Coordinate regulation of the individual ribosomal RNA operons in Escherichiacoli

Vol.

68,

BIOCHEMICAL

No. 3, 1976

COORDINATE

A.

Department University

SUMMARY: timesof tions of equally.

When Escherichia 40 minutes or relaxed control

There RNA in

are,

(r-RNA) the

undergoes by

F14

of

in

coli (3)

an

The

with

(7)

growth

scription

rRNA

can

in

Copyright All rights

5s

rRNA

Hall

61801

steady state up conditions, rRNA cistrons

of

genes

of

two

23s

of

two

of

been

position

organized a single

these

operons

of

the

the

5s

gene

presented

have

dictates

mapped --E.

genes

appears

to

factor

determined have

general

each

coli

electron

F prime

rRNA

that

been

by

the

operons

which

between

DNA of

for

6-7

molecule

identified to

ribosomal

in

differ

rRNA

additional

has

are

operons

and

An

copies

which

16~

with doubling or under condiare transcribed

6-7

as

sequences of

these during used

individual relation

in

Four

by Jarry

in

equivalence

(4).

whether

oligonucleotides transcripts

transcribed

Map

We have

the

are

(6).

rates,

reported of

Burrill Illinois

been

lo-

location

rRNA

“transcrip-

operon.

and

as

and

location

8).

independent

strain.

These

evidence

(8,

examined

rRNA

the

and

We have

A-

2).

location

analysis

low

131

approximately

mapping

cistrons

be

COLI

Kaplan

Urbana,

(1,

of

(5).

consistent

a rel

k.

heteroduplex

other

high

estimates,

analysis

recently

Samuel

coli is growing in hours, durinq shift of RNA.synthesis, all

cleavage

transductional

ton”

5s

most

6 strains

is

cated

20

maturational

and

INDIVIDUAL

ESCHERICHIA

and

of Microbiology, of Illinois,

16~-23~-5~

microscope

by

by

transductional

K12

Morgan

IN

COMMUNICATIONS

19,1975

genes

order

RESEARCH

OF THE

RNA OPERONS

Edward

December

BIOPHYSICAL

REGULATION

RIBOSOMAL

Received

AND

operons shift

the and

sequence Rosset

operons. to

0 I976 by Academic Press, Inc. of reproduction in any form reserved.

be of

the

transcribed

and

under

(10)

other used individual 969

coordinately relaxed

heterogeneity

The

each

principle

transcription

up,

are

in

relative and to

conditions within

order

to

those

determine rRNA

in

E.

&

distinguish

molarity to

at

of that

whether operons.

Kl2 tran-

fractional

occur there Published

in

al? is

an data

BIOCHEMICAL

Vol. 68, No. 3, 1976

(10)

indicate

conditions ine ential

that where

extreme

all

cistrons

growth

is

physiological

transcription

are optimum

states of

AND BIOPHYSICAL

individual

expressed or

in

near order

approximately optimum. to

rRNA

RESEARCH COMMUNICATIONS

We have

determine

operons

equally

whether

under

sought or

to not

examprefer-

exists.

