10 Q β Replicase

Qp R e p l i c e THOMAS BLUMENTHAL

I. Introduction . . . . . . . . . . . . . . . 11. Purification and Properties . . . . . . . . . A. Enzyme Assay . . . . . . . . . . . . B. Purification . . . . . . . . . . . . . . C. Structure of the Enzyme . . . . . . . . 111. Catalytic Properties . . . . . . . . . . . . A. The Reactions Catalyzed . . . . . . . . B. Initiation with Heterologous Templates . C. Initiation with Homologous Template . . D. Functions of S1 and Host Factor . . . . E. Role of EF-Tu.Ts in Initiation . . . . . F. Inhibitors of Initiation . . . . . . . . . G . Elongation . . . . . . . . . . . . . . H. Termination . . . . . . . . . . . . .

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267 269 269 270 270 273 273 273 275 276 277 278 278 279

I. Introduction

The small plus-strand RNA phages that infect Escherichia coli (i.e., QP, R17, f2, MS2) contain single-stranded RNA genomes 3600-4500 nucleotides in length. Replication of viral RNA is catalyzed by enzymes called RNA replicases, assembled after infection. RNA replication is accomplished by production of a single-stranded minus strand, which is then copied by the replicase to produce replicas of the infecting viral RNA. All RNA synthesis is initiated with a 5'-GTP and proceeds in a 5' + 3' direction. The first RNA replicase to be described and studied was isolated from 267 THE ENZYMES. VOL. X V Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-1?-1?2715-4

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E. cofi infected with the Group I phage MS2 ( 1 , 2). However, the enzyme made after infection with the Group I11 phage, QP, was found to be more stable and hence was chosen for in-depth study ( 3 ) . Partially purified QP replicase preparations were found to have a strong preference for homologous RNA ( 4 ) , but they could also copy synthetic RNA templates rich in cytidylate ( 5 , 6). Furthermore, the enzyme was able to make infectious product RNA in great excess over added template (7, 8). Homogeneous QP replicase is composed of four nonidentical subunits of molecular weights 70,000, 65,000, 45,000, and 35,000 (9, 10). Only the 65,000 MW polypeptide is phage-coded. The other three, all host-coded, are taken from the protein biosynthesis apparatus. The largest is 30 S ribosomal protein S1 and the other two are the protein synthesis elongation factors Tu and Ts (1 1-13). An additional host-coded protein called host factor (HF), which is a hexamer of 12,500 MW subunits, is also required forin vitro QP RNA replication (14, 15). The replicase of a Group I RNA phage, f2,has also been purified. This enzyme contains the same three host polypeptides, although it apparently uses a different H F (16, 17). Since the host proteins were designed to function in protein biosynthesis, determination of the roles they play in phage RNA replication is obviously of great interest. Although QP replicase has been noted for its template specificity, several reports indicate that the enzyme can be used to make RNA copies of a great variety of RNAs under certain conditions (f8).Thus QP replicase is a potentially valuable tool for modern molecular biology. 1. Haruna, I . , Nozu, K., Ohtaka, Y., and Spiegelman, S. (1963). PNAS 50, 905. 2. Weissmann, C . , Simon, L . , and Ochoa, S. (1963). PNAS 49, 407. 3. Haruna, I., and Spiegelman, S. (1965). PNAS 54, 579. 4. Haruna, I . , and Spiegelman, S . (1965). PNAS 54, 1189. 5 . Eikhom, T. S., and Spiegelman, S. (1967). PNAS 57, 1833. 6. Hori, K., Eoyang, L., and Bannerjee, A. K. (1967). PNAS 57, 1790. 7. Pace, N. R . , and Spiegelman, S. (1%6). Science 153, 64. 8. Spiegelman, S., Haruna, I., Holland, I. B., Beaudreau, G . , and Mills, D. R. (1965). PNAS 54, 919. 9. Kamen, R . I. (1970). Nuture (London) 228, 527. 10. Kondo, M., Gallerani, R., and Weissmann, C. (1970). Nature (London) 228, 525. 1 1 . Inouye, H . , Pollack, Y., and Petre, J. (1974). EJB 45, 109. 12. Wahba, A. J . , Miller, M. J . , Niveleau, A . , Landers, T. A., Carmichael, G . G . , Weber, K., Hawley, D. A . , and Slobin, L. I. (1974). JBC 249, 3314. 13. Blumenthal, T., Landers, T. A . , and Weber, K. (1972). PNAS 69, 1313. 14. Franze de Fernandez, M. T., Eoyang, L., and August, J. T. (1968). Nutiire (London) 219, 588. 15. Franze de Fernandez, M. T., Hayward, W. S . , and August, J. T. (1972).JBC 247,824. 16. Federoff, N. V., and Zinder, N. (1971). PNAS 68, 1838. 17. Fedoroff, N . V., and Zinder, N. D. (1973). Nriture N e w Biol. 241, 105. 18. Blumenthal, T., and Carmichael, G. G . (1979). Annic. Rev. Biochern. 48, 525.

