Resistance of bacterial protein synthesis to double-stranded RNA

Vol.60,No.4,1974

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNKATIONS

RESISTANCE OF BACTERIAL PROTEIN SYNTHESIS TO DOUBLE-STRANDED RNA Gilbert

Jay,

William

R. Abrams

and Raymond Kaempfer

The Biological Laboratories Harvard University Cambridge, Massachusetts 02138 Received

September

3,1974

ABSTRACT Double-stranded RNA fails to inhibit the formation of translation initiation complexes on R17 bacteriophage RNA, overall synthesis of R17 proteins, or the ability of bacterial initiation factor IF-3 to prevent the association of 30s and 50s ribosomal subunits into single ribosomes. Yet, IF-3 can form complexes with double-stranded RNA. However, IF-3 binds to doublestranded RNA with lower apparent affinity than to either Rl7 RNA or 305 ribosomal subunits; this may explain the resistance of bacterial protein synthesis to double-stranded RNA. Double-stranded initiation very

of mannnalian

tion

(3,5).

factor

recently important

RNA is

to know if

We report required

for

formation

of ability

(11,lZ)

Abbreviation:

Copyright All rights

(B), here

tightly

that

has not bacterial

initiation

complexes

are

not

affected

initiation

association

RNA. 1357

RNA have

sensitive

protein

been detected

Loss of inhibition. been found

RNA (6),

it

to inhibition

directed

by f2 phage

in another

study

(4).

factor

IF-3,

the

(9,10),

binds

to dsRNA,

RNA, overall

synthesis

factor

of protein,

of 30s and 50s ribosomal

by dsRNA. Double-stranded

dsRNA, double-stranded

0 I974 by Academic Press, Inc. of reproduction in any form reserved.

of

RNA to ribosomes

the

of

or phage messenger

on R17 phage

to prevent

RNA (5).

is

complete

of an initia-

establishment

synthesis

lysates,

to give

addition

double-stranded

synthesis

inhibition

although

the

(6,7)

to one report

of messenger

IF-3

of

protein

attachment

of

with

of ribosomal

such

by the

of

reticulocyte

to double-stranded

regions

bacterial

according

inhibited

and the

forms

inhibitor

RNA are sufficient

is concomitant

RNase III-sensitive

in precursor

In rabbit

can be relieved

to bind

activity

by dsRNA. While

units

able

is a powerful

(l-5).

of double-stranded

is

factor

Because

synthesis

The inhibition

that

initiation

is

protein

low concentrations

inhibition

to RNase III,

RNA, sensitive

RNA appears

sub-

to possess

Vol. 60, No. 4,1974

multiple

BIOCHEMICAL

binding

sites

for IF-3,

than to R17 RNA. In particular, somal subunits protein

AND BIOPHYSICAL

but binding

of IF-3 to these sites

IF-3 has a higher apparent

than for dsRNA. This may explain

synthesis

RESEARCH COMMUNICATIONS

to inhibition

affinity

the resistance

by double-stranded

is weaker for 30s ribo-

of bacterial

RNA.

RESULTSAND DISCUSSION Initiation

Complex Formation.

As shown in Fig.

1A, the entry of 32P-labeled

R17 phage RNA into

70s initiation

ation

The amount of IF-3 added in this experiment

factor

IF-3.

that a decrease in IF-3 resulted

complexes is absolutely

in decreased formation

Thus, IF-3 is not in excess in the reaction Addition

of increasing

concentrations

(5) does not lead to a detectable II&&).

complex formation

the same preparation synthesis Ability

perceptibly

complexes.

of dsRNA from Penicillium

at 0.1 rig/ml , and completely

of IF-3 to Keep Ribosomal Subunits

chrysogenum

70s complexes (Fig.

1 rig/ml to 1 pg/ml,

In reticulocyte

of p. chrysogenum dsRNA inhibits

FRACTION

was chosen so

mixture.

of dsRNA tested,

is not inhibited.

upon initi-

of initiation

decrease in 32P entering

In the range of concentrations

tiation

dependent

lysates,

initiation

ini-

by contrast,

of protein

at 0.5 rig/ml (5).

Apart.

When "C-labeled

505

NUMBER

complex formation. 1 M-NH&l-washed FIG. 1. Effect of dsRNA on initiation riblosomes (1.25 A260 or 33 pmol), 32P-labeled Rl7amJS2RNA (0.3 A260), fMet-q (0.11 A260 or 180 pmol), 3 pg IF-2 and 1.3 vg IF-3 were incubated in the presence of the indicated amounts of $6 dsRNA (13) for 12 min at 37'C (151, and analyzed by sedimentation through sucrose gradients (15). Arrow: position of free RI.7 RNA.