METHODS All strains were grown at 37O C with aeration. E. coli K12 strain W3110 was grown on GRCLP salts (249 Tris-HCl, 6g KCl, 0.79 Na,SO,, 0.29 MgCl,, O.lg Na,HPO, per liter, pH 7.0) supplemented with carbon and nitrogen sources chosen to obtain the desired growth rates. Cells grown in GRCLP supplemented with 0.1% Casamino acids, 50 ug/ml tryptophan, 0.4% qlucose, and 20 mM NH.Cl i n GRCLP supplemented had a doubling time of 40 minutes. Ceils-grown witfi RNA was 0.4% glycerol and 0.2% histidine had a doubling t ime of 20 hours. purified from exponential cultures grown for at east two doublings in the presence of [32P]-orthophosphate. Cells labeled during shift up were initial y growing in GRCLP media supplemented with 0.4% glycerol, 0.2% histidine and shifted up by the simultaneous addition of [32P]-orthophosphate and Casam no acids to O.l%, tryptophan to.50 ug/ml,glucose to 0.4% and NH,Cl to 20 mM. A lag of approximately 63 minutes is observed before growth characteristic of this media begins. During this interval the rate of RNA accumulation increases in an approximately exponential fashion until the rate characteristic of the new growth rate is achieved at nearly 60 minutes following shift up (unpublished data). In [32P] labeling experiments RNA synthesis was terminated 40 minutes after shift up by addition of rifampicin to 50 yg/ml and the cells harvested by centrifugation. Relaxed rRNA was prepared from E. coli K12 strain LMUR (rel A-, RNase met-, ura-) growing in the Tow phosphate media of Maalde and Hana1-3 lys-, walt (11) supplemented with 0.4% glucose, 30 ug/ml uracil, and 50 ug/ml methionine and lysine. Starvation for methionine was induced following a one minute centrifugal collection, followed by a one minute centrifugal wash in icecold media without methionine, and resuspension in pre-warmed media without methionine. RNA accumulation resumes within 5 minutes and continues an additional 55 minutes with a doubling of the RNA content with little increase in the optical density or protein content of the culture (unpublished data). [32P]-orthophosphate was added 10 minutes after starvation for methionine was Labeling was terminated by pouring the culture over 0.4 volumes of induced. frozen , crushed M9 salts containing 0.4% glucose, 30 ug/ml uracil, and 50 ug/ml lysine, centrifuged one minute and resuspended in an equal volume of Cells were aerated an additional pre-warmed Mg salts media with methionine. 60 minutes and harvested by centrifugation. Growth resumes approximately 25 55 rRNA was isolated from 50s minutes after relieving methionine starvation. ribosomal subunits and was free of detectable contaminating RNA as determined by acrylamide gel electrophoresis. [32P] -5s rRNA was mixed with [3H]-uracil labeled 5s rRNA purified from strain LMUR grown on 20 ml of Mg media with 0.4% glucose, 15 pg/ml unlabeled uracil plus 2 MC[3H]-uracil, 50 pg/ml methionine and lysine and 1% Casamino acids (doubling time 40 minutes). The RNA was digested with RNase Tl and fingerprinted as described (12). Details of purification are similar to those which will be published elsewhere (Morgan and Kaplan, in preparation).

RESULTS The

relative

yield

of

individual

oligonucleotides

970

derived

from

5s

rRNA

BIOCHEMICAL

Vol. 68, No. 3, 1976

produced

under

presented

in

Table

use

of

the

relative

The comparing

the

synthesized

oligonucleotide

various

AND BIOPHYSICAL

physiological

RESEARCH COMMUNICATIONS

conditions

described

in

METHODS

are

1. a dual

label

technique

molarities

of

minimizes individual

the

possibility

oligonucleotides

Molarities of RNase Tl derived oligonucleotides of 5s under different conditions-relative

Molari in --E.

ty coli

20 hr ling

doubtime

.86

CG

5

UG

4

1.0

CCG;’

3.5

1.0

CAG

0.5

UAG

4

AUG”

