JOURNAL OF VIROLOGY, Aug. 1975, p. 228-236 Copyright © 1975 American Society for Microbiology
Vol. 16, No. 2 Printed in U.S.A.
Hexamer of Bacteriophage f2 Coat Protein as a Repressor of Bacteriophage RNA Polymerase Synthesis JADWIGA CHROBOCZEK AND WTODZIMIERZ ZAG0RSKI*I
Department of Protein Biosynthesis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Rakowiecka 36, 02-532, Warsaw, Poland Received for publication 10 March 1975
Formation of complex I between phage f2 RNA and coat protein, leading to repression of phage RNA polymerase synthesis, depends nonlinearly upon the concentration of the coat protein. Maximum formation of complex I was observed when six molecules of coat protein were bound to one molecule of RNA. RNase digestion of a glutaraldehyde-fixed complex left, as the products, coat protein oligomers. The heaviest, hexamers, predominated in the mixture. It was also shown that, in an ionic environment required for phage protein synthesis, coat protein at a concentration optimum for complex I formation exists in solution as a dimer. The results indicate that the translational repression of the RNA polymerase cistron is due to a cooperative attachment to phage template of three dimers of coat protein, forming a hexameric cluster on an RNA strand.
Coat protein of small RNA bacteriophage is known to react with phage RNA, forming what is called complex I. I'his complex behaves as a modulated messenger in the in vitro proteinsynthesizing system. Specific inhibition of the translation of the RNA polymerase is observed when phage RNA is incubated in the presence of a few moles of coat protein per 1 mol of RNA. The interaction between phage coat protein and homologous RNA can be regarded as a model system for studying regulation of gene expression on the translational level. This resulted in considerable attention being paid to the problem of complex I formation (for review of the problem, see reference 14). However, the mechanism of formation of complex I still remains an open question. The uncertainties are concerned with the molar ratio of protein to RNA in the complex as well as the mutual positioning of protein molecules present in complex I. Spahr et al. (11) suggest that in complex I only one molecule of coat protein is strongly bound to the RNA strand and that the attachment of additional portions of coat protein is associated with processes of phage particle formation. Results from other laboratories, however, are different. Sugiyama and Nakada (15) assumed that 1 mol of RNA specifically binds several (less than 10) moles of coat protein. Eggen and Nathans (7) suggested that full repression of RNA polymerase synthesis occurs when 3 to 4 mol of coat protein is bound to the phage template. Ward et ' Present address: Biophysics Laboratory, University of Wisconsin, Madison, Wis. 53706.
al. (16) reported that the repression is due to the attachment of six molecules of coat protein to one RNA strand. Also, the problem of coat protein distribution along the phage RNA strand in complex I is still open to discussion. It was suggested by Hohn (8) that the six molecules of coat protein present in complex I are bound at several sites on the RNA strand, whereas Ward et al. (16) assumed that the coat protein molecules in complex I are attached at a single site of the RNA molecule. It is evident that different evaluations of the number of coat protein molecules present in complex I per one RNA molecule lead to different proposals concerning the mechanism of complex formation. A protein/RNA molar ratio of 1:1 in the complex would suggest that the ability of recognition of the proper site on the RNA strand is an intrinsic property of the coat protein monomer. In contrast, results showing that RNA specifically binds several protein molecules may suggest that oligomeric forms of coat protein are involved in recognition of a specific fragment of the RNA molecule. In this paper we present results showing that, under conditions favoring complex I formation, coat protein exists in solution in the form of dimers. On the other hand, in the complex, we found hexamers of coat protein attached to RNA strands. On this basis, we suggest that the formation of complex I is due to the cooperative attachment of three coat protein dimers to the phage template. This leads to the formation of a hexameric cluster of the coat protein molecules on the RNA strand.
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MATERIALS AND METHODS General. The methods of isolation of phage f2 and phage RNA and the conditions of incorporation of amino acids in the Escherichia coli cell-free system were described previously (6, 20, 21). RNA concentration was determined by its optical density at 260 nm using an extinction coefficient equal to 24 ml/mg per cm (18). The [140 lserine- or [3H ]uridine-labeled phage was prepared by the method of Steitz (12). Coat protein was isolated by the acetic acid extraction method as described by Eggen and Nathans (7). The extinction coefficient value at 280 nm of 1.1 mi/mg per cm was used to measure the concentration of phage coat protein (13). The homogeneity of each of the RNA preparations was checked by analytical ultracentrifugation in 5 mM Tris-hydrochloride buffer (pH 7.2) containing 0.2 M NaCl. f2 RNA (70 to 80%) was found to sediment in the 27S region. Sedimentation analysis of formaldehyde-treated f2 RNA in formaldehyde-sucrose gradients (11) also gave the same results. The following procedures were performed with buffer A (64.2 mM Tris-hydrochloride [pH 7.6], 14.7 mM magnesium acetate, 27.6 mM NH4Cl, 30 mM KCl, and 7 mM 2-mercaptoethanol): formation of complexes between coat protein and phage RNA, nucleolytic treatment of complexes, gradient analysis, Sephadex gel filtration and sedimentation analysis of coat protein. This buffer provides optimum salt conditions for phage protein synthesis in vitro. Formation of complexes between coat protein and f2 RNA. The 14C-labeled coat protein and 3H-labeled or unlabeled phage RNA at various concentrations were incubated in buffer A for 20 min at 25 C. Complexes were stabilized, by the method of Baltimore and Huang (3), by incubation for 10 min at 0 C in the presence of 2.5% glutaraldehyde. The mixtures were then layered upon 4.5 ml of linear (5 to 20%) sucrose gradients prepared in buffer A. Centrifugation was carried out for 100 min at 48,000 rpm and 4 C in an SW50 rotor in a Spinco model L ultracentrifuge. Gradients were fractionated from the bottom of the tube, and the radioactivity of the fractions was measured on filter paper disks (Whatman 3MM) as described previously (6). Treatment with nucleolytic enzymes and gel filtration. The gradient fractions containing the RNA-protein complexes were pooled and treated with 10 U of RNase T2 (Calbiochem) and 1 mg of snake venom Puretoxin (Miami Serpentarium Laboratory, Miami, Fla.). After 20 min of incubation at 37 C, the mixture was layered on the top of a Sephadex gel G-100 column (2.1 by 65 cm) equipped with a water jacket and cooled by running tap water (14 C). The column was equilibrated previously with buffer A. Gel filtration. Chromatography was carried out with the same buffer at a flow rate of 12 ml/h. Fractions of 1.2 ml were collected. The radioactivity in 1-ml samples of each fraction was determined by using the toluene-Triton X-100-based scintillation mixture of Patterson and Greene (9). The molecular weight of the coat protein oligomers was calculated from a calibration curve obtained by separate runs of: alkaline phosphatase from calf mucosa (Sigma Chem-
229
ical Co.), molecular weight 80,000; bovine serum albumin, fraction V (Serva, Heidelberg, Germany), molecular weight 67,000; pepsin, crystallized twice (Fluka), molecular weight 35,000; RNase from bovine pancreas, crystallized five times (Calbiochem), molecular weight 13,700. Aggregation of coat protein in solution. Coat protein aggregation was analyzed by gel filtration on a Sephadex G-100 column and checked by sedimentation analysis. Solutions of '4C-labeled coat protein in different concentrations, ranging up to 0.7 mg per ml of buffer A, were submitted to gel filtration under the conditions described above. The molecular weight for peak fractions was estimated and plotted as a function of coat protein concentration in the sample applied to the column. Sedimentation analysis was performed with 1.5 mg of coat protein in 1 ml of buffer A in a Spinco model E analytical ultracentrifuge. Molecular weight was determined by equilibrium ultracentrifugation for 5 h at 36,000 rpm and 20 C by the method of Yphantis (19).
