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• W2006021908 abstract "The coat proteins of the RNA bacteriophages Qβ and MS2 are specific RNA binding proteins. Although they possess common tertiary structures, they bind different RNA stem loops and thus provide useful models of specific protein-RNA recognition. Although the RNA-binding site of MS2 coat protein has been extensively characterized previously, little is known about Qβ. Here we describe the isolation of mutants that define the RNA-binding site of Qβ coat protein, showing that, as with MS2, it resides on the surface of a large β-sheet. Mutations are also described that convert Qβ coat protein to the RNA binding specificity of MS2. The results of these and other studies indicate that, although they bind different RNAs, the binding sites of the two coat proteins are sufficiently similar that each is easily converted by mutation to the RNA binding specificity of the other. The coat proteins of the RNA bacteriophages Qβ and MS2 are specific RNA binding proteins. Although they possess common tertiary structures, they bind different RNA stem loops and thus provide useful models of specific protein-RNA recognition. Although the RNA-binding site of MS2 coat protein has been extensively characterized previously, little is known about Qβ. Here we describe the isolation of mutants that define the RNA-binding site of Qβ coat protein, showing that, as with MS2, it resides on the surface of a large β-sheet. Mutations are also described that convert Qβ coat protein to the RNA binding specificity of MS2. The results of these and other studies indicate that, although they bind different RNAs, the binding sites of the two coat proteins are sufficiently similar that each is easily converted by mutation to the RNA binding specificity of the other. INTRODUCTIONThe coat proteins of the RNA bacteriophages play dual roles in the viral life cycle. In addition to serving as the major structural proteins of the virus particles, they act as translational repressors of viral replicase synthesis. This latter function is the result of coat protein interaction with an RNA stem loop which contains the replicase ribosome-binding site. The coat protein of bacteriophage MS2 is the most intensively studied of the RNA phage coat proteins. Its binding target on viral RNA has been thoroughly characterized (1Romaniuk P.J. Olwary P. Wu H.-N. Stormo G. Uhlenbeck O.C. Biochemistry. 1987; 26: 1563-1568Crossref PubMed Scopus (156) Google Scholar), coat protein itself has been subjected to detailed genetic analysis of its RNA binding function (2Peabody D.S. J. Biol. Chem. 1990; 265: 5684-5689Abstract Full Text PDF PubMed Google Scholar, 3Peabody D.S. EMBO J. 1993; 12: 595-600Crossref PubMed Scopus (136) Google Scholar, 4Lim F. Peabody D.S. Nucleic Acids Res. 1994; 22: 3748-3752Crossref PubMed Scopus (65) Google Scholar, 5Peabody D.S Ely K.R. Nucleic Acids Res. 1992; 20: 1649-1655Crossref PubMed Scopus (75) Google Scholar, 6Lim F. Spingola M. Peabody D.S. J. Biol. Chem. 1994; 269: 9006-9010Abstract Full Text PDF PubMed Google Scholar), and x-ray structures of the coat protein in both the free and RNA-bound forms are available (7Valegard K. Murray J.B. Stockley P.G. Stonehouse N.J. Liljas L. Nature. 1994; 371: 623-626Crossref PubMed Scopus (319) Google Scholar, 8Valegard K. Liljas L. Fridborg K. Unge T. Nature. 1990; 345: 36-41Crossref PubMed Scopus (332) Google Scholar, 9Ni C.-Z. Syed R. Kodandapani R. Wickersham J. Peabody D.S. Ely K.R. Structure. 1995; 3: 255-263Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The coat proteins of related phages are less well characterized, but, since some bind different RNAs, they provide opportunities to understand the basis of RNA binding specificity. The RNA binding targets of the coat proteins of MS2 and Qβ are shown in Fig. 1. The two coat proteins are about 25% identical in amino acid sequence and possess highly similar tertiary structures. Thus they utilize a common structural framework to bind structurally distinct RNAs.We previously reported genetic analyses of the MS2 coat protein RNA-binding site utilizing a two-plasmid system in which coat protein expressed from one plasmid (pCT119) translationally represses synthesis of a replicase-β-galactosidase fusion protein from the second plasmid (pRZ5). We constructed an equivalent two-plasmid system for Qβ coat protein in order to similarly dissect its RNA-binding site. Here we describe this system and its application in identifying amino acid residues important for the interaction of Qβ coat protein with its RNA. We also describe the isolation and characterization of specificity mutations that confer to Qβ coat protein the ability to bind the MS2 translational operator.DISCUSSIONOur genetic approach to dissection of the Qβ RNA-binding site relies on translationally repressing the synthesis of a hybrid replicase-β-galactosidase protein. We initially constructed pRZQ for this purpose. It contains a synthetic version of