.a2

1.0

.93 .81

1.1

1.1

1.6 .15

UCUG

.Vl

.74

1.0

error

in

produced

rRNA to

doubtime

1.0

.93

CCUG

40’ ling

1.2

CCAGA

of

from

a standard

shift up 1.0 1.1 .Vl 1.1 -99 1.2 .a7

RNA

relaxed RNA .vo 1.1 .Vl

.95 1.2 1.2

.92

1.3

1.3

1.2

1.2

1.4

1.1

.89

AAACG

1.1

1.2

AACUG

1.1

1.1

1.3

1.4

AACUCAG

1.1

1.0

1.3

1.3

ACCCCUAG

1.0

1.1

1.0

1.1

UCCCACCUG

1.2

1.3

1.3

1.2

UCUCCCCAUG

0.8-0.9

.80

.87

1.1

UCUCCUCAUG

0.1-0.2

.a5

.74

1.3

.Vl

.97 1.3

Table I. The molarities of 32P labeled oligonucleotides derived from 5s rRNA produced under various conditions relative to a f3H]-uracil labeled oligonucleotide from a 5s rRNA standard is compared to determine equivalancy of The 32P/3H ratios of individual oligoexpression of individual rRNA operons. nucleotides were multiplied by a correction for the pyrimidine/phosphate ratio (uracil labels uracil and cytosine equally) and divided by an average of similarly corrected values for CCG, AUG and CCAG. The approximate equal synthesis of rRNA from all cistrons under all conditions is indicated by values near 1.0 for oligonucleotides present in all, a few, or one 5s rRNA cistrons. Molarities of oligonucleotides present in E. -coli are those determined by Jarry & Rosset (1).

971

Vol. 68, No. 3, 1976

5s

rRNA

BIOCHEMICAL

labeled

general

under

consistent

find

that

within

with

may

other

the

results

be

due

to

in

relative

to due

to

preparation

the

in relative

other

rRNA

which

remains

variation

in

to

the

constant,

the

and

variable

CCAUG.

of

In

any

our

hands

that

the

of

case,

the

of

We

cleavage

oligonucleotide

expression

in

conditions.

enzyme

indicating

levels

(I). in

digestion

non-ideal

is

Rosset

amounts

the

contain

of

oligonucleotides

Jarry

on

due

molarity

spots

any

5s

in

depending

production

of

by

produced

and

spurious

present

fluctuations

is

RESEARCH COMMUNICATIONS

Recovery presented

CCAUG

RNA

sequences

not

conditions.

oligonucleotide

a given

This

various

AND BIOPHYSICAL

despite

32P/3H

ratio

fluctuation

individual

is

rRNA

cis-

trons. It into

was

necessary

ribosomes

cedure

in

used

the

chase,

into

new

rRNA

produced

release

5s

rRNA

the

uptake of

rRNA

5s rRNA of

and

since

is

synthesis

of

5s

rRNA,

incorporated

are

unable

in

the

RNA

incorporated rRNA

into

detect

isolated,

into

new

has

occurred

incorporation of

ribosomes

after

any

the

significant

conclude

after

the

from

after

proportion

we

RNA

pro-

cells

with

into to

The the

a substantial

we

synthesis label

5s

conditions

purity.

[32Pl

since is

relaxed

necessary

unpackaged

conditions

(13)

the

under

significant

However,

relaxed

produced of

pre-existing

possible.

equal

5S

obtain

preferential

an

relaxed

that

release

all

of

cistrons

under

conditions. We have

RNA

in

p.

detected

pressed

with

Jarry

and

Rosset

equally

in

contains

vidual

-P.

c.

two

the

be

maintained

e5S

are

not

at sequences

genes at

heterogeneous

i.

Both

mirabilis

5s

the

relevant

(1).

heterogeneous mirabilis

of

sequences

well

also

to

These

agree

by

some

containing

data).

data

mined

also

mirabilis

published

pears

is

significant

starvation,

Our

turnover

of

if

to

starvation

indication

chase

eliminate

under

of

even

order

should but

to

to

different

each

sequences

t-RNA present

genes in

heterogeneity E. high and other growth

972

on

p.

5s

rRNA

and

low

growth

map

genes

rates

to

of g.

coli

(unpublished

E.

coli

5s

Fl4

(un-

factor 5s

rRNA.

positions appear

rates.

stoichiometry and

F’

mirabilis

and

coli

of

deterto

be

p.

mirabilis

synthesis

of

5s

rRNA data).

genes

ex-

indiap-

Vol. 68, No. 3,1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS DISCUSSION

Several tence

of

alternative

multiple

scriptional of

from

individual

to

rRNA,

give

which

rRNA

rate;

tion

to

least

it

is

found

to

be

ceT1

growth

dictates

bably of

all

that

fractional

tribution

and

cation at the

rRNA

operons

all

the

reduces

different

preferential

conditions

changes

ponential

increase

synthesis

are

accumulation

tein

messenger

least

some

RNA.

coordinate

control A-

has

not

been

with

defect. clarified,

proteins Coordinate of

of

are RNA

control

protein Although this

or

synthesis the data

and

mechanism indicates

973

the

by

rRNA of that

of

in

to

occurs

at

rate.