1358

BIOCHEMICALAND BIOPHYSICALRESEARCHCOMMUNICATIONS

Vol.60,No.4,1974

ribosomal units,

subunits

(Fig.

they associate

to form single

however, association iting

is inhibited

affect

only partially.

(Fig.

this

activity

dsRNA by itself

of IF-3 (Fig.

does not influence

in an assay specific Rl7 RNA-Birected hibitory

effect

protein

S30 extract,

IF-3 (15),

2IJ). In a control the association

we fail

Protein

Synthesis.

synthesis

containing

of ribosomal

to detect

(Fig.

directed

subunits

mixture 25),

a lim-

fails

an effect

effectiveness

initiation

of protein

equal to that

As seen in Fig.

concentrations

of dsRNA, or

might be affected,

synthesis

not affected,

in a reticulocyte

we measEscherichia

including

lysate

of p. chrysogenum dsRNA (5; W.A. and R.K.,

3, the rate and extent

even though at the highest

: hu )B h

this

with an unpub-

of amino acid incorporation

concentration

Thus,

that an in-

by R17 RNA in a preincubated

synthesis

to

of dsRNA.

To examine the possibility

components for protein

is

it is seen that

of 30s and 50s subunits.

complex formation

all

experiment,

in the presence of 40 to 200 pg/ml of dsRNA from phage $6 (13);

RNA inhibits

lished).

In this

the association

30s sub-

In the presence of IF-3,

of dsRNA in the reaction

for IF-3,

than initiation

29.

2c)(11,12).

might be seen only at much higher

that a step other ured overall

Inclusion

with an excess of unlabeled

ribosomes (Fig.

amount of IF-3 was added, so that

prevented

coli

2A) are incubated

of dsRNA used there

are is

+lF-3 + dsRNA

50s Ill

OJ

IO

20

30

10

20

30 D FRACTION

20 NUME

FIG. 2. Effect of dsRNA on the inhibition of ribosomal subunit association by IF-3. "C-labeled 50s ribosomal subunits were incubated with SlOOB (ll), a supernatant highly enriched for 305 subunits, as described (15). SlOOBwas omitted in (A). IF-3 (3 pg) and $6 daRNA (5 ng; 50 rig/ml) were present as indicated. Analysis by centrifugation (15).

1359

Vol.60,No.4,

1974

approximately

one molecule

mixture.

This

needed

to inhibit

slightly caused ated ation

BIOCHEMICAL

during

is less

Even

seen

an effect

thesis.

This

if

is

not

protein

filters

a saturation

case.

between (Fig. binding

It

the

S30 extract

to dsRNA.

still

rate

free

Even though

curve

of

0

3

may be incub-

reaction

mixture,

degrad-

too

that

small

the

dsRNA is

to cause

one should

therefore,

that

it

is

inhibit

capable

inhib-

still

have

protein

syn-

the

failure

by their (5).

9

bacterial

of binding

retention The figure

66 dsRNA as a function

6 Time

degradation dsRNA is

however,

retained

32P-7abeled

that

when the

dsRNA does not

tested,

dsRNA is not

than

due to dsRNA degradation.

and dsRNA are detected

44);

this

of R77 RNA-dependent

is not

reaction

46 dsRNA is only

be argued

may be concluded,

at any concentration IF-3

to the

in the

greater

Much of for

to occur,

IF-3

times

fragments

initial

the

10'

a).

addition

It could

were

of

Labeled

action,

to TCA-precipitable

IF-3

synthesis

Complexes lose

of

its b).

dsRNA on the

of dsRNA to inhibit Binding

(curve

such degradation of

(curve

nuclease

RESEARCH COMMUNICATIONS

molecule

completely.

incubation

RNase before

degraded

ition.

the

pronounced

progressively

lysate

strand-specific

pancreatic

every

of dsRNA is more than

a reticulocyte

by single with

of dsRNA for

concentration

degraded

AND BIOPHYSICAL

to

IF-3.

on nitrocelluillustrates of increasing

12

(min)

FIG. 3. Effect of dsRNA on R17 RNA translation. ["4C]amino acid incorporation in a preincubated S30 (containing 100 pg ribosomes), with (0) or without(o) R17 RNA (0.4 Aca0) was as described (15). Before incubation, $6 dsRNA was f$ded: P 40 j.Ig/ml (A), 100 pg/ml (A), and 200 pg/ml (v). Curve a: TCA-precipitable in a parallel reaction mixture containing 100 pg 32P-labeled $6 d&WA and lacking [ 'C]amino acids. Curve b: as curve 5, except that the 32P-labeled 46 dsRNAcontaining mixture was preincubated for 5 min at 37'C with 50 vg/ml RNase A before addition of 530.

