Translational repression of f2 protein synthesis

Vol. 30, No. 3, 1968

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TRANSLATIONAL REPRESSION OF f2 PROTEINSYNTHESIS* Richard Ward, Mette Strand and RaymondC. Valentine Department of Biochemistry, University of California Berkeley, California 94720

ReceivedJanuary 13, 1968

Recently,

we have proposed a mechanismwhich may explain how bac-

teriophage f2 regulates its protein a&,

1967).

synthesis after

Sugiysma and Nakada (1967)

originally

system for MS2. The model is shown in Fig. 1. is that virus shell protein

(Ward -et

proposed a similar The essence of the model

subunits made early after

with each other to form an active viral

infection

infection

ccnnbine

repressor molecule which binds to the

RNAgenomeshutting off phage genes which are no longer needed in

the infection. repression"

We have called this kind of regulation since it occurs at the messenger level.

In the experiments described below translational studied in extracts as repressor.

using f2 RNAas polycistronic

The major findings were:

was active

as repressor.

teriophage QS was inactive

repression was

messenger and f2 capsid

1) Histidine

shut off by about six molecules of capsid. tion protein

"translational

2) Viral

genes were completely capsid but not matura-

3) Capsid from the unrelated bac-

as repressor, whereas capsid from the related

viruses MS2 and fr was fully a region of the RNAprotecting

active.

4) Capsid, as repressor, bound to

about 0.5% of the molecule from ribo-

nuclease digestion.

*Research supported by NIH grant ~108086 and NSF grant GB7036. 310

Vol. 30, No. 3, 1968

Fig. 1.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Proposed model for translational repression. Capsid (circles) binds to RNAgenane (thread) shutting off "histidine" genes. Translational repression may work closely with polarity in this system.

Results System. --The cell-free

Assay for Repression Using the Cell-free of E. coli and assay mixture for protein scribed by Capecchi (1%). The viral ticles

synthesis were similar

extract

to those de-

An RNase-less strain was used (Capecchi, 1966).

messengerRNAand capsid were extracted

fram purified

The RNAphages

according to the method of Sugiyama and Nskada (1967).

Q3, MS2, and fr were prepared by a similar procedure.

All

radioactive

acids were obtained from NewEngland Nuclear Corp. Virus protein tion was determined by calorimetric

and optical

f2 par-

amino

concentra-

density methods as was RNA

and cell protein. In our assay we take advantage of the fact that f2 messenger RNAdirects the incorporation

of radioactive

histidine

into all viral

induced proteins

except the coat which does not contain this smino acid (Capecchi, 1.966); sid synthesis was followed by measuring arginine incorporation In other words we follow genetic translation 'histidine-containing"

genes by radioactive

cap-

into protein.

of the coat gene versus the incorporation.

We have found

this to be a suitable assay although by this technique it is not possible to

311

Vol. 30, No. 3, 1968

distinguish

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the various histidine-containing

Strong Repression of Ristidine ment (Ward et al.,

lgf$'),

shut off histidine

incorporation

was less drasticslly

affected.

proteins one frcxn the other.

Genes with Capsid.--In

an earlier

experi-

we were able to show that capsid differentially while f2 directed

arginine incorporation

We wondered if it would be possible to completely shut off the histidine genes with capsid. drastic

inhibition

In certain of histidine

experiments we had earlier incorporation.

We felt

observed a more that the failure

to

obtain canplete repression in earlier

experiments might be due to partially ++ To or to improper Mg concentrations.

degraded capsid or RNApreparation insure faithful

translation

of the f2 messagewe used freshly

prepared viral

RRAand capsid, and carefully

checked the Mg* levels of the assay mixtures.

Under these conditions little

f2-directed

when capsid was added as repressor (Fig.

histidine 2).

incorporation

took place

A mechanismto account for this

very strong repression with capsid is discussed below.

I 0

Fig. 2.

2

I

I

I

4 6 8 pg Coat Protein

1 10

Shutting off the histidine genes with capsid. Each assay tube in th&s experiment contained 28.0 p&of RNA and either 2.8 x 16 cpm C1 -histidine or 1.4 x lo5 cpm C -arginine. Coat protein was freshly prepared. The tubes were incubated at 37O C for llminutes and precipitated with cold 54 trichloroacetic acid. The radioactive protein samples were collected by filtration on cellulose-nitrate filter pads and counted using a gas flow counter. 313

Vol. 30, No. 3, 1968

8lOCHEMlCAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

As shownin Fig. 2 very small amounts of f2 capsid were active. was of interest

to calculate

the number of capsid subunits per RNAmole-

cule which must be added to shut off the histidine

genes. Weused molecu-

lsx weight values of 1.1 x 106 for RNAand 15,800 for capsid. periment shown (Fig.

