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In this article we studied, by nuclear magnetic resonance relaxation measurements, the disassembly of a virus particle-the MS2 bacteriophage. MS2 is one of the single-stranded RNA bacteriophages that infect Escherichia coli. At pH 4.5, the phage turns to a metastable state, as is indicated by an increase in the observed nuclear magnetic resonance signal intensity upon decreasing the pH from 7.0 to 4.5. Steady-state fluorescence and circular dichroism spectra at pH 4.5 show that the difference in conformation and secondary structure is not pronounced if compared with the phage at pH 7.0. At pH 4.5, two-dimensional (15)N-(1)H heteronuclear multiple quantum coherence (HMQC) spectrum shows approximately 40 crosspeaks, corresponding to the most mobile residues of MS2 coat protein at pH 4.5. The (15)N linewidth is approximately 30 Hz, which is consistent with an intermediate with a rotational relaxation time of 100 ns. The average spin lattice relaxation time (T(1)) of the mobile residues was measured at different temperatures, clearly distinguishing between the dimer and the equilibrium intermediate. The results show, for the first time, the presence of intermediates in the process of dissociation of the MS2 bacteriophage.

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A method was developed to screen bacteria for synthesis of mutant proteins with altered assembly and solubility properties using bacteriophage MS2 coat protein as a model self-associating protein. Colonies expressing coat protein from a plasmid were covered with an agarose overlay under conditions that caused the lysis of some of the cells in each colony. The proteins thus liberated diffused through the overlay at rates depending on their molecular sizes. After transfer of the proteins to a nitrocellulose membrane, probing with coat protein-specific antiserum revealed spots whose sizes and intensities were related to the aggregation state of coat protein. The method was employed in the isolation of assembly defective mutants and to find soluble variants of an aggregation-prone coat protein mutant.

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BACKGROUND: The X-ray structure of the MS2 coat protein-operator RNA complex reveals the existence of quasi-synmetric interactions of adenosines -4 and -10 in pockets formed on different subunits of the coat protein dimer. Both pockets utilize the same five amino acid residues, namely Val29, Thr45, Ser47, Thr59, and Lys61. We call these sites the adenosine-binding pockets. RESULTS: We present here a heterodimer complementation analysis of the contributions of individual A-pocket amino acids to the binding of A-4 and A-10 in different halves of the dimer. Various substitutions of A-pocket residues were introduced into one half of single-chain coat protein heterodimers where they were tested for their abilities to complement Y85H or T91I substitutions (defects in the A-4 and A-10 half-sites, respectively) present in the other dimer half. CONCLUSIONS: These experiments provide functional tests of interactions predicted from structural analyses, demonstrating the importance of certain amino acid-nucleotide contacts observed in the crystal structure, and showing that others make little or no contribution to the stability of the complex. In summary, Val29 and Lys61 form important stabilizing interactions with both A-4 and A-10. Meanwhile, Ser47 and Thr59 interact primarily with A-10. The important interactions with Thr45 are restricted to A-4.

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PP7 is a single-strand RNA bacteriophage of Pseudomonas aeroginosa and a distant relative to coliphages like MS2 and Qbeta. Here we show that PP7 coat protein is a specific RNA-binding protein, capable of repressing the translation of sequences fused to the translation initiation region of PP7 replicase. Its RNA binding activity is specific since it represses the translational operator of PP7, but does not repress the operators of the MS2 or Qbeta phages. Conditions for the purification of coat protein and for the reconstitution of its RNA binding activity from disaggregated virus-like particles were established. Its dissociation constant for PP7 operator RNA in vitro was determined to be about 1 nm. Using a genetic system in which coat protein represses translation of a replicase-beta-galactosidase fusion protein, amino acid residues important for binding of PP7 RNA were identified.

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A prominent feature of the interaction of MS2 coat protein with RNA is the quasi-symmetric insertion of a bulged adenine (A-10) and a loop adenine (A-4) into conserved pockets on each subunit of the coat protein dimer. Because of its presence in both of these adenine-binding pockets, Thr(45) is thought to play an important role in interaction with RNA on both subunits of the dimer. To test the significance of Thr(45), we introduced all 19 amino acid substitutions. However, we were initially unable to determine the effects of the mutations on RNA binding because every substitution compromised the ability of coat protein to fold correctly. Genetic fusion of coat protein subunits reverted these protein structural defects, allowing us to show that the RNA binding activity of coat protein tolerates substitution of Thr(45), but only on one or the other subunit of the dimer. Single-chain heterodimer complementation experiments suggest that the primary site of Thr(45) interaction with RNA is with A-4 in the translational operator. Either contact of Thr(45) with A-10 makes little contribution to stability of the RNA-protein complex, or the effects of Thr(45) substitution are offset by conformational adjustments that introduce new, favorable contacts at nearby sites.