1360

Vol.60,No.4,1974

amounts ated

of

IF-3.

by the

addition trol

BIOCHEMICAL

That

finding

of

that

IF-3,

(data

not

Nearly

IF-3

ionic

labeled

R17 RNA (Fig.

.than with

IF-3

(14). the

obtained

is

the

needed

is

to reach

2% of

is conducted

with

from

4A,

half-maximal

complex

RNA is

the

untreated

completely

during

formation,

Fig.

the

indic-

RNase before

dsRNA can enter

complex

evident

in

pancreatic

is within

input

initiation

when binding It

with

R17 RNA is degraded of

for

44).

species

respectively The data

amount

retained

by contrast,

Affinities

3 equimolar

daltons,

radioactivity

treated

regions

complexes

the conthis

with

a proportion

an equal

amount

however,

that

formation

comof

"P-

3 to 4

with

R17 RNA

$6 dsRNA.

Relative of

to double-stranded

when dsRNA is

conditions

to

less

is

three-quarters

parable

times

that

the

shown);

treatment. under

binding

AND BIOW-IYSICAL RESEARCH COMMUNICATIONS

of

of dsRNA and R17 RNA for with

molecular

(13); Fig.

the

molecular

44 show that

of R17 RNA retained

A

weights

at any

on filters

of

weight IF-3

IF-3.

The $6 dsRNA consists

2.2x106,

2.8x10',

and 4.5~10~

of R17 RNA is

1.0~10~

concentration

up to saturation,

exceeds

that

of

$6 dsRNA.

daltons

Considering

RI7 RNA

0 IF-3

added (pg)

20

40 60 80 RNA added (pg)

100

4. Relative affinities of dsRNA and R17 RNA for IF-3. (A): freshly prepared, 32P-labeled $6 dsRlU (1.44 pg; 27,062 cpm) or R17 RNA (1.47 pg; 13,818 CPSI) were incubated for 12 min at 37°C with increasing amounts of purified IF-3 (15), in 0.05 M-Tris (pH 7.8), 0.05 M-NH&l, 0.005 M-Mg(OAc)z, 0.001 M-GTP and 0.016 M-2-mercaptoethanol. Samples were diluted with cold buffer A (0.05 M-Tris (pH 7.8), 0.05 M-N&&l, 0.005 M-Mg(OAc)a), and passed through Millipore HA 0.45 nm filters, washing extensively with buffer A. Filters were dried and analyzed for radioactivity. (B): 2 ug IF-3 (90 pmol), 12.4 ug 32P-labeled RI.7 RNA (12.4 pmol), and increasing amounts of 96 dsRNA or R17 RNA were incubated and analyzed as for Fig. 4&. FIG.

1361

Vol. 60, No. 4, 1974

the

fact

than

that

that

the

of

average

IF-3

er apparent

affinity

In the limiting

RNA or $6 dsRNA. cording

for

of

IF-3 it

Taking

into

R17 RNA, however,

account the

and $6 dsRNA compete If

we assume

binding

site

competes

is

as well

ent

affinity

for

IF-3,

for each

data

of

equally

that filled

for

32P-labeled

weight

that

(Fig.

weaker

than

4A), the

then

great-

with

on a molar

a

unlabeled

R17 and ac-

less

between

effect-

$6 dsRNA and

basis

on filters

once

a molecule.of

R17 RNA (Fig.

4E),

yet

subunits

more mol-

effectively

that

implies

3%

of

fact

site(s)

at

R17 RNA

IF-3.

the

IF-3

amounts

difference

with

of

that

exhibits

+6 dsRNA competes

is retained

as a molecule

IF-3

R17 RNA competes

an RNA molecule IF-3,

greater

Fig..4!

10 times

shows that

increasing

48 indicate for

of

of about

basis

molecular

well

curves

times

R17 RNA was incubated

of

unlabeled

Fig.

three

$6 dsRNA.

on a weight

the

the

result

presence

that

while

from

This

45

in the seen

to expectation,

ively.

Fig.

