a), JE14373 (b) and a str+ recombinant (c), respectively. Arrows indicate 30s protein-ST

rifampicin.

In contrast

corresponding

to 16s appeared

addition while

of rifampicin little,

stability

if

any,

gel

ribosomal

2d,

at

the nomenclature its

mobility

-,E

the

in Fig. coli

to amino

str

region

may suggest as described

that

in this acid

str

the restoration region

previously

is (17,

the

16) was found This

from the SlO,

to

to clearly

change

of SlO protein.

of normal gene for

3a and b.

SlO according

strain.

extracted

two-

of.70S

shown in Fig.

r-protein (ref.

mutant

pattern

were

substitution

of 70s r-proteins

showed

strains

with

was done with

The electrophoretic

by Wittmann

may be ascribed

in correlation

of r-proteins

3a and b, an acidic

r-proteins

the differential

strain.

analysis

and JE14373

mobility

pattern

rRNA

as 40 min after

showing

of r-proteins

(15).

electrophoretic

electrophoretic covering

alteration

from PA3092

smaller

10 min as well

of 23s rFNA occurred,

electrophoresis

by arrow

in the mutant,

e and f) and 50 to 60 % of 16s rI7NA degraded,

degradation

a possible

proteins

As indicated

strain

cells,

to be unstable

of rRNA stability,

dimensional

which

the parent

of 165 and 23s rl7NA in the mutant

changes

alter

with

(Fig.

To investigate the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in the The

recombinants

as shown in Fig.

SlO mutation

in this

3c,

mutant

18).

DISCUSSION With

respect

to regulatory

mechanism

160

for

stabilizing

stable

RNA, a mecha-

Vol. 75, No. 1, 1977

nism for

coordinate

was suggested

balancing

previously

coli

mutant

over

or stability

having

elements. sive

BIOCHEMICAL

activity

(10,

altered

11,

the isolated

ribonucleoprotein

3'-proximal

pieces

suggested

that

neighboring the existence

16s RNA in effect the

this

structure

of 30s ribosomal

independence

bound

a biochemical

Since ing

the E. -coli

finding

On the other Sl is

weakly that hand,

shown to bind

to know if

a Sl mutant

is

which

is

a mutant

Proteins

Sl,

4) showed

interactiaon target

subunit

were

which

might

to 5' proximal

half

Sl protein

shows altered

stability

25),

21)

and demonof

indirect to maintain

genetics

of ribosome

analysed.

streptomycin

to 3' end of 16s rRNA (24,

(ref.

Thus the lability

a few mutants

of altered

of

Sommer and Traut

from

that

S14 and S29 and

the primary

Concerning

reverted

S9,

region

S7 is one of main proteins

to 16s rRNA (ZZ), S4 binds

this

to the environmental

subparticles. only

that

(ref.

S3-SlO.

be ascribed

rRNA stability,

from

dimer

shows exten-

on protein-RNA

Recently,

S7, if

which

to 3'-proximal

in the 30s ribosomal

might

SlO on protein

affecting

S4 protein

(20). pair

strain

analysis

tum-

protein

S7, S9, SlO,

proteins

of a cross-linked

mutant

of altered

mutation

(2) *

protein

bound

contained

five

to the RNA molecule

identified strated

of these

own surrounding

and Brimacombe

Further

of 16s rRNA.

S7 protein

cross-linking

fragment

a case that

of 16s rRNA.

remain

by Yuki

is

RNA

an Escherichia

RNA, one may infer

digestion

proteins

as transfer

that

known nuclease

and transfer

study

here This

by its

the only

in partial

Another

We reported of 165 rRNA.

I is

S13 and S19 among 30s ribosome of 16s rRNA (3).

19).

be limited

ribosomes

be involved

16s and 23s rRNA as well

stability

ribonuclease

against

enzyme might

of both

of rRNA might

Since

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

An altered

dependence

be correlated

to

with

of 16s rRNA fragment was isolated it

(23).

must be interest-

of 16s r-RNA.

ACKNOWLEDGEMENT We want to express our sincere with temperature sensitive E. -coli critical reading of this manuscript. Aid from the Ministry of Education,

for supplying us thanks to Dr. Y. Hirota K12 mutants, and thank Dr. Y. Ikehara for This work was supported by Grant-inScience and Culture, Japan.

161

Vol. 75, No. 1, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

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