10.

QP REPLICASE

II.

Purification and Properties

269

A. ENZYME ASSAY

QP replicase is purified using a poly(C)-dependent poly(G) polymerase assay ( I 9). This assay measures incorporation of 3H- or 14C-labeledGTP into trichloroacetic acid-precipitable material. The assay mixture contains Mgz+(or MnZf),poly(C), GTP, and a source of QP replicase. EDTA, a sulfhydryl reducing agent, and glycerol are usually added as well. Rifampicin, DNase, and inorganic phosphate should be added during early stages in the enzyme purification to inhibit contaminating enzymatic activities. Although only three of the QP replicase subunits (11, Tu and Ts) are required for the poly(C)-dependent activity (20), enzyme containing equimolar amounts of all four subunits is in fact obtained from the purification (21). The HF is present only in small amounts in the purified enzyme. HF is thus purified separately by assay of stimulation of in vitro QP RNA replication (15). All of the subunits of QP replicase, in addition to HF, are required for in litro QP RNA replication. This activity can be monitored either by incorporation of 3H- or 14C-labelednucleotides into acid-precipitable material, or by the production of infectious RNA. The latter is measured by infection of E. coli spheroplasts. The Qp RNA-dependent assay is not used for routine purification of QP replicase because both excess QP RNA and excess HF inhibit the reaction (22). Thus the concentrations of both must be very carefully chosen and the enzyme preparation must be assayed at several concentrations to assure linearity. Furthermore, care must be taken to keep the ionic strength reasonably high to ensure that transcription of QP RNA rather than of contaminating 6 S RNAs (see below) is being measured. The number of active enzyme molecules in a preparation can be measured by incorporation of [Y-~’P]GTP.Aurintricarboxylic acid, an initiation inhibitor, can be added shortly after initiation to ensure that only first-round synthesis is assayed (23). The extinction coefficient (El,) of QP replicase has been reported to be 0.65 ( 1 3 , but results from this laboratory give a value of 1.0. If the latter value and a molecular weight of 215,000 are chosen to calculate enzyme concentration, it is found that 95% 19. 20. 21. 22. 23. 24.

Kamen, R. I. (1972). BEA 262, 88. Kamen, R . , Kondo, M . , Romer, W., and Weissmann, C. (1972). EJE 31, 44. Blumenthal, T. (1979). In “Methods in Enzymology,” Vol. 60,p. 628. Kondo, M . , and Weissmann, C. (1972). EJB 24, 530. Blumenthal, T., and Landers, T. A. (1973). BBRC 55, 680. Brown, S., and Blumenthal, T. (1976). P N A S 73, 1131.

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of the enzyme molecules initiate transcription of poly(C), whereas only 25% initiate in the in 1Jtt-o QP RNA replication assay (24). B. PURIFICATION

QP replicase is usually purified fromE. coli cells infected with QP phage containing an amber mutation late in the coat protein, QPam12 or QPamB86. These phage overproduce the enzyme because the coat protein normally serves as a repressor of translation of the replicase gene (15, 25). The standard purification utilizes a polyethylene glycol-dextran phase extraction procedure to remove nucleic acids, followed by chromatography on three successive ionic exchange columns: DEAEcellulose, phosphocellulose, and DEAE-Sephadex. If the enzyme is not sufficiently pure at that stage, sedimentation on glycerol gradients can serve as a final purification step. Upon analysis by SDS gel electrophoresis, purified enzyme preparations are found to contain equimolar amounts of the four subunits (21).

c.