RESULTS AND DISCUSSION Effect of coat protein concentration on the formation of complex I. As we showed previously, formation of complexes between coat protein and phage RNA is strongly influenced by ionic conditions in the incubation mixture (6). To assure that the complexes under study were those responsible for repression of RNA polymerase synthesis, we formed and analyzed complexes in an ionic environment needed for phage protein synthesis (21). The complexes were stabilized by glutaraldehyde treatment (17). This diminished possible dissociation of the complex due to extensive dilution accompanying Sephadex filtration. Under our experimental conditions, glutaraldehyde treatment did not promote the binding of additional portions of coat protein to phage RNA. On the other hand, glutaraldehyde-fixed complexes are more stable than unfixed (20). As previously reported (20), maximum repression was observed when about six molecules of coat protein were bound to one RNA strand in the complex. To analyze the kinetics of the formation of such multimeric complexes, we studied the dependence of the reaction on the concentration of RNA and protein in the incubation mixture. The formation of complex I illustrated in Fig. la was performed at an RNA concentration of 1.000 jg/ml and at a coat protein/RNA input molar ratio of 16. The molar ratio of the components in complex I was 6.4. In a parallel experiment, performed at an RNA concentration of 233 gg/ml and an input molar ratio of 20, only 0.9 mol of coat protein per 1 mol of RNA was recovered in complex I (Fig. lb). This
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Frac[ion no. FIG. 1. Complex formation between f2 coat protein and f2 RNA. The conditions for complex formation and the method of analysis of gradients are described in the text. (a) Phage 3H-labeled RNA (390 mg; specific activity 224 counts/lg per 10 min) was incubated with 80.1 ,ug of "4C-labeled coat protein (specific activity 426 counts/ltg per 10 min) in a total volume of 0.4 ml of buffer A at 25 C for 25 min. After incubation, the mixture was chilled in an ice bath, and the complex was fixed with glutaraldehyde and layered on the top of a sucrose gradient. Input molar ratio of coat protein to RNA is equal to 16. (b) 3H-labeled RNA (70 lg; specific activity 1,380 counts/lg per 10 min) was incubated with 17 Aig of "4C-labeled coat protein (specific activity 2,300 counts/ulg per 10 min) in a total volume of 0.3 ml of buffer A. Other details as in (a). Input molar ratio of coat protein to RNA is equal to 20. Symbols: 0, 3H label in RNA; *, 14C label in coat protein.
indicates that the formation of complex I depends strongly on the concentration of the components in the mixture and not only on the molar input ratio of coat protein to RNA. To test this assumption, we performed a series of experiments resembling those in Fig. 1, incubating RNA at two different concentrations with increasing amounts of coat protein. After gradient centrifugation, the molar ratios of the components in complex I were calculated and plotted versus the input molar ratios. The saturation curves obtained are shown in Fig 2a and b. The first portions of coat protein did not promote complex formation (Fig. 2a). The addition of further portions of coat protein initiated formation of complex I. From this point the curve in Fig. 2a sharply rises. It reaches a plateau at about six protein molecules per one molecule of RNA in complex I, the input molar ratio being about 20. The sigmoidal character of the curve suggests that complex I formation involves a cooperative binding of several coat protein molecules to one RNA strand. Since this type of interaction should show a pronounced concentration dependence, we performed the saturation experiments with approximately four-times-lower concentrations of RNA and coat protein, and the molar ratio of protein to RNA in the complex was plotted versus input
protein to RNA ratios (Fig. 2b). In this case the experimental points also fall on a sigmoidal curve; however, there is a lower slope throughout the entire region of input molar ratios. It can be seen that in Fig. 2b saturation is reached at a much higher input molar ratio of coat protein to RNA than that of the experiment performed at the higher RNA concentration (Fig. 2a). In contrast, when results are expressed as a function of coat protein concentration in the incubation mixture, it became evident that in both cases presented in Fig. 2 saturation was observed at about the same concentration of coat protein, i.e., at 200 to 250 ug/ml. This shows clearly that the formation of complex I occurs at defined ranges of protein concentration and that it depends not only on the input molar ratio of the protein to RNA, but it also depends strongly on the concentration of coat protein in the incubation mixture. We noted that, compared with curve 2a, curve 2b reaches a plateau at slightly higher amounts of coat protein in the complex. This can be due to the fact that in this experiment the plateau is observed at high input protein/ RNA ratios. Under such conditions, processes of the formation of phage-like particles may be initiated, leading to the enhancement of protein to RNA ratio in the complex. Next we showed that, similar to complex I
VOL. 16, 1975
REPRESSOR OF f2 RNA POLYMERASE SYNTHESIS
formation, the repression of the histidine incorporation in the phage RNA-directed cell-free system depends more on the concentration of coat protein in the incubation mixture than on the input molar ratio of components (see Fig. 3). At an RNA concentration of 280 gg/ml, maximum repression was observed at a molar ratio of about 60 for the components in the incubation mixture, whereas, at a higher concentration of RNA (equal to 800 ,ug/ml), the same effect was observed with a molar ratio of components of about 20. In both cases, maximum repression was observed at the same coat protein concentration of about 180,ug/ml. This resembles the effect of coat protein concentration upon formation of complex I. The slopes of repression curves (Fig. 3) are, however, different from those of complex formation (Fig. 2). We assume that the observed differences result from different techniques of analysis in both types of experiments. For example, gradient analysis of complex I was done at 4 C, whereas repression was measured at 37 C. Size of coat protein oligomers formed on
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231
the RNA strand. The sigmoidal character of the saturation curve (Fig. 2) implies that the coat protein molecules bound in complex I interact with each other, forming an oligomeric structure on a strand of RNA. To test this hypothesis, the following experiment was performed. Complex I, stabilized with glutaraldehyde and sedimented through a sucrose gradient, was treated with a mixture of the RNA-hydrolyzing enzymes, and the hydrolysate was filtered through Sephadex G-100 gel (Fig. 4). When complex I was formed at saturation of RNA with coat protein, the molar ratio of coat protein to RNA was about 6 (Fig. 4a). The gel filtration profile of the products of hydrolysis of the glutaraldehyde-fixed complex exhibits six peaks. The peaks represent various classes of "4C-labeled coat protein oligomers (Fig. 4b). From the calibration curve for the Sephaaex column, molecular weights for the peaks I-VI were established as follows: 19,000, 29,000, 32,000, 46,000, 61,000, and 76,000. In the next experiment (Fig. 4c), complex I was formed
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FIG. 2. Binding off2 coat protein to f2 RNA-saturation curves. The ratio of coat protein to RNA in the peak fraction of the sucrose gradients is plotted as a function of the concentration of coat protein as well as the input molar ratio of coat protein to RNA. (a) Portions of 160 ,ug each of RNA (specific activity 800 counts/ug per 10 min) were incubated -with appropriate amounts of coat protein (specific activity 1,090 counts/Mg per 10 min) in 0.2 ml of buffer A at 25 C for 20 min. (Final RNA concentration was 800 MAg/ml of buffer A.) After incubation and chilling in an ice bath, the complexes were fixed with glutaraldehyde and the mixture was layered on the top of a sucrose gradient. (b) Portions of 70 sg each of RNA (specific activity 1,380 counts/Mg per 10 min) were incubated with appropriate amounts of coat protein in 0.3 ml of buffer A at 25 C for 20 min. (Final RNA concentration was 233 Mg/ml of buffer A.) After fixation as in (a), the mixtures were layered on the top of a sucrose gradient. Coat protein had specific activity of 2,300 counts/Mg per 10 min in the experiments with an input molar ratio of coat protein to RNA from 5 to 20, and 1,900 counts/lg per 10 min in the experiments with an input molar ratio of coat protein to RNA from 30 to 100.