It

In the ex-

2) approximately 6 subunits of capsid per RNAwere

needed to ccanpletely shut off the histidine

genes. Wepropose that the

active repressor species is a capsid hexemer, although more information is needed on this point (see Sugiyama and Nskada, 1967). Capsid Not Maturation Protein is Repressor.--We next attempted to rule out the possibility

This possibility

repression. by intact

that maturation protein

(Steitz,

was involved in

1967)

existed since maturation protein was carried

virus as a minor capsid component (Roberts and Steitz,

Our "capsid" preparation the major shell protein. mutant particles (Table 1).

thus contained this protein

1967).

in addition to

Weprepared capsid from maturation deficient

(Sus-1) and found that it was fully

Since Steitz

has recently

(1967)

active

as repressor

shownthat mutant particles

of this type do not contain maturation protein we concluded that capsid itself

was the repressor species. Specific

Nature of the Repressor.--A number of experiments were carried

out to see if the interaction one.

of viral

capsid with viral

RNAwas a specific

Wewondered whether capsid from one species of phage would repress

RNAof another virus. coat proteins

Viruses were chosen which sre known to have different

(Weber and Konigsberg, 1967).

and RNAwere tested.

The results

Several canbinations of capsid

are shownin Table 2.

In summarizing the data of Table 2 we see that f2 and serologically related viruses (Scott,

1965) (MS2 end fr)

show strong cross-reactivity.

For example, shell fran phage MS2 reacts strongly with RNAfrom f2 and vice versa.

It was particularly

bey et al., activity

interesting

that the unrelated virus Q3 (Over-

1.966) showedno cross reaction - its coat showedno repressor

for the unrelated RNA. A similar type of species specificity 313

Vol. 30, No. 3, 1968

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table 1 Capsid Frcm Mutant Particles

(Sus 1) Missing The Maturation

Capsid

Ristidine Incorporated* CPM

w f2

0 0.5 1.2

1020 840 730 440 40 0

El 12:o sus 1”

Protein Was Active

0.71

68:

1.75 3.5 7.0

160 0

*Protocol as for Fig. 2, except each assay tube contained 37.6 pg of f2 RNA. m Sus 1 defective particles prepared and purified according to the procedure of L&dish -et al. (1965). -----m-

Table 2 Specificity

of Repressor

$ Inhibition

Source of -Protein (pg)

RNA bd f2 f2 f2 f2

MS-2

No inhibition

100 1CC

f2 MS-2 fr w MS-2 f2 f2 fr f2 f2

MS-2 Q9 fr fr TM? PolY u *

cl4 Ristidine Incorporation

93 9 100

4 0

f2 of Cl4 phenylalauine

incorporation

was observed.

has been shown for the RNApolymerases of these viruses (Raruna and Spiegelman, 1965).

We feel that these results plus those obtained by 314

of

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 30, No. 3, 1968

Sugiysma and Nskada (1967) establish the specific

nature of capsid-RNA

interaction. Viral

RNAJ3inding Site for Repressor.--We hoped to isolate

the RNA

binding site of the repressor by extensive degradation of the RNAin the presence of its repressor.

We reasoned that the bound repressor would pro-

tect this region fraan digestion by RNase. It labelled RNA (g2)

seemedmost convenient to use

as substrate ana follow digestion in the presence and

absence of capsid.

300 pg radioactive

In one experiment 30 pg of f2 capsid was incubated with viral

RNA(1.2 x 105 CPM). The ssmple was digested with

pancreatic RNase(40 cl@;)for 30 min at 37' C and assayed for TCA precipitable counts.

About 1.1s of the total

cating their

polynucleotide

counts remained precipitable

nature.

with TCA indi-

A portion of the capsid protected

ssm-

ple was sedimented in a sucrose gradient and ssmples were assayed for radioA peak sedimenting at about 5 S was observed.

activity.

