Biochimica et Biophysica Acta, 319 (1973) 304-311 ~) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA 97775

REVERSAL OF INHIBITION OF MEMBRANE-BOUND RNA SYNTHESIS IN

E S C H E R I C H I A C O L I INFECTED WITH R23

DAVID HUNT and MAMORU WATANABE

Department of Medicine and Biochemistry, University of Alberta, Edmonton, Alberta, T6G 2G3 (Canada)

(Received March 20th, 1973)

SUMMARY

1. Infection by RNA bacteriophage R23 causes a release of ribosomes from a membrane -DNA-RNA polymerase complex isolated from Escherichia coli. This re- lease may be responsible for the reduction in RNA synthetic capacity seen after phage infection.

2. The addition of ribosomes to such a membrane preparation restores the RNA synthetic capacity to uninfected levels. The ribosomes may exert their effect by binding to nascent RNA chains.

3. Exogenous RNA polymerase stimulates RNA synthesis by the membrane complex under conditions which probably prevent the initiation of new RNA chains. The mechanism of its effect may be analogous to that of ribosomes in this system.

INTRODUCTION

Investigations of the structural organization of bacterial cells indicate that the bacterial chromosome is attached to a portion of the cell membrane 1'2. Since the chromosomal DNA serves as the template for RNA synthesis, DNA and RNA may be expected to co-exist in a structural complex in association with the cell membrane during replication and transcription. Such a subcellular complex has been isolated by several groups of investigators 3- 5, and the bulk of the DNA-dependent RNA-form- ing capacity of the bacterial cell has been found in association with this fraction 4. This type of preparation provides a structurally-integrated system for investigation of any changes in the organization of bacterial RNA synthesis.

One of the factors involved in the control of bacterial RNA synthesis may be the supply of ribosomes available to bind to nascent RNA molecules 6'7. A significant proportion of the ribosomes present in the bacterial cell is normally found to be bound to a complex of cell membrane and DNA 8.

We have shown that the RNA synthetic capacity is greatly reduced in the cell membrane -DNA-RNA polymerase complex isolated from Escherichia coli infected with RNA bacteriophage R23 (ref. 9). This membrane complex can thus be used to

REVERSAL OF R23 INHIBITION OF RNA SYNTHESIS 305

study the mechanism of inhibition of host RNA synthesis characteristically seen after infection of intact cells. In this study, the effect of R23 on the ribosomes bound to the membrane-DNA-RNA polymerase complex was investigated. The results in- dicate that R23 infection causes a release of ribosomes from the membrane complex which may be responsible for the reduction in RNA synthetic capacity.

MATERIALS AND METHODS

[5-aH]UTP (spec. act. 17.1 Ci/mmole) and unlabeled nucleosides and nucleo- tides were purchased from Schwarz/Mann, Orangeburg, New York. Actinomycin D, was purchased from Merck Sharp and Dohme, West Point, Pennsylvania. Polyribo- inosinic acid and total yeast RNA were purchased from Miles Laboratories, Inc., Elkhart, Indiana.

RNA polymerase (DNA-dependent nucleoside triphosphate: RNA nucleo- tidyltransferase, EC 2.7.7.6) was prepared from exponential phase E. coli K38 by the method of Chamberlin and Berg I o. Ribosomes were prepared from E. coli K38 by the method of Brodie 1~.

Preparation of membrane complex Growth media for E. coli K38 and conditions for R23 infection have been des-

cribed 12'1a. For infected cultures, R23 was added at a multiplicity of infection of 50 phage/cell. The membrane complex was prepared as previously reported 9. Samples of the unfractionated cell lysate stage of the preparation were routinely examined microscopically and also plated to give an estimate of the number of viable cells present. Using both criteria, between 90 and 95 % of uninfected and R23-infected cells were lysed by the procedure.

Assay for RNA synthesis The assay for RNA synthesis has been previously described 9. Control incuba-

tions contained actinomycin D (10 #g/ml). The levels of UMP incorporation observed in the control incubations have been deducted in all the results presented, which thus represent actinomycin-sensitive RNA synthesis.