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Protein/NA, moLar rolhio in incubationnrnxture FIG. 3. Effect of concentration of f2 coat protein on phage-specific protein synthesis. (a) Portions of 36.2 Mg each of f2 RNA were incubated with appropriate amounts of coat protein for 20 min at 25 C in 120 Il of mixture containing: 0.03 Amol of guanosine 5'-triphosphate, 0.4 Mmol of adenosine 5'-triphosphate, 1.7 Mmol of phosphoenolpyruvate, 80 Mg of E. coli tRNA, 0.9 Mg of pyruvate kinase, 0.0125 Amol of each nonradioactive amino acid, 0.006 MCi of ["4C]histidine (specific activity 100 MCi/mmol) or 0.006 MCi of ["4C]alanine (specific activity 10 uCi/mmol), and salts as in buffer A. A 5-Ml volume (0.2 mg of protein) of E. coli S-30 extract was added, and each mixture was incubated for 30 min at 37 C. Samples were analyzed for ["4C]histidine and ["4C]alanine incorporation. Results are expressed as the percentage of histidine and alanine incorporation versus molar ratio of protein to RNA. In the absence of f2 RNA, the incorporation was 720 counts/min of ["4C]histidine and 108 counts/min of ["4C]alanine (blank values). In the absence of coat protein, the incorporation was 5,330 counts/min of ["4C]histidine and 1,230 counts/min of ["4C]alanine. Net incorporations in the absence of coat protein were taken as 100%)6. (b) Experimental procedure as in (a), but the incubation mixtures contained 100 Mg f2 RNA each. In the absence of coat protein, the incorporation was 9,440 counts/min of ["4C]histidine and 3,770 counts/min of ["4C]alanine per 125 ul of mixture. Symbols: 0, ["4C]histidine; 0, ["4CJalanine.
under conditions of partial saturation of RNA with coat protein. The molar ratio of coat protein to RNA in complex I was about 2, as determined by the density gradient method. Again, six classes of protein oligomers appeared as the products of RNase treatment of the complex (Fig. 4d). The molecular weights for the peaks I-VI were: 17,500, 26,500, 35,000, 44,000, 62,000, and 80,000. The molecular weight of the coat protein monomer is 13,700 (17). By dividing the series of molecular weights for peaks I-VI by 13,700, the following sequences of values are obtained: 1.3, 2.1, 2.3, 3.4, 4.4, and 5.5 for Fig. 4b; and 1.2, 1.9, 2.6, 3.2, 4.5, and 5.9 for Fig. 4d. From this, we conclude that peak VI represents the hexamer of coat protein. It is of importance that, among oligomers detected during the gel filtration, the hexamer predominates and represents the heaviest component found in the mixture (Fig. 4). This and the fact that the plateau value of the curves in Fig. 2 is equal to six indicate that, in complex I, six
molecules of protein are bound to one strand of RNA and they interact with each other. The distances between protein subunits in complex I are such that glutaraldehyde can cross-link coat protein molecules. The lighter peaks (I to V) found during Sephadex filtration tentatively represent the following oligomeric forms of coat protein: monomers, dimers, trimers, tetramers, and pentamers. We believe that the components lighter than hexamers found during gel filtration originate from incompletely crosslinked hexamers. The incomplete cross-linking can result in the appearance of association-dissociation phenomena during gel filtration. The observed deviations from the expected molecular weights of the coat protein oligomers may be attributed to these phenomena. The lack of complete cross-linking is due to the mild conditions of glutaraldehyde treatment. More drastic conditions of fixation (incubation at 37 C for 1 h) result, however, in precipitation of complex I. It should be emphasized that, when the solution of coat protein in buffer A was sub-
VOL. 16, 1975 VREPRESSOR OF f2 RNA POLYMERASE SYNTHESIS
jected to exactly the same procedure and analysis as the aforementioned (including fixation with glutaraldehyde), no components heavier than dimers were detected on Sephadex gels. The results (Fig. 4b) indicate that the six molecules of coat protein, which are present in complex I, are not randomly spread along the RNA molecule, but they form a hexamer attached to the RNA molecule at a single site. This contradicts the results of Hohn (8), who
233
observed that RNase treatment liberated from complex I only monomers of coat protein. The effect reported by Hohn can be attributed to the dissociation of unfixed coat protein hexamers after detachment from the RNA strand. When the molar ratio of components (as determined by density gradient analysis) was below 6, again the hexamers predominated among detected oligomers of coat protein. This indicates that there was no random distribution
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Fraction no. FIG. 4. Gel filtration of coat protein oligomers obtained after digestion of complex I with nucleolytic enzymes. (a) A 320-ug amount of unlabeled f2 RNA was incubated with 100 ,g of "C-labeled coat protein (specific activity 3,000 counts/gg per 10 min) in a final volume of 0.5 ml of buffer A. The incubation mixture was layered on the top of a sucrose gradient and centrifuged. Three gradients run in parallel were fractionated, and the parallel fractions were pooled. Absorbancy at 260 nm and radioactivity of each fraction were measured. (b) Fractions 12 to 20 from the gradient presented in (a) were pooled and treated with RNase, and the mixture was subjected to Sephadex gel filtration. (c) Experimental procedure as in (a), but 250 tig of RNA was incubated with 56Mg of coat protein. (d) Fractions 8 to 15 from the gradient presented in (c) were pooled and treated with RNase, and the mixture was subjected to Sephadex gel filtration. Symbols: 0, absorbancy at 260 nm; 0, "4C-labeled coat protein.
234
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CHROBOCZEK AND ZAG0RSKI
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FracLon no. FIG. 4c and d. of coat protein molecules between the RNA strands. There are RNA strands that are occupied by hexamers of coat protein and others that are naked. The naked RNA strands sediment at the same position as does complex I. Due to this the molar ratio of protein to RNA in fractions from the sucrose gradient is lowered. The above discussed mechanism of formation of complex I implies that full repression of RNA polymerase synthesis in vitro can be obtained when all RNA strands are saturated with the hexameric repressor. This supposition was confirmed by testing the template activity of complexes isolated directly from the density gradients. It was observed that full repression of the RNA polymerase synthesis is obtained only when the molar ratio of coat protein to RNA in complex I was about 6 (20). Behavior of coat protein in solution. Formation of complex I is observed at definite concentrations of coat protein (Fig. 2). Therefore, one can expect the existence of concentration-dependent aggregation of coat protein molecules in solution into oligomers, which bind to RNA, forming complex I. To check this supposition, the sedimentation behavior of coat pro-
tein in buffer A was studied in an analytical ultracentrifuge. At a concentration of 1,500 ,ug/ml, the coat protein behaves as a dimer with a molecular weight about 26,500 and a sedimentation coefficient equal to 2.55. The behavior of the coat protein at concentrations close to those used for formation of complex I (Fig. 2) was studied by gel filtration. The effect of varying coat protein concentrations on its molecular weight is shown in Fig. 5. When the concentration of coat protein in the sample applied on the column was below 200,ug/ml, the coat protein migrated as a diffuse band. The molecular weight for the peak fraction was between that of the dimer and the monomer. This behavior is typical for Sephadex filtration of a protein showing aggregation tendencies (2) and indicates the existence of a dynamic equilibrium between monomers and dimers in the solutions of coat protein. At coat protein concentrations higher than 200 ,ug/ml, coat protein migrates as a single band with a molecular weight from 26,000 to 27,000. Therefore, at the concentration at which the maximum of complex I formation occurs, coat protein exists in the form of a dimer.