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We have determined the crystal structures, at 2.8 A resolution, of two different RNA aptamers, each bound to MS2 coat protein. One of the aptamers contains a non-Watson-Crick base pair, while the other is missing one of the unpaired adenines that make sequence-specific contacts in the wild-type complex. Despite these differences, the RNA aptamers bind in the same location on the protein as the wild-type translational operator. Comparison of these new structures with other MS2-RNA complexes allows us to refine further the definition of the minimal recognition elements and suggests a possible application of the MS2 system for routine structure determination of small nucleic acid motifs.

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The crystal structure, at 2.8 A resolution, of an RNA aptamer bound to bacteriophage MS2 coat protein has been determined. It provides an opportunity to compare the interactions of MS2 coat protein and wild type operator with those of an aptamer, whose secondary structure differs from the wild type RNA in having a three-base loop (compared to a tetraloop) and an additional base pair between this loop and the sequence-specific recognition element in the stem. The RNA binds in the same location on the coat protein as the wild type operator and maintains many of the same RNA-protein interactions. In order to achieve this, the RNA stem loop undergoes a concerted rearrangement of the 3' side while leaving the 5' side and the loop interactions largely unchanged, illustrating the ability of RNA to present similar molecular recognition surfaces from distinct primary and secondary structures.

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An octapeptide sequence called Flag was inserted into the bacteriophage MS2 coat protein at two different locations and its effects on protein folding and virus assembly were determined. Assays of the translational repressor and capsid assembly functions of the recombinants show that when the peptide is inserted at its N-terminus coat protein folds properly into the form that binds RNA (i.e., the dimer), but is defective for capsid assembly. On the other hand, a recombinant protein which is expected to display the Flag insertion as a surface loop does not fold correctly and, as a consequence, is proteolytically degraded. Genetic fusion of the two subunits of the coat dimer results in a protein considerably more tolerant of these structural perturbations and mostly corrects the defects accompanying Flag peptide insertion. Increased resistance of the single-chain coat protein to urea denaturation indicates that the fused dimer is substantially more stable than wild type. Covalent joining of subunits of oligomers probably represents a general strategy for engineering increased protein stability.

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The coat proteins of the RNA phages MS2 and Qbetaare structurally homologous, yet they specifically bind different RNA structures. In an effort to identify the basis of RNA binding specificity we sought to isolate mutants that convert MS2 coat protein to the RNA binding specificity of Qbeta. A library of mutations was created which selectively substitutes amino acids within the RNA binding site. Genetic selection for the ability to repress translation from the Qbetatranslational operator led to the isolation of several MS2 mutants that acquired binding activity for QbetaRNA. Some of these also had reduced abilities to repress translation from the MS2 translational operator. These changes in RNA binding specificity were the results of substitutions of amino acid residues 87 and 89. Additional codon- directed mutagenesis experiments confirmed earlier results showing that the identity of Asn87 is important for specific binding of MS2 RNA. Glu89, on the other hand, is not required for recognition of MS2 RNA, but prevents binding of QbetaRNA.

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The coat protein of the RNA bacteriophage MS2 interacts with viral RNA to translationally repress replicase synthesis. This protein-RNA interaction is also thought to play a role in genome encapsidation. In this study the strength of the interaction was perturbed by constructing a recombinant genome containing a super-repressing coat mutation. Because replicase synthesis is prematurely repressed, the mutant produces plaques about five orders of magnitude less efficiently than wild-type. The few plaques obtained are second-site revertants of the original coat mutation and fall into two categories. Those of the first type contain nucleotide substitutions within the translational operator that reduce or destroy its ability to bind coat protein, showing that this interaction is not necessary for genome encapsidation. Revertants of the second type are double mutants in which one substitution converts the coat initiator AUG to AUA and the other substitutes an A for the G normally present two nucleotides upstream of the coat start codon. The mutation of the coat protein gene AUG to AUA, by itself, reduces coat protein synthesis to a few percent of the wild-type level. The second substitution destabilizes the coat initiator stem-loop and restores coat protein synthesis to within a few fold of wild-type levels.