No

which

These or

are

operons

also

a total

the

regulation

deproas

at

coordinately occurs is

of

exof

ribosomal

components

regulation

an

kinetics

dfstinguish

accumulation

repli-

operons

growth

of

dis-

rRNA

repression

individual

pro-

molarities

regulation

difficult

for

and

origin

of

translation

polymerase of

most

conditions

of

been

mandatory.

proposed

occurs.

release

possibilities and

up,

have

Symmetrical

dosage

RNA accumulation

a gradual

is

dictated

shift

are

necessary

relative

Coordinate

transcription

These

ribosomal (IS).

Rel

rate

autocatalytic

controlled

the

the

during

is

In

operon

tRNAs

for

the

clear.

rRNA

which

as

exist

RNA products yet

constant.

gene

of

occurs

in

relative rates.

rRNA

the

operon

here,

around

replication rates

consistent

and

in

of

nearly

operons

may operons,

different

this

studied

regula-

the

transcription

not

non-preferential

remains rRNA

transcription

repression

is

is of

one of

in

contain

which

two

least

transcription

of

chromosomal

widely

at

that

tran-

operons

may

exis-

a high

Multiple

6),

a way

the

off

different

regions

and

rRNA,

oligonucleotides clustering

the

“spacer”

transcription

under

probably

different

that

somehow shutting

(4,

in

(14)

with

for

ribosomes.

regulation

explain

provide

by

sequences

that

to

involved

3)

non-rRNA

co-transcribed

We feel

be

order to

regulated

non-homologous

and

may

heterogeneous

interesting

sometimes

1)

non-coordinately;

cellular

in

coli:

accumulation

appreciable

respect

--E.

operons

functionally

in

exist

in

multiple rRNA

involved

this

operons

operons

contain

at

2)

with

rise

somehow

possibilities

when

altered

rRNA

by

accumulation of

rRNA

is

un

BIOCHEMICAL

Vol. 68, No. 3, 1976

likely

to

be

complicated

by

AND BIOPHYSICAL

non-coordinate

RESEARCH COMMUNICATIONS

regulation

of

individual

rRNA

operons. Acknowledgment This ported by acknowledge

research was supported by N.I.H. N.I.H. pre-doctoral training grant C.R. Woese and E. Reichmann for

grant HD-03521. E.M. was GM 00510. We would like use of space and equipment.

supto

References 1.

Jarry,

2.

Pace,

3.

Doolittle,

4.

Ginsburg,

5.

Jarry,

B.,

6.

Deonier, Biol.,

89,

7. 8. 9.

B., B.,

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and

Pace,

W.F., D., and

N.R.

N.R.

(1970).

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R.S.,

M., Genet.,

Lee,

105,

142-149.

Nature, J.

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Molec. H.J.,

113, 43-50.

Genet.,

Bact.,

(1975).

(1973).

E.,

Gen.

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Birnbaum, 119,

Chem.,

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and

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126,

Genet.,

Davidson,

M.,

250,

N.

29-35.

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R.R.

(1975).

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Molec.

L.S.,

(1970).

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and

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11.

Maalde,

0.

and

Hanawalt,

and

Kaplan,

S.,

and

Pfister,

(1972).

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377-380.

Jarry,

F.

L.,

125-129.

458-462.

L.

Sanger,

J.

J.A.

Rosset,

10.

12.

(1971).

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Steitz,

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257,

Gorelic,

(1971).

R.

and and

R.C.,

Jaskunas, Nature, Unger, Gen.

and

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Browlee,

Genet., Molec.

(1971). P.

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G.G.

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381. 13.

Nakada,

14.

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15.

D.,

Anderson,

I.A.C.,

and

Mayasanik,

B.

9, 472-488.

Jaskunas,

E., M.

Dahlberg, In Press. S.R.,

J.E.,

Burgess,

L.,

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Jaskunas,

L.,

and

J.

(1964). S.R.,

Dennis,

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P.P.,

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