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REPRESSOR OF f2 RNA POLYMERASE SYNTHESIS
235
solution and binds cooperatively to a nucleic acid strand (1, 5). The specificity of the interacX 28 tion of phage coat protein dimers with RNA cseems, however, to be higher than that of 32-protein with DNA since coat protein binds to 24 .7 a) a single specific site on RNA (4). There seems to be a correlation of our model L 20 with the sequence of events in the phage life cycle. It is known that shortly after infection D 16 -t coat protein and phage RNA polymerase are produced in the host cell. The first portions of O 12 the synthesized coat protein do not exert any I II II I repressive activity. Only at a late period, when 100 200 300 400 500 600 700 the concentration of coat protein is probably Coa proleih concenLcion ,Lc/mL. sufficiently high to promote the formation of FIG. 5. Effect of concentration on the molecular coat protein dimers, does the translational weight of the coat protein, estimated by Sephadex gel repression mechanism start to operate. I0 9
filtration. 14C-labeled coat protein of specific activity 3,000 counts/lg per 10 min was used.
Comparing the curves presented in Fig. 2 and 5, one can see that the decrease of concentration of coat protein dimers in the solution is accompanied by a decrease in complex I formation. It should be noted that at concentrations of coat protein far exceeding those required for the complex I formation the oligomers heavier than dimers were not detected, either in Sephadex filtration or in the sedimentation analysis. It can be concluded, therefore, that, under conditions favoring complex I formation, hexamers of coat protein did not exist in the solution. The existence of stable coat protein dimers was observed at low pH by Rohrmann and Krueger (10). Under experimental conditions that favor the repression of the RNA polymerase gene, at neutral pH, coat protein dimers did not form phage shells nor did they precipitate, even at a high protein concentration. However, when 2-mercaptoethanol was omitted from the incubation mixture, coat protein precipitated (20). The conclusion from our work is that the formation of the complex responsible for repression of the RNA polymerase cistron proceeds via weak interaction of the coat protein dimer with a definite fragment of the RNA molecule. This results in an unstable attachment of the dimer to RNA. Stability is attained after cooperative attachment of two further dimers. Repression of the synthesis of RNA polymerase occurs only when a stable coat protein hexamer is formed on the RNA strand. The proposed mechanism of complex I formation resembles to some extent the interaction of "32-protein" of bacteriophage T4 with T4 RNA. This protein shows a pronounced affinity to single-stranded DNA. It exists as a dimer in
ACKNOWLEDGMENTS This work was supported by the Polish Academy of Sciences project no. 09.3.1 and by grant FG-Po-334 from the U.S. Department of Agriculture. Helpful discussions with P. Szafranski, A. S. Spirin, and Paul Kaesberg are gratefully acknowledged. We wish to thank E. Borkowska for technical assistance. LITERATURE CITED 1. Alberts, B. M., and L. Frey. 1970. T4 bacteriophage gene 32: a structural protein in the replication and recombination of DNA. Nature (London) 227:1313-1318. 2. Andrews, P. 1964. Estimation of the molecular weights of protein by Sephadex gel-filtration. Biochem. J.
91:222-233. 3. Baltimore, D., and A. S. Huang. 1968. Isopycnic separation of subcellular components from poliovirus-infected and normal HeLa cells. Science 162:572-574. 4. Bernardi, A., and P. F. Spahr. 1972. Nucleotide sequence at the binding site for coat protein on RNA of bacteriophage R17. Proc. Natl. Acad. Sci. U.S.A. 69:3033-3037. 5. Caroll, L. B., K. E. Neet, and D. A. Goldthwait. 1972. Self-association of gene-32 protein of bacteriophage T4. Proc. Natl. Acad. Sci. U.S.A. 63:2741-2744. 6. Chroboczek, J., M. Pietrzak, and W. Zag6rski. 1973. Specificity of formation of complexes between coat protein and bacteriophage f2 RNA. J. Virol.
12:230-240. 7. Eggen, K., and D. Nathans. 1969. Regulation of protein
8.
9.
10.
11. 12.
synthesis directed by coliphage MS2 RNA. II. In vitro repression by phage coat protein. J. Mol. Biol. 39:293-305. Hohn, T. 1969. Studies on a possible precursor in the self assembly of the bacteriophage f2. Eur. J. Biochem. 8:552-556. Patterson, M. S., and R. C. Greene. 1965. Measurement of low energy beta-emitters in aqueous solution by liquid scintillation counting of emulsions. Anal. Chem. 37:854-857. Rohrmann, G. F., and R. G. Krueger. 1970. Physical, biochemical, and immunological properties of coliphage MS2 particles. J. Virol. 6:269-279. Spahr, P. F., M. Faber, and R. F. Gesteland. 1969. Binding site on R17 RNA for coat protein. Nature (London) 222:455-458. Steitz, J. A. 1968. Identification of the A protein as a structural component of bacteriophage R17. J. Mol. Biol. 33:923-936.
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13. Sugiyama, T., R. R. Hebert, and K. A. Hartman. 1967. Ribonucleoprotein complexes formed between bacteriophage MS2 RNA and MS2 protein in vitro. J. Mol. Biol. 25:455-463. 14. Sugiyama, T., B. D. Korant, and K. K. Lonberg-Holm. 1972. RNA virus gene expression and its control. Annu. Rev. Microbiol. 26:467-502. 15. Sugiyama, T., and D. Nakada. 1968. Translational con-
trol of bacteriophage MS2 RNA cistrons by MS2 coat protein: polyacrylamide gel electrophoretic analysis of proteins synthesized in vitro. J. Mol. Biol. 31:431-439. 16. Ward, R., M. Strand, and R. C. Valentine. 1968. Translational repression of f2 protein synthesis. Biochem. Biophys. Res. Commun. 30:310-317. 17. Weber, K., and W. Konigsberg. 1967. Amino acid se-
J. VIROL. of the f2 coat protein. J. Biol. Chem. 242:3563-3578. Webster, R. E., D. L. Engelhardt, N. D. Zinder, and W. Konigsberg. 1967. Amber mutants and chain termination in vitro. J. Mol. Biol. 29:27-43. Yphantis, D. A. 1964. Equilibrium ultracentrifugation of dilute solutions. Biochemistry 3:297-317. Zag6rska, L., J. Chroboczek, and W. Zag6rski. 1975. Template activity of complexes formed between bacteriophage f2 RNA and coat protein. J. Virol. 15:509-514. Zag6rski, W., W. Filipowicz, A. Wodnar, A. Leonowicz, L. Zag6rska, and P. Szafraniski. 1972. The effect of magnesium-ion concentration on the translation of phage f2 RNA in a cell-free system of Escherichia coli. Eur. J. Biochem. 25:315-322. quence
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21.