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There are four groups of RNA bacteriophages with distinct antigenic and physicochemical properties due to differences in surface residues of the viral coat proteins. Coat proteins also play a role as translational repressor during the viral life cycle, binding an RNA hairpin within the genome. In this study, the first crystal structure of the coat protein from a Group II phage GA is reported and compared to the Group I MS2 coat protein. The structure of the GA dimer was determined at 2.8 A resolution (R-factor = 0.20). The overall folding pattern of the coat protein is similar to the Group I MS2 coat protein in the intact virus (Golmohammadi R, Valegård K, Fridborg K, Liljas L. 1993, J Mol Biol 234:620-639) or as an unassembled dimer (Ni Cz, Syed R, Kodandapani R. Wickersham J, Peabody DS, Ely KR, 1995, Structure 3:255-263). The structures differ in the FG loops and in the first turn of the alpha A helix. GA and MS2 coat proteins differ in sequence at 49 of 129 amino acid residues. Sequence differences that contribute to distinct immunological and physical properties of the proteins are found at the surface of the intact virus in the AB and FG loops. There are six differences in potential RNA contact residues within the RNA-binding site located in an antiparallel beta-sheet across the dimer interface. Three differences involve residues in the center of this concave site: Lys/Arg 83, Ser/Asn 87, and Asp/Glu 89. Residue 87 was shown by molecular genetics to define RNA-binding specificity by GA or MS2 coat protein (Lim F. Spingola M, Peabody DS, 1994, J Biol Chem 269:9006-9010). This sequence difference reflects recognition of the nucleotide at position -5 in the unpaired loop of the translational operators bound by these coat proteins. In GA, the nucleotide at this position is a purine whereas in MS2, it is a pyrimidine.

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The coat proteins of the RNA bacteriophages Qbeta 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 Qbeta. Here we describe the isolation of mutants that define the RNA-binding site of Qbeta coat protein, showing that, as with MS2, it resides on the surface of a large beta-sheet. Mutations are also described that convert Qbeta 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.

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The coat protein of bacteriophage MS2 functions as a symmetric dimer to bind an asymmetric RNA hairpin. This implies the existence of two equivalent RNA binding sites related to one another by a 2-fold symmetry axis. In this view the symmetric binding site defined by mutations conferring the repressor-defective phenotype is a composite picture of these two asymmetric sites. In order to determine whether the RNA ligand interacts with amino acid residues on both subunits of the dimer and in the hope of constructing a functional map of the RNA binding site, we performed heterodimer complementation experiments. Taking advantage of the physical proximity of their N- and C-termini, the two subunits of the dimer were genetically fused, producing a duplicated coat protein which folds normally and allows the construction of the functional equivalent of obligatory heterodimers containing all possible pairwise combinations of the repressor-defective mutations. The restoration of repressor function in certain heterodimers shows that a single RNA molecule interacts with both subunits of the dimer and allows the construction of a functional map of the binding site.

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BACKGROUND: The coat protein in RNA bacteriophages binds and encapsidates viral RNA, and also acts as translational repressor of viral replicase by binding to an RNA hairpin in the RNA genome. Because of its dual function, the MS2 coat protein is an interesting candidate for structural studies of protein-RNA interactions and protein-protein interactions. In this study, unassembled MS2 coat protein dimers were selected to analyze repressor activity and virus assembly. RESULTS: The crystal structure of a mutant MS2 coat protein that is defective in viral assembly yet retains repressor activity has been determined at 2.0 A resolution. The unassembled dimer is stabilized by interdigitation of alpha-helices, and the formation of a 10-stranded antiparallel beta-sheet across the interface between monomers. The substitution of arginine for tryptophan at residue 82 results in the formation of two new inter-subunit hydrogen bonds that further stabilize the dimer. Residues that influence RNA recognition, identified by molecular genetics, were located across the beta-sheet. Two of these residues (Tyr85 and Asn87) are displaced in the unliganded dimer and are located in the same beta-strand as the Trp-->Arg mutation. CONCLUSIONS: When compared with the structure of the coat protein in the assembled virus, differences in orientation of residues 85 and 87 suggest conformational adjustment on binding RNA in the first step of viral assembly. The substitution at residue 82 may affect virus assembly by imposing conformational restriction on the loop that makes critical inter-subunit contacts in the capsid.

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The coat protein of the RNA bacteriophage MS2 is a specific RNA binding protein that represses translation of the viral replicase gene during the infection cycle. As an approach to characterizing the RNA-binding site of coat protein we have isolated a series of coat mutants that suppress the effects of a mutation in the translational operator. Each of the mutants exhibits a super-repressor phenotype, more tightly repressing both the mutant and wild-type operators than does the wild-type protein. The variant coat proteins were purified and subjected to filter binding assays to determine their affinities for the mutant and wild-type operators. Each protein binds the operators from 3 to 7.5-fold more tightly than normal coat protein. The amino acid substitutions seem to extend the normal binding site by introducing new interactions with RNA.

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The coat proteins of RNA phages MS2 and GA are specific RNA-binding proteins which function to encapsidate viral RNA and to translationally repress synthesis of the viral replicase. The two proteins have highly homologous amino acid sequences, yet they show different RNA binding specificities, recognizing RNA stem-loop structures which differ primarily in the nucleotide sequences of their loops. We sought to convert MS2 coat protein to the RNA binding specificity of GA through the introduction of GA-like amino acid substitutions into the MS2 coat protein RNA-binding site. The effects of the mutations were determined by measuring the affinity of the coat protein variants for RNA in vitro and by measuring translational repression in vivo. We found five substitutions that affect RNA binding. One dramatically reduces binding of MS2 coat protein to both operators. Three others compensate for this defect by nonspecifically strengthening the interaction. Another substitution accounts for the ability to recognize the differences in the RNA loop sequence.