the wild-type Qβ operator. Unfortunately, pRZQ was not susceptible to efficient translational repression by Qβ coat protein expressed from pQCT119. We did not know how to explain this behavior, but noticed the presence of two AUG triplets (at −9 and −4 in Fig. 1B) in addition to the replicase initiation codon (at +1). Since translational repression is the result of competition between coat protein and ribosomes for binding of the operator RNA, we speculated that the extra AUGs might somehow tip the balance in favor of ribosome binding. This was the reasoning behind the construction of pRZQ5, which contains nucleotide substitutions that preserve the required RNA secondary structure, but destroy the two extra AUGs (see Fig. 1). These changes confer the desired capacity for translational repression. This is not the result of any difference in affinity of the two operator RNAs for coat protein, since their binding behaviors in vitro are indistinguishable (Fig. 2).Do these observations imply that the extra AUGs are bound by ribosomes? N-terminal sequence analysis of the replicase protein suggests that the AUG at +1 is the bona fide translation initiation codon (19Weiner A.M. Platt T. Weber K. J. Biol. Chem. 1972; 247: 3242-3251Abstract Full Text PDF PubMed Google Scholar). Although the AUG at −9 is in the replicase reading frame and could potentially lead to the synthesis of a protein three amino acids longer, we are unaware of any evidence for the existence of such an elongated product in Qβ-infected cells. Since pRZQ produces about 50% more β-galactosidase than pRZQ5 2F. Lim, M. Spingola, and D. S. Peabody, unpublished observations. it is possible that the −9 AUG is utilized for translation initiation in this system. On the other hand, the AUG at −4 cannot produce an elongated protein, since it resides in the −1 reading frame and is followed, after two codons, by a nonsense triplet. Since pRZQ5 simultaneously inactivates both of the extra AUGs, we cannot distinguish their relative contributions to inhibition of repression. Moreover, we do not know whether the behavior of pRZQ means that the operator is poorly repressed in intact Qβ RNA in infected cells. Placement of the operator in the unusual context of pRZQ may have altered its function, perhaps somehow making the extra AUGs more susceptible to ribosome binding.We previously reported the identification of amino acid residues making up the RNA binding site of MS2 coat protein. Their side chains reside on one surface of a large β-sheet. The location and make-up of the RNA-binding site was confirmed by the x-ray structure of the MS2 RNA-protein complex (7Valegard K. Murray J.B. Stockley P.G. Stonehouse N.J. Liljas L. Nature. 1994; 371: 623-626Crossref PubMed Scopus (319) Google Scholar). In the current study we applied a similar genetic strategy to identify the amino acid constituents of the Qβ RNA-binding site. Given that MS2 and Qβ coat proteins are evolutionary relatives and possess homologous amino acid sequences, we assumed that Qβ coat protein would have high structural similarity to MS2, and would probably utilize a similar β-sheet surface for RNA binding. These expectations were confirmed by the mutational analyses reported here and by the x-ray structure of Qβ coat protein which Lars Liljas and colleagues kindly made available to us while this manuscript was in preparation. Table I shows the nucleotide and amino acid substitutions that resulted in the repressor-defective phenotype. Table II and Fig. 4 show that these substitutions also caused defects in RNA binding in vitro. As with MS2, the RNA-binding site mainly resides on the surface of the coat protein β-sheet, although some loop residues are also implicated. Most of the essential Qβ amino acids occupy positions that are structurally equivalent to important RNA-binding site residues of MS2 coat protein. The degree of conservation of these residues is shown in Table I which lists, in parentheses, the equivalent MS2 residues in those cases where a clear structural homologue is readily identified. Nearly all these amino acids are conserved between the two proteins. Fig. 5B shows the locations of the amino acid residues identified by genetic analysis as important for RNA binding by MS2 coat protein. A similar map of the Qβ-binding site, based on the studies reported here, is shown in Fig. 5A. Fig. 5C is a superposition of Fig. 5, A and B, and shows the degree of amino acid identity in residues required for RNA binding by the two proteins. The conserved aspects of the two sites may represent portions of coat protein structure required for binding of structurally similar parts of the operators.It is possible, for several reasons, that the Qβ RNA-binding site includes residues not identified by this analysis. For example, some types of mutations could be absent in our mutational library. However, sequence analysis of 32 mutations in 21 repressor-defective assembly-defective mutants revealed a wide spectrum of different substitution