Vol. 16, No. 2 Printed in U.S.A.
Hexamer of Bacteriophage f2 Coat Protein as a Repressor of Bacteriophage RNA Polymerase Synthesis JADWIGA CHROBOCZEK AND WTODZIMIERZ ZAG0RSKI*I
Department of Protein Biosynthesis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Rakowiecka 36, 02-532, Warsaw, Poland Received for publication 10 March 1975
Formation of complex I between phage f2 RNA and coat protein, leading to repression of phage RNA polymerase synthesis, depends nonlinearly upon the concentration of the coat protein. Maximum formation of complex I was observed when six molecules of coat protein were bound to one molecule of RNA. RNase digestion of a glutaraldehyde-fixed complex left, as the products, coat protein oligomers. The heaviest, hexamers, predominated in the mixture. It was also shown that, in an ionic environment required for phage protein synthesis, coat protein at a concentration optimum for complex I formation exists in solution as a dimer. The results indicate that the translational repression of the RNA polymerase cistron is due to a cooperative attachment to phage template of three dimers of coat protein, forming a hexameric cluster on an RNA strand.
Coat protein of small RNA bacteriophage is known to react with phage RNA, forming what is called complex I. I'his complex behaves as a modulated messenger in the in vitro proteinsynthesizing system. Specific inhibition of the translation of the RNA polymerase is observed when phage RNA is incubated in the presence of a few moles of coat protein per 1 mol of RNA. The interaction between phage coat protein and homologous RNA can be regarded as a model system for studying regulation of gene expression on the translational level. This resulted in considerable attention being paid to the problem of complex I formation (for review of the problem, see reference 14). However, the mechanism of formation of complex I still remains an open question. The uncertainties are concerned with the molar ratio of protein to RNA in the complex as well as the mutual positioning of protein molecules present in complex I. Spahr et al. (11) suggest that in complex I only one molecule of coat protein is strongly bound to the RNA strand and that the attachment of additional portions of coat protein is associated with processes of phage particle formation. Results from other laboratories, however, are different. Sugiyama and Nakada (15) assumed that 1 mol of RNA specifically binds several (less than 10) moles of coat protein. Eggen and Nathans (7) suggested that full repression of RNA polymerase synthesis occurs when 3 to 4 mol of coat protein is bound to the phage template. Ward et ' Present address: Biophysics Laboratory, University of Wisconsin, Madison, Wis. 53706.
al. (16) reported that the repression is due to the attachment of six molecules of coat protein to one RNA strand. Also, the problem of coat protein distribution along the phage RNA strand in complex I is still open to discussion. It was suggested by Hohn (8) that the six molecules of coat protein present in complex I are bound at several sites on the RNA strand, whereas Ward et al. (16) assumed that the coat protein molecules in complex I are attached at a single site of the RNA molecule. It is evident that different evaluations of the number of coat protein molecules present in complex I per one RNA molecule lead to different proposals concerning the mechanism of complex formation. A protein/RNA molar ratio of 1:1 in the complex would suggest that the ability of recognition of the proper site on the RNA strand is an intrinsic property of the coat protein monomer. In contrast, results showing that RNA specifically binds several protein molecules may suggest that oligomeric forms of coat protein are involved in recognition of a specific fragment of the RNA molecule. In this paper we present results showing that, under conditions favoring complex I formation, coat protein exists in solution in the form of dimers. On the other hand, in the complex, we found hexamers of coat protein attached to RNA strands. On this basis, we suggest that the formation of complex I is due to the cooperative attachment of three coat protein dimers to the phage template. This leads to the formation of a hexameric cluster of the coat protein molecules on the RNA strand.
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MATERIALS AND METHODS General. The methods of isolation of phage f2 and phage RNA and the conditions of incorporation of amino acids in the Escherichia coli cell-free system were described previously (6, 20, 21). RNA concentration was determined by its optical density at 260 nm using an extinction coefficient equal to 24 ml/mg per cm (18). The [140 lserine- or [3H ]uridine-labeled phage was prepared by the method of Steitz (12). Coat protein was isolated by the acetic acid extraction method as described by Eggen and Nathans (7). The extinction coefficient value at 280 nm of 1.1 mi/mg per cm was used to measure the concentration of phage coat protein (13). The homogeneity of each of the RNA preparations was checked by analytical ultracentrifugation in 5 mM Tris-hydrochloride buffer (pH 7.2) containing 0.2 M NaCl. f2 RNA (70 to 80%) was found to sediment in the 27S region. Sedimentation analysis of formaldehyde-treated f2 RNA in formaldehyde-sucrose gradients (11) also gave the same results. The following procedures were performed with buffer A (64.2 mM Tris-hydrochloride [pH 7.6], 14.7 mM magnesium acetate, 27.6 mM NH4Cl, 30 mM KCl, and 7 mM 2-mercaptoethanol): formation of complexes between coat protein and phage RNA, nucleolytic treatment of complexes, gradient analysis, Sephadex gel filtration and sedimentation analysis of coat protein. This buffer provides optimum salt conditions for phage protein synthesis in vitro. Formation of complexes between coat protein and f2 RNA. The 14C-labeled coat protein and 3H-labeled or unlabeled phage RNA at various concentrations were incubated in buffer A for 20 min at 25 C. Complexes were stabilized, by the method of Baltimore and Huang (3), by incubation for 10 min at 0 C in the presence of 2.5% glutaraldehyde. The mixtures were then layered upon 4.5 ml of linear (5 to 20%) sucrose gradients prepared in buffer A. Centrifugation was carried out for 100 min at 48,000 rpm and 4 C in an SW50 rotor in a Spinco model L ultracentrifuge. Gradients were fractionated from the bottom of the tube, and the radioactivity of the fractions was measured on filter paper disks (Whatman 3MM) as described previously (6). Treatment with nucleolytic enzymes and gel filtration. The gradient fractions containing the RNA-protein complexes were pooled and treated with 10 U of RNase T2 (Calbiochem) and 1 mg of snake venom Puretoxin (Miami Serpentarium Laboratory, Miami, Fla.). After 20 min of incubation at 37 C, the mixture was layered on the top of a Sephadex gel G-100 column (2.1 by 65 cm) equipped with a water jacket and cooled by running tap water (14 C). The column was equilibrated previously with buffer A. Gel filtration. Chromatography was carried out with the same buffer at a flow rate of 12 ml/h. Fractions of 1.2 ml were collected. The radioactivity in 1-ml samples of each fraction was determined by using the toluene-Triton X-100-based scintillation mixture of Patterson and Greene (9). The molecular weight of the coat protein oligomers was calculated from a calibration curve obtained by separate runs of: alkaline phosphatase from calf mucosa (Sigma Chem-
229
ical Co.), molecular weight 80,000; bovine serum albumin, fraction V (Serva, Heidelberg, Germany), molecular weight 67,000; pepsin, crystallized twice (Fluka), molecular weight 35,000; RNase from bovine pancreas, crystallized five times (Calbiochem), molecular weight 13,700. Aggregation of coat protein in solution. Coat protein aggregation was analyzed by gel filtration on a Sephadex G-100 column and checked by sedimentation analysis. Solutions of '4C-labeled coat protein in different concentrations, ranging up to 0.7 mg per ml of buffer A, were submitted to gel filtration under the conditions described above. The molecular weight for peak fractions was estimated and plotted as a function of coat protein concentration in the sample applied to the column. Sedimentation analysis was performed with 1.5 mg of coat protein in 1 ml of buffer A in a Spinco model E analytical ultracentrifuge. Molecular weight was determined by equilibrium ultracentrifugation for 5 h at 36,000 rpm and 20 C by the method of Yphantis (19).