Treatments of ribosomes Heat-inactivated ribosomes were heated for 5 min at 100 °C. Pronase-treated ribosomes were incubated for 60 min at 37 °C with 500 pg/ml

of pronase.

RESULTS

Changes in composition of membrane complex following R23 infection The DNA-membrane-RNA polymerase complex was isolated and examined

following disruption of cells with lysozyme-EDTA. This gentle process of lysis pre- serves the complex in a state similar to that present in vivo 4. After lysis of uninfected cells and removal of the membrane complex by centrifugation, very little A260 nm- absorbing material was left in the supernatant. However, when cells infected with R23 were lysed and the membrane complex removed, between 40 and 50 ~ of the total

306 D. HUNT, M. WATANABE

A26 0 nm-absorbing ma te r i a l r e m a i n e d in the s u p e r n a t a n t (Tab le 1). Th is /126 0 nm- a b s o r b i n g ma te r i a l was no t D N A (as d e t e r m i n e d by the d i p h e n y l a m i n e reac t ion) ,

in a g r e e m e n t wi th o u r p rev ious s tudies 9.

TABLF 1

EFFECT OF R23 INFECTION ON THE APPEARANCE OF A26o ,m-ABSORBING MATERIAL IN THE SUPERNATANT

Cells were lysed as described for the preparation of membrane complex. The A26o n,, of the super- natant was measured following removal of the complex by centrifugation and the value calculated as a percentage of the A26o am of the unfractionated lysate.

0.3

0 (uninfected) 6 5 47

10 48 20 46 30 45 45 49 60 42

70 of total A26o nm in supernatant

W h e n the s u p e r n a t a n t f r o m cells in fec ted wi th R23 was ana lyzed on a sucrose

dens i ty g rad ien t , a sha rp p e a k was o b s e r v e d wh ich c o i n c i d e d w i t h the p e a k p r o d u c e d

by r i b o s o m e s p r e p a r e d f r o m un in f ec t ed cells (Fig. 1). Th is p e a k r ep resen ted a b o u t

0.2

0.1

i 2 4 6 8

Bot tom

Time after infection (rain)

/

10 12 14 16 18 20 Top

FRACTION NUMBER

Fig. 1. Sedimentation distribution of E. coli ribosomes and supernatant from R23-infvcted cells. Supernatant was obtained from cells infected for 30 rain with R23 after cell lysis and removal of the membrane complex. A 0.l-ml sample of the supernatant (O), or of ribosomes prepared from £. coil (O), was layered on a 5-ml 5-20 70 sucrose density gradient containing 0.02 M Tris-HCI, pH 8.0, 0.01 M MgCI2, and 0.05 M KC1. The gradients were centrifuged simultaneously at 20 °C for 1.5 h at 40 000 rev./min in an SW 56 rotor. After centrifugation, the tubes were punctured, 20 frac- tions were collected, and the A26o nm of each fraction was recorded.

REVERSAL OF R23 INHIBITION OF RNA SYNTHESIS 307

40 % of the .4260 ,m-absorbing material present in the infected supernatant; the re- mainder stayed at the top of the gradient. No polysomes or ribosomal subunits were detected under the conditions used. In the ease of supernatant from uninfected cells, no peak was observed (not shown). The release of ribosomes from the membrane com- plex following R23 infection was rapid, being fully established by 5 rain of infection (Table I).

Effect o f added ribosomes on R N A synthesis by membrane complex When 40/~g of E. coli ribosomes (an amount approximately equivalent to the

ribosomes released from the same quantity of infected membrane complex as was present in the reaction mixture) were added to the membrane complex from uninfected cells, RNA synthesis was stimulated by 15-20 % (Table II). In contrast, the RNA synthetic activity of the membrane complex from R23-infected cells was stimulated approximately 100 % by the addition of ribosomes. This restored the RNA synthetic capacity of the infected complex to the level of the uninfected complex.

TABLE II

EFFECT OF RIBOSOMES ON RNA SYNTHESIS BY MEMBRANE COMPLEX

Actinomycin-sensitive [3H]UMP incorporation was assayed as described previously using mem- brane complex from uninfected cells or cells infected for 30 min with R23. Ribosomes prepared from E. coli (40#g) were added to some tubes as indicated.