STRUCTURE OF THE

ENZYME

1, Sirhirnit Ident$cation

The phage-coded polypeptide (subunit 11) has been identified as the product of the replicase (or synthetase) gene (9, 10). All of the other three subunits are present before infection. The largest polypeptide has been identified as ribosomal protein S1, while the two smaller ones are the protein synthesis elongation factors Tu and Ts. These identifications were based on identity of molecular properties, such as molecular weight and amino acid sequence, as well as on functional interchangeability with these polypeptides isolated from uninfected cells ( I 1 -13). The f 2 replicase contains three host-coded polypeptides with molecular weights identical to those of QP replicase (16);they are presumed to be the same polypeptides. A variety of experiments demonstrate that all four subunits are integral parts of QP replicase and not contaminants: (1) The four subunits co-electrophorese in nondenaturing solvents but separate into four bands when electrophoresed in the presence of urea (26); (2) all four polypeptides co-chromatograph with the RNA synthesis activity throughout the purification (21);( 3 ) S l is required for in vitro QP RNA replication (20);(4) the 25. Palmenberg, A . , and Kaesberg, P. (1973). J . Virol. 11, 603. 26. Karnen, R. I. (1975). In "RNA Phages" (N. Zinder, ed.), p. 203. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

10. QP REPLICASE

27 1

elongation factors, acting as the EF-Tu.Ts complex (see below) are required to maintain the phage-coded polypeptide in an active conformation (27); ( 5 ) the rate of recovery of enzymatic activity during renaturation of QP replicase denatured in urea is controlled by the rate of renaturation of EF-Tu. Addition of native EF-Tu allows rapid recovery of activity (28); (6) replacement of EF-Tu in QP replicase with antibiotic-resistant EF-Tu from a mutant bacterial strain results in the formation of an unstable enzyme (29); (7) replacement of EF-Ts in QP replicase with EF-Ts from Bcrcillirs stecirotliermophilus results in the formation of QP replicase with several altered properties (30). The functions performed by the host polypeptides in RNA synthesis will be discussed in later sections. 2. Molecular Weight The sum of the molecular weights of the four subunits determined by SDS gel electrophoresis is 215,000. When the four polypeptides are covalently cross-linked by the bifunctional protein cross-linking reagent dimethylsuberimidate, a protein of approximately 215,000 molecular weight is seen on SDS gels (31). Furthermore the replicase activity elutes from sizing columns as if it had a molecular weight in this range (23).However, when QP replicase is analyzed by glycerol gradient sedimentation it behaves as if it had a molecular weight closer to 130,000. The low sedimentation value (approximately 7 S) is probably the result of an oblate shape conferred by ribosomal protein S1. An altered form of QP replicase lacking S1 also has a sedimentation constant of 7 S which is commensurate with the sum of its subunit molecular weights (145,000) (23).

3. Subunit Relationships Several lines of evidence suggest that QP replicase is composed of a relatively loose complex of two tighter subcomplexes, S1-I1 and EFTu.Ts. THe two subcomplexes appear to be bound to each other by nonionic interactions. If the enzyme is dialyzed into a low ionic strength buffer and then sedimented on glycerol gradients, it dissociates into the two subcomplexes (14). Furthermore, if QP replicase is treated with dimethyl suberimidate, covalent complexes of both the two subspecies, along with the complex of all four subunits, are found. As the ionic strength of the cross-linking mixture is increased, the amount of the large complex is increased at the expense of the two subcomplexes (31). 27. Landers, T. A., Blumenthal, T., and Weber, K. (1974). JBC 249, 5801. 28. Blumenthal, T., and Landers, T. A. (1975). Bicicliemistry 15, 422. 29. Blumenthal, T., Saari, B . , Van der Meide, P. H., and Bosch, L. (1980). JBC 255, 5300. 30. Stringfellow, L. E., and Blumenthal, T. (1982). To be submitted. 31. Young, R . A., and Blumenthal, T. (1975). JBC 250, 1829.