RESULTS AND DISCUSSION Effect of coat protein concentration on the formation of complex I. As we showed previously, formation of complexes between coat protein and phage RNA is strongly influenced by ionic conditions in the incubation mixture (6). To assure that the complexes under study were those responsible for repression of RNA polymerase synthesis, we formed and analyzed complexes in an ionic environment needed for phage protein synthesis (21). The complexes were stabilized by glutaraldehyde treatment (17). This diminished possible dissociation of the complex due to extensive dilution accompanying Sephadex filtration. Under our experimental conditions, glutaraldehyde treatment did not promote the binding of additional portions of coat protein to phage RNA. On the other hand, glutaraldehyde-fixed complexes are more stable than unfixed (20). As previously reported (20), maximum repression was observed when about six molecules of coat protein were bound to one RNA strand in the complex. To analyze the kinetics of the formation of such multimeric complexes, we studied the dependence of the reaction on the concentration of RNA and protein in the incubation mixture. The formation of complex I illustrated in Fig. la was performed at an RNA concentration of 1.000 jg/ml and at a coat protein/RNA input molar ratio of 16. The molar ratio of the components in complex I was 6.4. In a parallel experiment, performed at an RNA concentration of 233 gg/ml and an input molar ratio of 20, only 0.9 mol of coat protein per 1 mol of RNA was recovered in complex I (Fig. lb). This
230
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CHROBOCZEK AND ZAG0RSKI
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Frac[ion no. FIG. 1. Complex formation between f2 coat protein and f2 RNA. The conditions for complex formation and the method of analysis of gradients are described in the text. (a) Phage 3H-labeled RNA (390 mg; specific activity 224 counts/lg per 10 min) was incubated with 80.1 ,ug of "4C-labeled coat protein (specific activity 426 counts/ltg per 10 min) in a total volume of 0.4 ml of buffer A at 25 C for 25 min. After incubation, the mixture was chilled in an ice bath, and the complex was fixed with glutaraldehyde and layered on the top of a sucrose gradient. Input molar ratio of coat protein to RNA is equal to 16. (b) 3H-labeled RNA (70 lg; specific activity 1,380 counts/lg per 10 min) was incubated with 17 Aig of "4C-labeled coat protein (specific activity 2,300 counts/ulg per 10 min) in a total volume of 0.3 ml of buffer A. Other details as in (a). Input molar ratio of coat protein to RNA is equal to 20. Symbols: 0, 3H label in RNA; *, 14C label in coat protein.
indicates that the formation of complex I depends strongly on the concentration of the components in the mixture and not only on the molar input ratio of coat protein to RNA. To test this assumption, we performed a series of experiments resembling those in Fig. 1, incubating RNA at two different concentrations with increasing amounts of coat protein. After gradient centrifugation, the molar ratios of the components in complex I were calculated and plotted versus the input molar ratios. The saturation curves obtained are shown in Fig 2a and b. The first portions of coat protein did not promote complex formation (Fig. 2a). The addition of further portions of coat protein initiated formation of complex I. From this point the curve in Fig. 2a sharply rises. It reaches a plateau at about six protein molecules per one molecule of RNA in complex I, the input molar ratio being about 20. The sigmoidal character of the curve suggests that complex I formation involves a cooperative binding of several coat protein molecules to one RNA strand. Since this type of interaction should show a pronounced concentration dependence, we performed the saturation experiments with approximately four-times-lower concentrations of RNA and coat protein, and the molar ratio of protein to RNA in the complex was plotted versus input
protein to RNA ratios (Fig. 2b). In this case the experimental points also fall on a sigmoidal curve; however, there is a lower slope throughout the entire region of input molar ratios. It can be seen that in Fig. 2b saturation is reached at a much higher input molar ratio of coat protein to RNA than that of the experiment performed at the higher RNA concentration (Fig. 2a). In contrast, when results are expressed as a function of coat protein concentration in the incubation mixture, it became evident that in both cases presented in Fig. 2 saturation was observed at about the same concentration of coat protein, i.e., at 200 to 250 ug/ml. This shows clearly that the formation of complex I occurs at defined ranges of protein concentration and that it depends not only on the input molar ratio of the protein to RNA, but it also depends strongly on the concentration of coat protein in the incubation mixture. We noted that, compared with curve 2a, curve 2b reaches a plateau at slightly higher amounts of coat protein in the complex. This can be due to the fact that in this experiment the plateau is observed at high input protein/ RNA ratios. Under such conditions, processes of the formation of phage-like particles may be initiated, leading to the enhancement of protein to RNA ratio in the complex. Next we showed that, similar to complex I
VOL. 16, 1975
REPRESSOR OF f2 RNA POLYMERASE SYNTHESIS
formation, the repression of the histidine incorporation in the phage RNA-directed cell-free system depends more on the concentration of coat protein in the incubation mixture than on the input molar ratio of components (see Fig. 3). At an RNA concentration of 280 gg/ml, maximum repression was observed at a molar ratio of about 60 for the components in the incubation mixture, whereas, at a higher concentration of RNA (equal to 800 ,ug/ml), the same effect was observed with a molar ratio of components of about 20. In both cases, maximum repression was observed at the same coat protein concentration of about 180,ug/ml. This resembles the effect of coat protein concentration upon formation of complex I. The slopes of repression curves (Fig. 3) are, however, different from those of complex formation (Fig. 2). We assume that the observed differences result from different techniques of analysis in both types of experiments. For example, gradient analysis of complex I was done at 4 C, whereas repression was measured at 37 C. Size of coat protein oligomers formed on
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231
the RNA strand. The sigmoidal character of the saturation curve (Fig. 2) implies that the coat protein molecules bound in complex I interact with each other, forming an oligomeric structure on a strand of RNA. To test this hypothesis, the following experiment was performed. Complex I, stabilized with glutaraldehyde and sedimented through a sucrose gradient, was treated with a mixture of the RNA-hydrolyzing enzymes, and the hydrolysate was filtered through Sephadex G-100 gel (Fig. 4). When complex I was formed at saturation of RNA with coat protein, the molar ratio of coat protein to RNA was about 6 (Fig. 4a). The gel filtration profile of the products of hydrolysis of the glutaraldehyde-fixed complex exhibits six peaks. The peaks represent various classes of "4C-labeled coat protein oligomers (Fig. 4b). From the calibration curve for the Sephaaex column, molecular weights for the peaks I-VI were established as follows: 19,000, 29,000, 32,000, 46,000, 61,000, and 76,000. In the next experiment (Fig. 4c), complex I was formed
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FIG. 2. Binding off2 coat protein to f2 RNA-saturation curves. The ratio of coat protein to RNA in the peak fraction of the sucrose gradients is plotted as a function of the concentration of coat protein as well as the input molar ratio of coat protein to RNA. (a) Portions of 160 ,ug each of RNA (specific activity 800 counts/ug per 10 min) were incubated -with appropriate amounts of coat protein (specific activity 1,090 counts/Mg per 10 min) in 0.2 ml of buffer A at 25 C for 20 min. (Final RNA concentration was 800 MAg/ml of buffer A.) After incubation and chilling in an ice bath, the complexes were fixed with glutaraldehyde and the mixture was layered on the top of a sucrose gradient. (b) Portions of 70 sg each of RNA (specific activity 1,380 counts/Mg per 10 min) were incubated with appropriate amounts of coat protein in 0.3 ml of buffer A at 25 C for 20 min. (Final RNA concentration was 233 Mg/ml of buffer A.) After fixation as in (a), the mixtures were layered on the top of a sucrose gradient. Coat protein had specific activity of 2,300 counts/Mg per 10 min in the experiments with an input molar ratio of coat protein to RNA from 5 to 20, and 1,900 counts/lg per 10 min in the experiments with an input molar ratio of coat protein to RNA from 30 to 100.