Source of membrane complex [3HI UMP incorporated (pmoles/h )

Control d- Ribosomes

Uninfected cells 970 1140 R23-infected ceils 455 990

The time course of RNA synthesis observed with the membrane complex from uninfected cells was typical of in vitro RNA synthetic systems. The rate of syn- thesis was initially rapid, but decreased and reached a plateau after about 60 min (Fig. 2). The addition of ribosomes to the uninfected membrane complex mildly stimulated RNA synthesis but did not alter the pattern of the time course. The kinet- ics of RNA synthesis by the membrane complex from R23-infected cells was distinctly different. The initial rate of synthesis was slower, UMP incorporation continued for a longer period, and the overall level of synthesis was reduced. When ribosomes were added to this complex, the time course of RNA synthesis was dramatically altered so that it became identical to that observed with the uninfected membrane complex (Fig. 2).

Ribosomes which had been heat-inactivated or treated with the proteolytic enzyme pronase gave no stimulation of RNA synthesis by uninfected or R23-infected membrane complex.

Effect o f exogenous R N A polymerase on R N A synthesis by membrane complex When purified'E, eoli RNA polymerase is added to the isolated membrane

complex, an increased level of RNA synthesis is observed, and a larger increase is

308 D. HUNT, M. WATANABE

."7" 1000

8O0

~ 600

2 8 400 Z

,°°, A'°' '° ' ' '" UNINFECTED

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,N EC,ED ..........................

i , i i 30 60 90 120

MINUTES

Fig. 2. Effect of ribosomes on RNA synthesis by the membrane complex uninfected and R23-infected cells. The time course of actinomycin-sensitive [all ]UMP incorporation was determined, as described in the text, for membrane complex from uninfected ceils and from cells infected for 30 min with R23, in the absence or presence of 40/tg of E. coli ribosomes. /x, uninfected, plus ribosomes; O, uninfected, control; A, infected, plus ribosomes; O, infected, control.

detected with infected than with uninfected complex 9. Addi t ion o f r ibosomes did not

increase the s t imulat ion due to exogenous R N A polymerase (Table III) .

TABLE III

EFFECT OF VARIOUS ADDITIONS ON RNA SYNTHESIS BY EXOGENOUS RNA POLY- MERASE ON THE MEMBRANE COMPLEX

Actinomycin-sensitive [3H]UMP incorporation was assayed as described previously using mem- brane complex from uninfected cells or cells infected for 30 min with R23. Purified E. coil RNA polymerase (12/~g) and ribosomes prepared from E. coli (40/~g) were added to some tubes as indi- cated. In some cases, the RNA polymerase was pre-incubated for 5 min at 37 °C in the presence of poly(I) (20 #g) or total yeast RNA (50/~g) before the membrane complex was added to start the reaction. Such a pre-incubation in itself had no effect on the activity of the enzyme.

Additions [all] UMP incorporated (pmoles/h )

Uninfected R23-infected membrane membrane complex complex

None 985 480 Poly(I) (10/zg) 1002 473 Ribosomes 1120 995 RNA polymerase 2251 3350 Ribosomes ÷ RNA polymerase 2235 3280 RNA polymerase pre-incubated with Poly(I) 2160 3240 RNA polymerase pre-incubated with RNA 1650 1830

St imulat ion by added R N A polymerase o f R N A synthesis by the infected

membrane complex could be due to an increased number o f ini t iat ion sites on the D N A template. The effect o f an inhibi tor o f R N A chain init iat ion on the s t imulat ion

was therefore examined. Poly(I) inhibits R N A chain init iat ion by complexing R N A

REVERSAL OF R23 INHIBITION OF RNA SYNTHESIS 309

polymerase molecules before they bind to the DNA template, but it has no effect on chain elongation 14. Addition of poly(I) alone had no effect on the RNA synthetic activity of the membrane complex from uninfected or R23-infected cells (Table III). In addition, pre-incubation of exogenous RNA polymerase with poly(I) did not af- fect the stimulation of RNA synthesis produced by the enzyme, indicating that stimu- lation of RNA synthesis by exogenous polymerase was unlikely to be due to a greater number of initiation sites.