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The presence of RNA, particularly QP RNA, has been found to alter the relationship of the two subcomplexes. Sedimentation of QP replicase in the presence of RNA leads to release of the EF-Tu. Ts, whereas the S1-I1 complex remains bound to the RNA (10, 27). Also the presence of QP RNA completely prevents the covalent attachment of EF-Tu . Ts to S1-I1 by dimethyl suberimidate (31). However the EF-Tu Ts is not fully dissociated either in the presence of RNA (32) or during RNA synthesis, as indicated by continued sensitivity to antibodies to EF-Tu and EF-Ts following initiation (33). Thus, the following equation describes the subunit relationships of QP replicase as we currently understand them: 3 . 1 1 + Tu.Ts

Salt

RNA

S1.11.Tu.T~

where the S 1 * I1 + Tu . Ts represents a less tightly associated, rather than a fully dissociated, complex. A II*Tu.Ts complex can be isolated from QP replicase preparations. This enzyme will transcribe poly(C) but not QP RNA. Since the QP RNA replication activity can be regained by simply mixing S1 with II.Tu.Ts, S1 is not an important structural element of QP replicase (20). 4. Structurul Role of EF-Tu ' Ts

Neither S 1-11 nor EF-Tu .Ts alone has detectable RNA polymerizing activity. Furthermore if the two complexes are mixed together, only a small amount of QP replicase activity is recovered (14). However, substantial amounts of enzyme can be reconstituted if the S1-I1 is denatured in urea and then renatured in buffer containing EF-Tu-Ts (28). If the denatured S1-I1 is renatured in the absence of EF-Tu'Ts and then the EF-Tu.Ts is added, the activity is not regained (27). Thus the EF-TueTs is apparently involved in maintenance of enzyme structure. The rate of renaturation of denatured QP replicase is controlled by the rate of renaturation of EF-Tu. EF-Tu-dependent GDP binding and RNA synthetic activities are regained in parallel (28). If nondenatured EF-Tu or EF-Tu-Ts complex is added to renaturing QP replicase at the onset of renaturation, the enzymatic activity is recovered rapidly, even at low temperature, and all of the enzyme formed contains the exogenous EF-Tu (or EF-Tu-Ts) in place of the endogenous EF-Tu. This technique has been used to test the effects of a variety of alterations of the elongation factors on QP replicase reconstitution and activity. It has been found that when EF-Tu.Ts complexes, which are more stable than the endogenous 32. Blumenthal, T., Young, R. A . , and Brown, S. (1975). JBC 251, 2740. 33. Carmichael, G. G . , Landers, T. A . , and Weber, K. (1976). JBC 251, 2744.

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273

EF-Tu-Ts, are inserted by this technique large increases in both the rate and extent of reconstitution are observed. When covalently cross-linked EF-Tu-Ts or E. coli EF-Tu complexed with B. stearothermophilus EF-Ts (which form a very tight complex) are used, more than 50% of the initial QP replicase activity can be recovered (24, 30). Two additional experiments have also suggested that EF-Tu- Ts is important in stabilization of QP replicase structure. First, Sl-I1 forms large aggregates ( 2 1 1 S ) in the absence of EF-Tu . Ts (14). Second, if the EF-Tu is replaced by an antibiotic resistant mutant EF-Tu, the enzyme formed is extremely unstable even though the mutant EF-Tu is not itself more unstable than wild-type EF-Tu (29). 111.