types (not shown). Therefore, it seems unlikely that any amino acid residue whose identity is crucial for RNA binding is missing from this analysis, but, of course, we cannot rule out the possible existence of mutational cold spots which would lead to under-representation of certain mutants. It is also possible that certain residues play a dual role, functioning both in RNA binding and in protein folding or stability. Mutants with substitutions at these sites will not have passed our screen for capsid formation. Although we obtained multiple isolates of all but one of the repressor-defective mutants, it is also possible that the mutant library contains repressor defects not yet isolated. For these reasons the RNA-binding site could be more extensive than is indicated by the present set of repressor-defective mutations.Unconserved amino acids within the two binding sites presumably account for their differing RNA binding specificities. We isolated four specificity mutants of Qβ coat protein based on their abilities to repress the MS2 operator. Three of them contained multiple amino acid substitutions. This was a consequence of the high mutation rate of the error-prone PCR method we used in generating the mutant library. We restricted our analyses to residues 91 and 65. The D91N,A114G and T18S,N22Y,D91N mutants had only the D91N substitution in common, and, of the affected amino acids, Asp91 was the only one present on the surface of the β-sheet where the RNA-binding site resides. The single mutant D91N was constructed to test this assertion, and we found that the translational repressor activity of both of the original mutants was conferred by the D91N substitution alone (Table IV). Similarly, Q65H possessed the translational repressor properties of the T29S,Q65H double mutant. For these reasons only the D91N and Q65H single substitutions were characterized further. Both mutants possess slightly increased affinities for the normal binding target of Qβ coat protein, the Qβ translational operator. This is evident both in the translational repression data presented in Table IV and in the in vitro binding affinities of the proteins for Qβ operator RNA (Fig. 6 and Table V). Thus, D91N binds the Qβ operator 2-fold more tightly than does wild-type Qβ coat protein. Meanwhile, Q65H shows an 8-fold improvement in binding of this RNA. The most dramatic effects, however, are found in the increased affinities for the MS2 operator. D91N and Q65H, respectively, bind the MS2 operator RNA 20-fold and 33-fold more tightly than does wild-type Qβ coat protein.The effects of the D91N substitution are easily rationalized. Asp91 of Qβ coat protein occupies a position which is structurally homologous to Asn87 of MS2 coat protein. Genetic and structural studies identified Asn87 of MS2 as an important site of interaction with RNA (6Lim F. Spingola M. Peabody D.S. J. Biol. Chem. 1994; 269: 9006-9010Abstract Full Text PDF PubMed Google Scholar, 7Valegard K. Murray J.B. Stockley P.G. Stonehouse N.J. Liljas L. Nature. 1994; 371: 623-626Crossref PubMed Scopus (319) Google Scholar). It forms a hydrogen bond with the −5 uridine residue in the MS2 operator. On the other hand, Asp91 of Qβ is apparently not required for binding of the Qβ operator, since no substitutions at this site were found in our extensive collection of repressor-defective Qβ coat mutants, and because the D91N substitution itself clearly does not impair binding to Qβ RNA. It is striking that converting Asp91 to its MS2 counterpart dramatically improves activity for the MS2 operator. In fact, Qβ-D91N binds MS2 RNA nearly as well as does MS2 coat protein itself. Clearly, the RNA binding surface of Qβ coat protein is sufficiently similar to that of MS2 that it readily accommodates MS2 RNA, and a single amino acid substitution converts Qβ coat protein to a good repressor of MS2.The effects of the Q65H substitution are not as easily understood. Gln65 in Qβ coat protein occupies a position which is structurally homologous to Thr59 of MS2. Substitutions like T59S or T59A result in repressor defects in MS2 coat protein (3Peabody D.S. EMBO J. 1993; 12: 595-600Crossref PubMed Scopus (136) Google Scholar, 6Lim F. Spingola M. Peabody D.S. J. Biol. Chem. 1994; 269: 9006-9010Abstract Full Text PDF PubMed Google Scholar). However, the MS2 mutant T59Q is not repressor-defective for the MS2 operator.2 Although it is clear from the crystal structure of the MS2 coat protein-RNA complex that Thr59 makes contact with RNA, the contact apparently does not involve the side chain (7Valegard K. Murray J.B. Stockley P.G. Stonehouse N.J. Liljas L. Nature. 