Although poly(I) had no effect on stimulation of RNA synthesis by exogenous RNA polymerase, pre-incubation of the enzyme with RNA, on the other hand, markedly inhibited its stimulatory effect on RNA synthesis (Table III).

Characteristics of RATA synthesis by membrane complex The inability of poly(I) to inhibit RNA synthesis by the membrane complex

from both uninfected and infected cells suggested that the membrane-bound RNA polymerase was so strongly complexed to the DNA within this complex that it was not susceptible to poly(I) and/or that the RNA synthesis observed represented chain elongation only. In support of the latter possibility, NaC1, at a concentration of 0.8 M, had no effect on RNA synthesis by the membrane complex. NaCI at this concen- tration is known to also inhibit RNA chain initiation, but not chain growth ~s'16.

DISCUSSION

Infection with R23 has been shown to markedly inhibit RNA synthetic capa- city in intact cells t 2, t 7 and in a DNA-membrane-RNA polymerase complex isolated from E. coli by gentle lysis 9. Phage infection did not release DNA or detectable amounts of RNA polymerase 9, but did result in dissociation of 40-50 ~o of the RNA from the membrane complex. About 40 ~ of the material released was ribosomes; the nature of the remainder has not been established. The ribosomes appear to be re- leased from the complex as free 70-S monosomes, and studies are in progress to in- vestigate any possible changes in their structure.

When ribosomes prepared from E. coli were added to the membrane complex from uninfected cells, RNA synthesis was stimulated 15-20 ~o (Table II), whereas addition of ribosomes to membrane complex from infected cells stimulated RNA synthesis 100 ~ and restored the RNA synthetic capacity to the level and pattern characteristic of the complex from uninfected cells. Thus, the loss of ribosomes from the membrane appeared to be directly responsible for the reduction in RNA synthesis in the infected complex, and the pronounced stimulatory effect of added ribosomes on RNA synthesis by the infected complex was probably the result of the initial loss of ribosomes from the complex following infection. The total reversibility of the in- hibition of RNA synthesis suggested that no other factors were involved, at least to an important degree, and that the alterations were not permanently disruptive to the cell.

It has been postulated that ribosomes bind nascent RNA chains and thereby reduce the blocking of the template by the RNA product which would otherwise occur in an integrated RNA synthetic system 6'7. Thus, the addition of ribosomes to a deoxyribonucleoprotein complex from E. coli stimulates RNA synthesis 6. The RNA made in the presence of ribosomes is of considerably higher average molecular

310 D. HUNT, M. WATANABE

weight than that made in their absence, and a significant portion of the nascent RNA is released from the complex as r ibosome-RNA aggregates 6' 18

Such a mechanism could also be the basis for the stimulatory effect of ribo- somes seen in the present studies. RNA synthesis in uninfected complexes was stimu- lated by a similar amount at each time point examined, suggesting that the added ribosomes allowed the RNA chains to grow slightly longer (Fig. 2). The loss of ribo- somes from infected complexes would halt RNA synthesis by preventing removal of nascent RNA chains from the template.

Ribosomes which have been heat-treated or digested with the proteolytic enzyme pronase did not stimulate RNA synthesis by uninfected or R23-infected membrane fraction. Studies are in progress to determine whether intact ribosomes are needed to produce the observed stimulation of RNA synthesis, or whether, possibly, a protein component of the ribosome is the active fraction.

The results suggested that the stimulatory effect of exogenous RNA polymerase had a similar basis or locus of action to the ribosomes. Ribosomes did not increase the stimulation of RNA synthesis due to exogenous RNA polymerase, suggesting that they might both be acting by a common mechanism. RNA synthesis might be stimulated through an enhancement of chain initiation or elongation. The lack of effect of inhibitors of chain initiation suggested that RNA synthesis in the membrane preparation used is mainly, if not entirely, the result of extension of previously initi- ated chains, and that little or no re-initiation occurs in this system. Although it is possible that the close structural arrangement of molecules within the membrane complex could reduce, or even nullify completely, the normal effects of the inhibitors, such a conclusion is in agreement with the results of Pettijohn et al. 19'2°. These authors found that RNA synthesis by an isolated protein-DNA complex occurred exclusively as an extension of nascent RNA chains which had been initiated in vivo. Attempts to measure directly the levels of RNA chain initiation by the membrane complex using D,-32p]GTP have been unsuccessful, thus far, because of extremely high non-specific binding of GTP to the complex.