Catalytic Properties

A. THEREACTIONS CATALYZED

QP replicase catalyzes synthesis of RNA in response to RNA templates. The enzyme initiates synthesis with GTP at or near the 3' end of the template and makes a complete complementary copy of the RNA by Watson-Crick base-pairing (34-36). An RNA primer can substitute for GTP to allow initiation (37). QP replicase has also been reported to be capable of autocatalytic RNA synthesis in the absence of template or primer, but since this reaction has not been studied in any detail it will not be considered further here (38). B. INITIATION WITH HETEROLOGOUS TEMPLATES Efficient in vitro QP RNA replication requires the presence of all four polypeptides of QP replicase as well as the host factor. However, the 11.Tu.T~complex by itself has the capability of transcribing QP RNA in addition to most other RNA species tested, but it does so at reduced efficiency (20, 39). With all templates the initiating nucleoside triphosphate is GTP. ITP, even at very high concentration, cannot substitute for GTP (37). Surprisingly, the amount of GTP required for a maximal initia34. August, J. T., Bannerjee, A. K . , Eoyang, L., Franze de Fernandez, M . T., Hori, K., Kuo, C. H., Rensing, U., and Shapiro, L. (1968). C S H S Q B 33, 73. 35. Billeter, M. A., Dahlberg, J. E., Goodman, H. M . , Hindley, J., and Weissman, C. (1969). C S H S Q E 34, 635. 36. Spiegelman, S . , Pace, N. R., Mills, D. R . , Levisohn, R., Eikhom, T. S . , Taylor, M. M . , Peterson, R . L . , and Bishop, D. H. L. (1968). C S H S Q E 33, 101. 37. Feix, G., and Hake, H. (1975). EERC 65, 503. 38. Sumper, M., and Luce, R. (1975). PNAS 72, 162. 39. Blumenthal, T., and Hill, D. (1980).JBC 255, 11713.

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tion rate varies widely depending on the template (40). Templates that are selected against by QP replicase, such as f2 RNA and 16 S rRNA, require much higher GTP concentration for initiation than favored templates like synthetic polymers containing cytidylate. It has long been known that MnZ+ions reduce the template specificity of Qp replicase (4. 41). Since Mn2+also reduces the GTP initiation requirement for all templates (40), Mn2+ may reduce template specificity by allowing initiation under more stringent conditions. QP replicase may manifest a high template specificity by forming complexes with heterologous RNA species that fail to initiate because of failure to form an efficient initiation site for GTP. Mn’+ions might reduce the template specificity by forming a complex with GTP that is more efficiently incorporated than is Mg2+.GTP with unfavored templates. Monovalent cations produce an effect opposite to that produced by Mn‘+ ions. That is, they increase the GTP requirement for initiation with all templates (40). This effect is presumably a result of the tighter association between S1.11 and EF-lb.Ts at higher ionic strength as mentioned previously. Thus it appears that a looser complex between the two subcomplexes favors formation of an efficient initiation site by QP replicase and template. In the reactions discussed above it is presumed that initiation occurs at or near the 3‘ end of the template, but this has not been demonstrated. However, several experiments have shown that a stretch of C residues at (or one base away from) the 3’ end is necessary for transcription of both homologous and synthetic templates (42, 43). Transcription of oligo(C) is prevented if poly(A) is ligated to the 3’ end, but the presence of a single 3’ G residue is not inhibitory (42). Apparently any RNA molecule can be transcribed by QP replicase if oligo(C) is added to the 3’ end (42). Clearly the base sequence at the 3’ end of the template is critically important to the initiation reaction, but the precise nature of the sequence restrictions has not been elucidated. The GTP-dependent initiation reaction can be bypassed by addition of a primer as short as a dinucleotide complementary to the template ( 3 7 , 4 0 ) . In the presence of an appropriate primer, GTP can be entirely replaced by ITP, which will not substitute for GTP in initiation (37). It is not known whether this technique allows internal initiation. Since primer-dependent synthesis is very efficient with seemingly any RNA template, this is a particularly good method for producing cRNA using QP replicase. Nearly 40. 41. 42. 43.

Blumenthal, T. (1980). PNAS 77, 2601. Palmenberg, A., and Kaesberg, P. (1974). PNAS 71, 1371. Feix, G . , and Sano, H. (1975). EJB 58, 59. Kuppers, B . , and Sumper, M. (1975). PNAS 72, 2640.

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275

QP REPLICASE

full length copies of 9 S globin RNA have been prepared using oligo(U) or oligo(dT) as primers complementary to the 3‘ poly(A) (44, 45).

c.