232
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CHROBOCZEK AND ZAG0RSKI
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Protein/NA, moLar rolhio in incubationnrnxture FIG. 3. Effect of concentration of f2 coat protein on phage-specific protein synthesis. (a) Portions of 36.2 Mg each of f2 RNA were incubated with appropriate amounts of coat protein for 20 min at 25 C in 120 Il of mixture containing: 0.03 Amol of guanosine 5'-triphosphate, 0.4 Mmol of adenosine 5'-triphosphate, 1.7 Mmol of phosphoenolpyruvate, 80 Mg of E. coli tRNA, 0.9 Mg of pyruvate kinase, 0.0125 Amol of each nonradioactive amino acid, 0.006 MCi of ["4C]histidine (specific activity 100 MCi/mmol) or 0.006 MCi of ["4C]alanine (specific activity 10 uCi/mmol), and salts as in buffer A. A 5-Ml volume (0.2 mg of protein) of E. coli S-30 extract was added, and each mixture was incubated for 30 min at 37 C. Samples were analyzed for ["4C]histidine and ["4C]alanine incorporation. Results are expressed as the percentage of histidine and alanine incorporation versus molar ratio of protein to RNA. In the absence of f2 RNA, the incorporation was 720 counts/min of ["4C]histidine and 108 counts/min of ["4C]alanine (blank values). In the absence of coat protein, the incorporation was 5,330 counts/min of ["4C]histidine and 1,230 counts/min of ["4C]alanine. Net incorporations in the absence of coat protein were taken as 100%)6. (b) Experimental procedure as in (a), but the incubation mixtures contained 100 Mg f2 RNA each. In the absence of coat protein, the incorporation was 9,440 counts/min of ["4C]histidine and 3,770 counts/min of ["4C]alanine per 125 ul of mixture. Symbols: 0, ["4C]histidine; 0, ["4CJalanine.
under conditions of partial saturation of RNA with coat protein. The molar ratio of coat protein to RNA in complex I was about 2, as determined by the density gradient method. Again, six classes of protein oligomers appeared as the products of RNase treatment of the complex (Fig. 4d). The molecular weights for the peaks I-VI were: 17,500, 26,500, 35,000, 44,000, 62,000, and 80,000. The molecular weight of the coat protein monomer is 13,700 (17). By dividing the series of molecular weights for peaks I-VI by 13,700, the following sequences of values are obtained: 1.3, 2.1, 2.3, 3.4, 4.4, and 5.5 for Fig. 4b; and 1.2, 1.9, 2.6, 3.2, 4.5, and 5.9 for Fig. 4d. From this, we conclude that peak VI represents the hexamer of coat protein. It is of importance that, among oligomers detected during the gel filtration, the hexamer predominates and represents the heaviest component found in the mixture (Fig. 4). This and the fact that the plateau value of the curves in Fig. 2 is equal to six indicate that, in complex I, six
molecules of protein are bound to one strand of RNA and they interact with each other. The distances between protein subunits in complex I are such that glutaraldehyde can cross-link coat protein molecules. The lighter peaks (I to V) found during Sephadex filtration tentatively represent the following oligomeric forms of coat protein: monomers, dimers, trimers, tetramers, and pentamers. We believe that the components lighter than hexamers found during gel filtration originate from incompletely crosslinked hexamers. The incomplete cross-linking can result in the appearance of association-dissociation phenomena during gel filtration. The observed deviations from the expected molecular weights of the coat protein oligomers may be attributed to these phenomena. The lack of complete cross-linking is due to the mild conditions of glutaraldehyde treatment. More drastic conditions of fixation (incubation at 37 C for 1 h) result, however, in precipitation of complex I. It should be emphasized that, when the solution of coat protein in buffer A was sub-
VOL. 16, 1975 VREPRESSOR OF f2 RNA POLYMERASE SYNTHESIS
jected to exactly the same procedure and analysis as the aforementioned (including fixation with glutaraldehyde), no components heavier than dimers were detected on Sephadex gels. The results (Fig. 4b) indicate that the six molecules of coat protein, which are present in complex I, are not randomly spread along the RNA molecule, but they form a hexamer attached to the RNA molecule at a single site. This contradicts the results of Hohn (8), who
233
observed that RNase treatment liberated from complex I only monomers of coat protein. The effect reported by Hohn can be attributed to the dissociation of unfixed coat protein hexamers after detachment from the RNA strand. When the molar ratio of components (as determined by density gradient analysis) was below 6, again the hexamers predominated among detected oligomers of coat protein. This indicates that there was no random distribution
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Fraction no. FIG. 4. Gel filtration of coat protein oligomers obtained after digestion of complex I with nucleolytic enzymes. (a) A 320-ug amount of unlabeled f2 RNA was incubated with 100 ,g of "C-labeled coat protein (specific activity 3,000 counts/gg per 10 min) in a final volume of 0.5 ml of buffer A. The incubation mixture was layered on the top of a sucrose gradient and centrifuged. Three gradients run in parallel were fractionated, and the parallel fractions were pooled. Absorbancy at 260 nm and radioactivity of each fraction were measured. (b) Fractions 12 to 20 from the gradient presented in (a) were pooled and treated with RNase, and the mixture was subjected to Sephadex gel filtration. (c) Experimental procedure as in (a), but 250 tig of RNA was incubated with 56Mg of coat protein. (d) Fractions 8 to 15 from the gradient presented in (c) were pooled and treated with RNase, and the mixture was subjected to Sephadex gel filtration. Symbols: 0, absorbancy at 260 nm; 0, "4C-labeled coat protein.
234
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CHROBOCZEK AND ZAG0RSKI
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FracLon no. FIG. 4c and d. of coat protein molecules between the RNA strands. There are RNA strands that are occupied by hexamers of coat protein and others that are naked. The naked RNA strands sediment at the same position as does complex I. Due to this the molar ratio of protein to RNA in fractions from the sucrose gradient is lowered. The above discussed mechanism of formation of complex I implies that full repression of RNA polymerase synthesis in vitro can be obtained when all RNA strands are saturated with the hexameric repressor. This supposition was confirmed by testing the template activity of complexes isolated directly from the density gradients. It was observed that full repression of the RNA polymerase synthesis is obtained only when the molar ratio of coat protein to RNA in complex I was about 6 (20). Behavior of coat protein in solution. Formation of complex I is observed at definite concentrations of coat protein (Fig. 2). Therefore, one can expect the existence of concentration-dependent aggregation of coat protein molecules in solution into oligomers, which bind to RNA, forming complex I. To check this supposition, the sedimentation behavior of coat pro-
tein in buffer A was studied in an analytical ultracentrifuge. At a concentration of 1,500 ,ug/ml, the coat protein behaves as a dimer with a molecular weight about 26,500 and a sedimentation coefficient equal to 2.55. The behavior of the coat protein at concentrations close to those used for formation of complex I (Fig. 2) was studied by gel filtration. The effect of varying coat protein concentrations on its molecular weight is shown in Fig. 5. When the concentration of coat protein in the sample applied on the column was below 200,ug/ml, the coat protein migrated as a diffuse band. The molecular weight for the peak fraction was between that of the dimer and the monomer. This behavior is typical for Sephadex filtration of a protein showing aggregation tendencies (2) and indicates the existence of a dynamic equilibrium between monomers and dimers in the solutions of coat protein. At coat protein concentrations higher than 200 ,ug/ml, coat protein migrates as a single band with a molecular weight from 26,000 to 27,000. Therefore, at the concentration at which the maximum of complex I formation occurs, coat protein exists in the form of a dimer.