When purified exogenous RNA polymerase, with or without pre-incubation with poly(I), was added to the membrane complex, an increased level of RNA syn- thesis was observed, especially in R23-infected complexes. Since poly(I) blocks the DNA binding site of the enzyme 14, the stimulation could not be due to an increase in initiation of new RNA chains by the polymerase. Pre-incubation of enzyme with RNA, on the other hand, markedly inhibited the stimulatory effect on RNA synthesis. Such a pre-incubation would presumably block the site on the enzyme which binds to the nascent RNA chain. Thus, the enhancement of RNA synthesis produced by exogenous RNA polymerase in uninfected and infected complexes was probably due to a stimulation of synthesis of chains already initiated. The reason for the greater stimulatory effect in infected complexes remains to be clarified.

In conclusion, the inhibition of RNA synthetic capacity of the membrane com- plex isolated from E. coli following R23 infection appears to be due primarily to the release of ribosomes occurring immediately after infection. The rapidity and revers- ibility of these changes suggest that there is an architectural alteration consequent to adsorption of the phage particle to the cell membrane, which in turn leads to changes in host metabolism.

REVERSAL OF R23 INHIBITION OF RNA SYNTHESIS 311

ACKNOWLEDGEMENTS

This invest igat ion was supported by G r a n t M A 2822 from the Medical Re- search Counci l of Canada , Ottawa, Canada.

Dr M a m o r u Watanabe is an Associate, Medical Research Counci l of Canada, Ottawa, Canada.

The authors gratefully acknowledge the expert technical assistance of Miss Chris t ina Chew.

REFERENCES

1 Jacob, F., Ryter, A. and Cuzin, F. (1966) Proc. R. Soc. London B Biol. Sci. 164, 267-278 2 Ryter, A. (1968) Bacteriol. Rev. 32, 39-54 3 Tremblay, G. Y., Daniels, M. J. and Schaechter, M. (1969) J. Mol, Biol. 40, 65-76 4 Rouvi~re, J., Lederberg, S., Granboulan, P. and Gros, F. (1969) J. Mol. Biol. 46, 413-430 5 Smith, D. W. and Hanawalt, P. C. 0967) Biochim. Biophys. Acta 149, 519-531 6 Shin, D. H. and Moldave, K. (1966) J. Mol. Biol. 21,231-245 7 Schaechter, M. and McQuillen, K. (1966) J. Mol. Biol. 22, 223-233 8 Varricchio, F. (1972) d. Bacteriol. 109, 1284-1294 9 Hunt, D., Saito, Y. and Watanabe, M. (1971) J. Biol. Chem. 246, 4151-4156

10 Chamberlin, M. and Berg, P. (1962) Proe. Natl. ,4cad. ScL U.S. 48, 81-94 11 Brodie, A. F. (1962) in Methods in Enzymology iColowick, S. P. and Kaplan, N. O., eds), Vol. V,

pp. 57-61, Academic Press, New York 12 Watanabe, M., Watanabe, H. and August, J. T. (1968) J. Mol. Biol. 33, 1-20 13 Watanabe, H. and Watanabe, M. (1968) J. Virol. 2, 1400-1407 14 Hirschbein, L., Dubert, J. M. and Babinet, C. (1967) Eur. d. Biochem. 1, 135-140 15 Richardson, J. (1966) d. Mol. Biol. 21, 83-114 16 Fuchs, E., Millette, R., Zillig, W. and Walter, G. (1967) Eur. J. Biochem. 3, 183-193 17 Watanabe, H. and Watanab¢, M. (1971) Can. d. MicrobioL 17, 461-466 18 Jones, O. W., Dieckmann, M. and Berg, P. (1968) J. Mol. Biol. 31, 177-189 19 Pettijohn, D. E., Clarkson, K., Kossman, C. R. and Stonington, O. G. (1970) J. Mol. Biol. 52,

281-300 20 Pettijohn, D. E. (1969) Biochem. Biophys. Res. Commun. 34, 541-547