1994; 371: 623-626Crossref PubMed Scopus (319) Google Scholar). The effects of the Q65H substitution on RNA binding by Qβ coat protein suggest that histidine in this position establishes new contacts with the two RNAs, substantially increasing affinity for both the Qβ and MS2 operators.We have shown that Qβ coat protein easily acquires binding activity for MS2 RNA by mutation. In work being reported elsewhere we show that MS2 coat protein acquires the ability to bind the Qβ operator with similar ease. Thus, the MS2 and Qβ coat proteins are sufficiently similar that their RNA-binding specificities are readily interconverted. Perhaps this is not unexpected given the similarities in the amino acid sequences of their RNA-binding sites. However, some may find it surprising that these similar proteins bind operators with the large apparent structural differences illustrated in Fig. 1. Compared to MS2, the Qβ operator requires a longer base paired stem, a smaller loop, and is relatively indifferent to deletion of the bulged A. In MS2 the critical nature of the bulge is easily reconciled with the structure of the protein-RNA complex where quasi-symmetric interactions between the two halves of the dimer and the As at −4 and −10 are observed. The amino acid residues (Val29, Thr45, Ser47, and Lys61) of MS2 coat protein that form the binding sites for these two As are conserved in Qβ, thus satisfying one of the important requirements for interaction with MS2 RNA. However, given the relative dispensability of the Qβ bulged A and the apparent difference in its spatial relationship to the loop A, the quasi-symmetry of these interactions is likely abolished in the Qβ coat protein-RNA complex. We suspect that the interaction of coat protein with the loop A is conserved between the two phages, but the interaction with the bulged A probably is not. Other contacts must be formed to compensate for this loss. The differences in makeup of the RNA-binding site of the two coat proteins presumably reflect this fact. On the other hand, the similarities of the binding sites might not reflect any significant similarities in the precise modes of interaction of the two RNAs with their respective coat proteins. Experiments currently in progress should determine whether the nature of the interaction with the loop A is a conserved feature in the RNA-protein complexes of MS2 and Qβ. INTRODUCTIONThe coat proteins of the RNA bacteriophages play dual roles in the viral life cycle. In addition to serving as the major structural proteins of the virus particles, they act as translational repressors of viral replicase synthesis. This latter function is the result of coat protein interaction with an RNA stem loop which contains the replicase ribosome-binding site. The coat protein of bacteriophage MS2 is the most intensively studied of the RNA phage coat proteins. Its binding target on viral RNA has been thoroughly characterized (1Romaniuk P.J. Olwary P. Wu H.-N. Stormo G. Uhlenbeck O.C. Biochemistry. 1987; 26: 1563-1568Crossref PubMed Scopus (156) Google Scholar), coat protein itself has been subjected to detailed genetic analysis of its RNA binding function (2Peabody D.S. J. Biol. Chem. 1990; 265: 5684-5689Abstract Full Text PDF PubMed Google Scholar, 3Peabody D.S. EMBO J. 1993; 12: 595-600Crossref PubMed Scopus (136) Google Scholar, 4Lim F. Peabody D.S. Nucleic Acids Res. 1994; 22: 3748-3752Crossref PubMed Scopus (65) Google Scholar, 5Peabody D.S Ely K.R. Nucleic Acids Res. 1992; 20: 1649-1655Crossref PubMed Scopus (75) Google Scholar, 6Lim F. Spingola M. Peabody D.S. J. Biol. Chem. 1994; 269: 9006-9010Abstract Full Text PDF PubMed Google Scholar), and x-ray structures of the coat protein in both the free and RNA-bound forms are available (7Valegard K. Murray J.B. Stockley P.G. Stonehouse N.J. Liljas L. Nature. 1994; 371: 623-626Crossref PubMed Scopus (319) Google Scholar, 8Valegard K. Liljas L. Fridborg K. Unge T. Nature. 1990; 345: 36-41Crossref PubMed Scopus (332) Google Scholar, 9Ni C.-Z. Syed R. Kodandapani R. Wickersham J. Peabody D.S. Ely K.R. Structure. 1995; 3: 255-263Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The coat proteins of related phages are less well characterized, but, since some bind different RNAs, they provide opportunities to understand the basis of RNA binding specificity. The RNA binding targets of the coat proteins of MS2 and Qβ are shown in Fig. 1. The two coat proteins are about 25% identical in amino acid sequence and possess highly similar tertiary structures. Thus they utilize a common structural framework to bind structurally distinct RNAs.We previously reported genetic analyses of the MS2 coat protein RNA-binding site utilizing a two-plasmid system in which coat protein expressed from one plasmid (pCT119) translationally represses synthesis of a replicase-β-galactosidase fusion protein from the second plasmid (pRZ5). We constructed an equivalent two-plasmid system for Qβ coat protein in order to similarly dissect its RNA-binding site. Here we describe this system and its application in identifying amino acid residues important for the interaction of Qβ coat protein with its RNA. We also describe the isolation and characterization of specificity mutations that confer to Qβ coat protein the ability to bind the MS2 translational operator." @default.
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