RESEARCH COMMUNICATIONS

of $6 dsRNA is

on filters

R17 RNA than of

It

retention

of 46 dsRNA.

experiment

amount

weight

can be calculated

causes

of R17 RNA than

AND BIOPHYSICAL

molecular

R17 RNA, it

half-saturation ecules

BIOCHEMICAL

that

exhibits

dsRNA possesses

a single 46 dsRNA a lower

multiple

appar-

binding

on R17 RNA.

added (pml)

subunits for IF-3. 2 pg IF-3 (9Opmol),2.5 vg 32P-labeled 46 dsRNA (0.8 pmol), and increasing amounts of 30s ribosomal subunits (1 M-NH&l-washed)(15) were incubated and analyzed as for Fig. 44. FIG.

5.

Relative

affinities

of dsRNA and 30s ribosomal

1362

BIOCHEMICALAND BIOPHYSICALRESEARCHCOMMUNICATIONS

Vol.60,No.4,1974

Relative

Affinities

experiment

of Fig.

sufficient

to retain

30s ribosomal

5, 32P-labeled

decreasing

IF-3 prefers

greater

30s subunits

We conclude that initiation,

formation resistance affinity

is related

affinity

protein

synthesis

that

to dsRNA. During

dsRNA is capable of binding for neither

of R17 RNA are affected that

is resistant

of R17 RNA to preformed fMet-tRNAm30S

show that while

to the fact

subunit,

of IF-3 for 30s subunits

for dsRNA. We have found similarly

bacterial

over dsRNA. By contrast,

initiation

to IF-3, complex

by dsRNA. Most likely, IF-3 possesses greater

for R17 RNA than for dsRNA and, in particular,

the 30s ribosomal factor

Since no more than one IF-3 molecule

is not accompanied by inhibition,

nor translation

is observed at low

over R17 RNA (G.J. and R.K., in preparation).

IF-3 mediates the binding

binding

with an amount of IF-3

competition

the apparent

than that

bacterial

complexes (17). Our results this

(16),

In the

In the presence of increasing

Effective

over IF-3.

can be bound per 30s subunit

IF-3.

amounts of 32P are bound. Thus, 30s subunits

dsRNA for IF-3.

of added 30s subunits

must be significantly

$6 dsRNA was incubated

50% of the RNA on filters.

subunits,

compete with labeled ratios

of dsRNA and 30s Ribosomal Subunits for

prefers

its

the apparent

natural

site,

the mammalian initiation

(5) binds dsRNA two orders of magnitude more tightly

than R17 RNA (W.A.

and R.K., unpublished).

We thank Dr. Anne Vidaver for phage +6 and its host strain, and Dr. G. D. Novelli for tRNAFt. Supported by Grant GM-19333 from the US Public Health Service.

REFERENCES Hunt, T. & Ehrenfeld, E. (1971) Nature New Biol. 230, 91-94. :: Ehrenfeld, E. & Hunt, T. (1971) Proc. Nat. Acad. sci. USA 68, 1075-1078. 3. Hunter, A., Hunt, T., Jackson, R. & Robertson, H. (1972) insynthese, Struktur und Funktion des Haemoglobins, eds. Martin, H. & Nowicki, L. (Lehmanns, Munich), 133-145. Robertson, H. & Mathews, M. (1973) Proc. Nat. Acad. Sci. USA 70 225-229 2: Kaempfer, R. & Kaufman, J. (1973) Proc. Nat. Acad. Sci. USA 7K'1222-1226. 6. Dunn, J. & Studier, F. (1973) Proc. Nat. Acad. Sci. USA70, z96-3300. 7. Nikolaev, N., Silengo, L. & Schlessinger, D. (1973) Proc. Nat. Acad. Sci. USA, 7&, 3361-3365.

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BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Chao, J., Chao, L. & Speyer, J. (1971) Biochem. Biophys. Res. Comnun. 45, 1096-1102. Iwasaki, K., Sabol, S., Wahba, A. & Ochoa, S. (1969) Arch. Biochem. Biophys. 125, 542-547. Revel, M., Herzberg, M. & Greenshpan, H. (1969) Cold Spring Harbor Symp. Quant. Biol. 34, 261-275. Kaempfer, R. (1971). Proc. Nat. Acad. Sci. USA 68, 2458-2462. Kaempfer, R. (1972). J. Mol. Biol. 71, 583-598. A. & Van Etten, J. (1973) J. Mol. Biol. 78-, 617-625. Semancik, J., Vidaver, Gesteland, R. & Boedtker, H. (1964) J. Mol. Biol. S, 496-507. Jay, G. & Kaempfer, R. (1974) J. Mol. Biol. 82, 193-212. Sabol, S. & Ochoa, S. (1971) Nature New Biol. 234, 233-236. Jay, G. & Kaempfer, R. (1974) Proc. Nat. Acad.Sci. USA, in press.

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