INITIATION

WITH

HOMOLOGOUS TEMPLATE

QP replicase has been reported to bind tenfold more tightly to QP RNA than to nonhomologous RNA molecules (46). Furthermore, the enzyme has been shown to bind to internal regions on QP RNA, and there is some evidence that this internal binding is a prerequisite for RNA replication (47, 48). Weissmann (49) has hypothesized that the process of replication begins with binding of the replicase to two internal RNA binding sites, termed “M” and “ S . ” According to this scheme the secondary structure of the RNA causes the 3’ end of the template to be correctly positioned for initiation of RNA synthesis. Although both biochemical and electron microscopic evidence have been adduced in support of this model, it has not been unequivocally demonstrated that binding of QP replicase to the internal regions of the RNA is actually involved in replication [see Ref. (18)for a more detailed consideration of this problem]. It has been shown, however, that the penultimate C residue at the 3‘ end of QP RNA is required for efficient replication (50). In addition to QP RNA, QP replicase can replicate a variety of 6 S RNAs (51-5.3). These molecules, ranging in size from 91-220 nucleotides, are found in QP-infected cells and as contaminants of QP replicase preparations. They have unique sequences with no apparent homology to QP RNA or to each other, but all have a short stretch of C residues near the 3‘ end. These residues have been shown to be required for replication (54). Like QP RNA the 6 S RNAs contain extensive secondary structure. QP replicase binds to internal regions of these RNAs ( 5 3 , but there is no evidence that it must do so to initiate replication. 44. Feix, G. (1976). Nature (London) 259, 593. 45. Vournakis, J. N . , Carmichael, G. G., and Efstratiadis, A. (1976). BBRC 70, 774. 46. Silverman, P. M. (1973). ABB 157, 234. 47. Meyer, F., Weber, H . , and Weissmann, C. (1981). J M B , in press. 48. Vollenweider, H . J., Koller, T., Weber, H . , and Weissmann, C. (1976).JMB 101, 367. 49. Weissmann, C. (1974). FEES Leu. 43, 10. 50. Rensing, U . , and August, J. T. (1969). Nature (London) 224, 853. 51. Kacian, D. L., Mills, D. R . , Kramer, F., and Spiegelman, S. (1972). PNAS 69,3038. 52. Mills, D. R . , Kramer, F. R . , and Spiegelman, S. (1973). Science 180, 916. 53. Schaffner, W., Ruegg, K. J . , and Weissrnann, C. (1977). J M B 117, 877. 54. Mills, D. R . , Kramer, F. R., Dobkin, C . , Nishihara, T., and Cole, P. (1980). Bioclietnistry 19, 228. 55. Mills, D. R . , Kramer, F. R., Dobkin, C . , Nishihara, T., and Spiegelman, S. (1977).I n “Nucleic Acid-Protein Recognition” (H. J. Vogel, ed.), p. 533. Academic Press, New York.

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OF s1 A N D HOSTFACTOR D. FUNCTIONS