We are exploring

this peak to see if it contains the repressor-RNA canplex. experiment an identical

radioactive

In a control

RNA ssmple was processed as above except

the RNAwas digested with RNasefor 30 min before capsid was added. An RNase "core" of approximately 0.53 of the whole molecule was observed (Sober -et

".,

III several experiments we have found that prior addition of

1966).

capsid protected

a significantly

RNA/capsid ratio

that would minimize reconstitution

1967) particles It

of the RNA. We chose an

whose RNAwould be protected in their

of intact

Wetherefore

argue that repression protection

associated with repressor binding to the RNA. Wepropose that

cepsid es repressor binds to a specific

tentatively

we used in

experiment gave maximal repression when assayed in the pro-

tein synthesis assay above. is closely

virus (Hohn,

coat.

should also be pointed out that the RNA/capsid ratio

the protection

viral

larger portion

called the repon (see Fig. 1).

region of the RNAwe have The nature of the repon is

under investigation. Discussion.--In

one sense the infective

315

cycle of the minute phage f2

Vol. 30, No. 3, 1968

BIOCHEMICAL

may be regarded as a biosynthetic particles

as ultimate

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

pathway with virus canponents and finished

products of the pathway.

cycle in this context leads to someinteresting

Treatment of the infective speculation regarding the

nature of the control mechanismswhich operate to insure a balanced infective cycle.

Wehave described what we consider to be an important viral

regulatory

mechanismwhich operates in somerespects like a "feedback" switch. a viral

biosynthetic

of other viral

product,

serves as an inhibitor

components. We call this regulatory

repression since the control

Capsid,

to regulate synthesis switch translational

is exerted at the messengerRNAor translational

level. There might be other levels of control due to polarization et al.; --

or polarized

1966 and other articles

workers have studied polarity of f2.

Strong polarity

of the infection

reading of the phage message(see Zinder in CSHSymposium, Vol. 31, 1966).

of translation

effects

such as control

in vitro --

were reported.

appears to work closely with the polarity

These

using amber mutants

Translational

repression

mechanism. For example, in the

case of the capsid mutants of Zinder --et al. (1966) translation

of the histi-

dine genes was almost completely blocked by the nonsense cadon in the capsid gene - translation indicate

did not proceed beyond this point.

that all of the viral

repressor.

Our data (Fig.

2)

genes (except shell) respond to capsid as

If this is the case and the repressor is specifically

as we think on only one of the genes, then strong polarization must occur in neighboring genes. The net effect

located effects

is that repression of one

gene shuts off aSl of the remaining genes on the RNAstrand aa the result of the strong polarization Translational

of the message.

repression,

fran several viewpoints.

It

Powers - it allows certain needed. The "timing"

as a regulatory

mechanism, seemsattractive

endowsthe virus with clynsmic self-regulatory

genes to be shut off when they are no longer

of the switch is related to the smount of translation

or number of times the viral

messageis read, a fact which allows regulation 316

Vol. 30, No. 3, 1968

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to occur even when the infective

cycle may be slowed down because of environ-

mental conditions of the infected

host cell.

Comparedto the life

higher cells and even higher phages this type of regulation primitive.

It

sion of protein

is interesting

cycle of

at first

seems

to speculate though that translational

repres-

synthesis such as observed here may occur in higher forms

although as far as we're aware there are as yet no other examples of this method of regulation. References Capecchi, M. R. (1%) 2. Mol. Biol., 21, 173. (1965) --hoc. E. Acad. s., UJ., &, 579. Haruna, I., and Spiegelman,S. Hohn, T. (1967) Eur. J. Biochem., 2, lr Lodish, H. F., HorshiT K., and Zinxer, N.D. (1965) Virology, 2& 139. Overbey, L. R., Barlow, G. H., Doi, R. H., Jacob, M., and Spiegelman, S. (1966) 2. Bacterial., Roberts, J. W., and Steitz, U.S., (1967) Proc. Natl. Acad. g., Scott, D. W. Sober," H. 1416*~~~!sm~~' A., $%rfz* A., Latt S A., and Rushinsky , G* W* (1966) Biochemistr;, i, $08. ' ' ' Steitz, J. A. (1967) J. Mol. Biol., in press. proC. Natl. Acad. S&., U.S., a, 1744. Sugiyama, T., and Nakadg, D. (m) %chem. Bmys. Ward, R., Shive, K., and Valentine, R. C. (m) -Res. W. (1967) J. Biol. Chem.,(gi)35E70;d Zinder, N. D., Engelhardt, D. L., and WGbster, R. E. mHarbor Symposia, 2, 251.

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