VOL. 16, 1975
REPRESSOR OF f2 RNA POLYMERASE SYNTHESIS
235
solution and binds cooperatively to a nucleic acid strand (1, 5). The specificity of the interacX 28 tion of phage coat protein dimers with RNA cseems, however, to be higher than that of 32-protein with DNA since coat protein binds to 24 .7 a) a single specific site on RNA (4). There seems to be a correlation of our model L 20 with the sequence of events in the phage life cycle. It is known that shortly after infection D 16 -t coat protein and phage RNA polymerase are produced in the host cell. The first portions of O 12 the synthesized coat protein do not exert any I II II I repressive activity. Only at a late period, when 100 200 300 400 500 600 700 the concentration of coat protein is probably Coa proleih concenLcion ,Lc/mL. sufficiently high to promote the formation of FIG. 5. Effect of concentration on the molecular coat protein dimers, does the translational weight of the coat protein, estimated by Sephadex gel repression mechanism start to operate. I0 9
filtration. 14C-labeled coat protein of specific activity 3,000 counts/lg per 10 min was used.
Comparing the curves presented in Fig. 2 and 5, one can see that the decrease of concentration of coat protein dimers in the solution is accompanied by a decrease in complex I formation. It should be noted that at concentrations of coat protein far exceeding those required for the complex I formation the oligomers heavier than dimers were not detected, either in Sephadex filtration or in the sedimentation analysis. It can be concluded, therefore, that, under conditions favoring complex I formation, hexamers of coat protein did not exist in the solution. The existence of stable coat protein dimers was observed at low pH by Rohrmann and Krueger (10). Under experimental conditions that favor the repression of the RNA polymerase gene, at neutral pH, coat protein dimers did not form phage shells nor did they precipitate, even at a high protein concentration. However, when 2-mercaptoethanol was omitted from the incubation mixture, coat protein precipitated (20). The conclusion from our work is that the formation of the complex responsible for repression of the RNA polymerase cistron proceeds via weak interaction of the coat protein dimer with a definite fragment of the RNA molecule. This results in an unstable attachment of the dimer to RNA. Stability is attained after cooperative attachment of two further dimers. Repression of the synthesis of RNA polymerase occurs only when a stable coat protein hexamer is formed on the RNA strand. The proposed mechanism of complex I formation resembles to some extent the interaction of "32-protein" of bacteriophage T4 with T4 RNA. This protein shows a pronounced affinity to single-stranded DNA. It exists as a dimer in
ACKNOWLEDGMENTS This work was supported by the Polish Academy of Sciences project no. 09.3.1 and by grant FG-Po-334 from the U.S. Department of Agriculture. Helpful discussions with P. Szafranski, A. S. Spirin, and Paul Kaesberg are gratefully acknowledged. We wish to thank E. Borkowska for technical assistance. LITERATURE CITED 1. Alberts, B. M., and L. Frey. 1970. T4 bacteriophage gene 32: a structural protein in the replication and recombination of DNA. Nature (London) 227:1313-1318. 2. Andrews, P. 1964. Estimation of the molecular weights of protein by Sephadex gel-filtration. Biochem. J.
91:222-233. 3. Baltimore, D., and A. S. Huang. 1968. Isopycnic separation of subcellular components from poliovirus-infected and normal HeLa cells. Science 162:572-574. 4. Bernardi, A., and P. F. Spahr. 1972. Nucleotide sequence at the binding site for coat protein on RNA of bacteriophage R17. Proc. Natl. Acad. Sci. U.S.A. 69:3033-3037. 5. Caroll, L. B., K. E. Neet, and D. A. Goldthwait. 1972. Self-association of gene-32 protein of bacteriophage T4. Proc. Natl. Acad. Sci. U.S.A. 63:2741-2744. 6. Chroboczek, J., M. Pietrzak, and W. Zag6rski. 1973. Specificity of formation of complexes between coat protein and bacteriophage f2 RNA. J. Virol.
12:230-240. 7. Eggen, K., and D. Nathans. 1969. Regulation of protein
8.
9.
10.
11. 12.
synthesis directed by coliphage MS2 RNA. II. In vitro repression by phage coat protein. J. Mol. Biol. 39:293-305. Hohn, T. 1969. Studies on a possible precursor in the self assembly of the bacteriophage f2. Eur. J. Biochem. 8:552-556. Patterson, M. S., and R. C. Greene. 1965. Measurement of low energy beta-emitters in aqueous solution by liquid scintillation counting of emulsions. Anal. Chem. 37:854-857. Rohrmann, G. F., and R. G. Krueger. 1970. Physical, biochemical, and immunological properties of coliphage MS2 particles. J. Virol. 6:269-279. Spahr, P. F., M. Faber, and R. F. Gesteland. 1969. Binding site on R17 RNA for coat protein. Nature (London) 222:455-458. Steitz, J. A. 1968. Identification of the A protein as a structural component of bacteriophage R17. J. Mol. Biol. 33:923-936.
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13. Sugiyama, T., R. R. Hebert, and K. A. Hartman. 1967. Ribonucleoprotein complexes formed between bacteriophage MS2 RNA and MS2 protein in vitro. J. Mol. Biol. 25:455-463. 14. Sugiyama, T., B. D. Korant, and K. K. Lonberg-Holm. 1972. RNA virus gene expression and its control. Annu. Rev. Microbiol. 26:467-502. 15. Sugiyama, T., and D. Nakada. 1968. Translational con-
trol of bacteriophage MS2 RNA cistrons by MS2 coat protein: polyacrylamide gel electrophoretic analysis of proteins synthesized in vitro. J. Mol. Biol. 31:431-439. 16. Ward, R., M. Strand, and R. C. Valentine. 1968. Translational repression of f2 protein synthesis. Biochem. Biophys. Res. Commun. 30:310-317. 17. Weber, K., and W. Konigsberg. 1967. Amino acid se-
J. VIROL. of the f2 coat protein. J. Biol. Chem. 242:3563-3578. Webster, R. E., D. L. Engelhardt, N. D. Zinder, and W. Konigsberg. 1967. Amber mutants and chain termination in vitro. J. Mol. Biol. 29:27-43. Yphantis, D. A. 1964. Equilibrium ultracentrifugation of dilute solutions. Biochemistry 3:297-317. Zag6rska, L., J. Chroboczek, and W. Zag6rski. 1975. Template activity of complexes formed between bacteriophage f2 RNA and coat protein. J. Virol. 15:509-514. Zag6rski, W., W. Filipowicz, A. Wodnar, A. Leonowicz, L. Zag6rska, and P. Szafraniski. 1972. The effect of magnesium-ion concentration on the translation of phage f2 RNA in a cell-free system of Escherichia coli. Eur. J. Biochem. 25:315-322. quence
18.
19. 20.
21.