The requirement for both S1 and the host factor (HF) is specific for initiation of QP RNA replication (15, 20). 6 S RNA replication does not require these polypeptides, even though the replicase binds to internal regions of these RNA species (55). Thus it seems likely that S1 and H F perform sequence-specific functions in QP RNA replication. S1 in the absence of the other subunits has been shown to bind to the “S” site mentioned previously as well as another site close to the 3’ end of QP RNA (56,57). H F also binds to two sites on QP RNA, both A-rich. One is the site near the 3’ end to which S1 binds (56); the other is a site about 670 nucleotides from the 3‘ end, which does not correspond to any of the previously identified sites hypothesized to be important in QP RNA replication (56). However, none of the binding sites described has been directly implicated in RNA recognition by QP replicase. This apparently site-specific binding may be simply a reflection of the high affinities for homogeneous synthetic RNA polymers characteristic of these two proteins, S1 for polypyrimidines and HF for poly(A) (58, 59). The host factor is a hexamer of heat-stable 12,500 MW polypeptide (15), although recent evidence suggests it probably acts in QP RNA replication as a larger aggregate (60). It binds much more tightly to intact QP RNA than to QP RNA that has been fragmented with RNase TI. Furthermore the affinity for QP RNA is reduced in parallel with the loss of the tendency to aggregate as ionic strength is increased (60). Thus it seems likely that an H F aggregate binds to multiple sites in the folded structure of QP RNA. This binding could result in an alteration in the RNA secondary structure. Since H F causes a reduction in the requirement for GTP for initiation of transcription of QP RNA but not of other templates, it may do so by causing a specific change in the RNA secondary structure (39). These results suggest the possibility that template specificity may reside in the HF rather than in the replicase. Apparently, however, both components show specificity. It has recently been reported that the replicases produced by QP and by the closely related phage SP can efficiently replicate the 6 S RNAs from either QP- or SP-infected cells. However, in both cases, the replication of the heterologous 6 S RNA was much more easily inhibited by salt and low substrate concentration than was the replication of the homologous 6 S RNA (61). The most likely interpretation of these 56. Senear, A., and Steitz, J . A . (1975). JEC 251, 1902. 57. Goelz, S . , and Steitz, J. A. (1977). JBC 252, 5177. 58. Carmichael, G. G. (1975). JBC 250, 6160. 59. Carmichael, G. G . , Weber, K., Niveleau, A., and Wahba, A. (1975).JEC 250, 3607. 60. de Haseth, P L., and Uhlenbeck, 0. C. (1980). Biochemistry 19, 6146. 61. Fukemi, Y., and Haruna, I. (1979). Molec. Gen. Genet. 169, 173.

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results is that the binding site for the initiating GTP is better with the homologous enzyme-template complexes than with the heterologous complexes. Since the replication of these 6 S RNA is an HF-independent reaction, these results indicate that template specificity can be expressed by the replicase alone. E. ROLEOF EF-Tu.Ts

IN

INITIATION

While it is not hard to imagine why polypeptides such as S1 and HF, which bind to single-stranded RNA, are found in an RNA replicase, it is more difficult to explain the presence of protein synthesis elongation factors in RNA synthesizing enzymes. Nevertheless two hypotheses have been advanced, both of which predict that the EF-Tu and EF-Ts would function in the initiation reaction. First, since EF-Tu binds aminoacyltRNA and since the 3' end of the phage RNA where replication initiates resembles the cloverleaf structure characteristic of tRNAs, the EF-Tu might be involved in binding the enzyme to the 3' end of the template. Second, since EF-Tu binds GTP tightly and specifically and since QP replicase uses only GTP to initiate RNA synthesis, the EF-Tu might supply the GTP binding site for initiation (13, 27). The fact that the phage-coded polypeptide is capable of elongating preinitiated polynucleotide chains at normal rates in the absence of EF-Tu and EF-Tu is consistent with an involvement of the protein synthesis elongation factors in initiation of RNA synthesis (27). The question of whether the RNA andor GTP binding functions of EF-Tu are actually utilized in the RNA replication process has been approached by using the denaturation-renaturation scheme described previously to insert modified EF-Tu in QP replicase. It has been demonstrated that contrary to expectations, EF-Tu modified by several different techniques such that it can no longer bind aminoacyl-tRNA, nevertheless functions apparently normally in QP replicase (62, 63). Furthermore EFTu * TS complex that has been cross-linked covalently with dimethyl suberimidate has been shown to substitute for EF-Tu and EF-Ts in QP replicase (24). Thus the elongation factors must act as a complex in QP replicase. Since the enzyme containing the cross-linked EF-Tu . Ts complex lacked detectable GTP binding activity characteristic of the EF-Tu in normal replicase, this experiment was interpreted as evidence that the EF-Tu GTP binding site was not used to supply the initiating GTP. However, the finding that EF-Tu .Ts from the thermophilic bacteria Thermirs thermophilus is able to bind GTP, but considerably more weakly than the 62. Brown, S . , and Blurnenthal, T. (1976). JBC 251, 2749. 63. Blumenthal, T., Douglas, J., and Smith, D. (1977). PNAS 74, 3264.

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THOMAS BLUMENTHAL

EF-Tu ( 6 4 ) ,opens the possibility that the cross-linked E. coli EF-Tu . Ts in QP replicase could be binding the initiating GTP, but that the binding was not detected in the filter-binding assays used. Therefore the question of whether the initiation site on QP replicase is the hypothetical EF-Tu. Ts GTP binding site remains open. The fact that substitution of either the endogenous EF-Ts with EF-Ts from Bcrcilliis srerrrotherrnophilus or of the endogenous EF-Tu.Ts with cross-linked EF-Tu*Ts results in an altered Ki for competitive inhibition of initiation by GDP, circumstantially implicates the EF-Tu.Ts complex as at least a component of the initiation site (30, 65).

F. INHIBITORS O F INITIATION Polyanions such as polyethylene sulfonate (66), aurintricarboxylic acid (23) [in this case a polymeric contaminant is the actual inhibitor (67)], and poly(U) (22, 68, 6 9 ) inhibit QP replicase initiation by competing with the template for binding to the enzyme. These polymers do not inhibit the elongation of preinitiated RNA chains. GDP and ppGpp, which cannot substitute for GTP in the initiation reaction, act as competitive inhibitors of initiation but not of elongation (65). Other ligands that interact with EF-Tu, such as TPCK and kirromycin, do not inhibit QP replicase, although kirromycin does inhibit renaturation of denatured enzyme (62). G. ELONGATION In spite of the fact that QP replicase catalyzes the production of a product RNA strand wholly complementary to the template RNA strand, in the homologous reaction at least, the product behaves as a fully singlestranded molecule (70). If the enzyme is removed a double-stranded product-template molecule is formed. Thus during the elongation reaction the product and the template are maintained as single-stranded entities. Presumably both the enzyme and intrastrand hydrogen bonding serve as barriers to annealing of product and template. Multistranded structures are found both in QP-infected cells and in Arai, K . , Arai, N . , Nakamura, S., and Kaziro, Y. (1978). EJB 92, 521. Blumenthal, T. (1977). BBA 478, 201. Kondo, M . , and Weissmann, C. (1972). BBA 59, 41. Gonzalez, R . G., Blackburn, B. J., and Schleich, T. (1980). BBA 562, 534. 68. Hori, K. (1973). J B Tokyo 74, 273. 69. Kondo, M. (1976). BJ 155, 461. 70. Weissmann, C . , Feix, G . , and Slor, H . (1968). CSffSQB 33, 83.

64. 65. 66. 67.

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replication reactions in vitro. These structures consist of a single plus or minus strand and several nascent strands (71, 72). However, these complex structures are not prerequisites for QP replicase-catalyzed RNA replication. Replication of a 6 S RNA molecule, MDV-1, can be carried out by a single enzyme molecule (73). The rate of MDV-1 elongation is variable. There are several discrete sites on the RNA where QP replicase pauses. These sites correspond to regions that have the ability to form 3’-terminal hairpin structures (74). Pausing could be due to formation of hairpin structures in either the nascent product or the portion of the template just copied, or both. QP replicase has a relatively high rate of base-pairing errors, about 1.6 transition mutations per doubling (75). This corresponds to a misreading frequency of 10-3-10-4. The lack of a proofreading function is presumably responsible for the high error rate. H. T E R M I N A T I O N Even though all RNA synthesis begins with GTP at the 5’ end, the 3’ end of all product strands in both QP RNA and 6 S RNA replication is an A residue (50, 76). This A must be added posttranscriptionally and is presumably integral to the termination process. The enzyme will not adenylate free completed plus or minus strands from which the terminal A has been removed (76).

71. 72. 73. 2038. 74. 75. 76.

Feix, G . , Slor, H., and Weissmann, C. (1967). P N A S 57, 1401. Hori, K. (1970). BBA 217, 394. Dobkin, C., Mills, D. R . , Kramer, F. R . , and Spiegelman, S . (1979). Biochemistry 18,

Mills, D. R . , Dobkin, C . , and Kramer, F. R . (1978). Cell 15, 541. Domingo, E., Sabo, D., Taniguchi, T., and Weissrnann, C. (1978). Cell 13, 735. Weber, H., and Weissmann, C. (1970). J M B 51, 215.