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The RNA bacteriophages of E. coli specifically encapsidate a single copy of the viral genome in a protein shell composed mainly of 180 molecules of coat protein. Coat protein is also a translational repressor and shuts off viral replicase synthesis by interaction with a RNA stem-loop containing the replicase initiation codon. We wondered whether the translational operator also serves as the viral pac site, the signal which mediates the exclusive encapsidation of viral RNA by its interaction with coat protein. To test this idea we measured the ability of lacZ RNA fused to the translational operator to be incorporated into virus-like particles formed from coat protein expressed from a plasmid. The results indicate that the operator-lacZ RNA is indeed encapsidated and that nucleotide substitutions in the translational operator which reduce the tightness of the coat protein-operator interaction also reduce or abolish encapsidation of the hybrid RNA. When coat protein is expressed in excess compared to the operator-lacZ RNA, host RNAs are packaged as well. However, elevation of the level of operator-lacZ RNA relative to coat protein results in its selective encapsidation at the expense of cellular RNAs. Our results are consistent with the proposition that this single protein-RNA interaction accounts both for translational repression and viral genome encapsidation.

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The coat protein of the RNA bacteriophage MS2 binds a specific stem-loop structure in viral RNA to accomplish encapsidation of the genome and translational repression of replicase synthesis. In order to identify the structural components of coat protein required for its RNA binding function, a series of repressor-defective mutants has been isolated. To ensure that the repressor defects were due to substitution of binding site residues, the mutant coat proteins were screened for retention of the ability to form virus-like particles. Since virus assembly presumably requires native structure, this approach eliminated mutants whose repressor defects were secondary consequences of protein folding or stability defects. Each of the variant coat proteins was purified and its ability to bind operator RNA in vitro was measured. DNA sequence analysis identified the nucleotide and amino acid substitutions responsible for reduced RNA binding affinity. Localization of the substituted sites in the three-dimensional structure of coat protein reveals that amino acid residues on three adjacent strands of the coat protein beta-sheet are required for translational repression and RNA binding. The sidechains of the affected residues form a contiguous patch on the interior surface of the viral coat.

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The coat protein of the RNA bacteriophage MS2 is a translational repressor and interacts with a specific RNA stem-loop to inhibit translation of the viral replicase gene. As part of an effort to dissect genetically its RNA binding function, mutations were identified in the coat protein sequence that suppress mutational defects in the translational operator. Each of the mutants displayed a super-repressor phenotype, repressing translation from the wild-type and a variety of mutant operators better than did the wild-type coat protein. At least one mutant probably binds RNA more tightly than wild-type. The other mutants, however, were defective for assembly of virus-like particles, and self-associated predominantly as dimers. It is proposed that this assembly defect accounts for their super-repressor characteristics, since failure to assemble into virus-like particles elevates the effective concentration of repressor dimers. This hypothesis is supported by the observation that deletion of thirteen amino acids known to be important for assembly of dimers into capsids also resulted in the same assembly defect and in super-repressor activity. A second class of assembly defects is also described. Deletion of two amino acids from the C-terminus of coat protein resulted in failure to form capsids, most of the coat protein having the apparent molecular weight expected of trimers. This mutant (dl-8) was completely defective for repressor activity, probably because of an inability to form dimers. These results point out the inter-dependence of the structural and regulatory functions of coat protein.

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The coat protein of bacteriophage MS2 is a translational repressor. It inhibits the synthesis of the viral replicase by binding a specific RNA structure that contains the replicase translation initiation region. In order to begin a genetic dissection of the repressor activity of coat protein, a two-plasmid system has been constructed that expresses coat protein and a replicase-beta-galactosidase fusion protein from different, compatible plasmids containing different antibiotic-resistant determinants. The coat protein expressed from the first plasmid (pCT1) represses synthesis of a replicase-beta-galactosidase fusion protein encoded on the other plasmid (pRZ5). Mutations in the translational operator or in coat protein result in constitutive synthesis of the enzyme. This permits the straightforward isolation of mutations in the coat sequence that affect repressor function. Because of the potential importance of cysteine residues for RNA binding, mutations were constructed that substitute serines for the cysteine residues normally present at positions 46 and 101. Both of these mutations result in translational repressor defects. Chromatographic and electron microscopic analyses indicate that the plasmid-encoded wild-type coat protein forms capsids in vivo. The ability of the mutants to adopt and/or maintain the appropriate conformation was assayed by comparing them to the wild-type protein for their ability to form capsids. Both mutants exhibited evidence of improper folding and/or instability as indicated by their aberrant elution behavior on a column of Sepharose CL-4B. Methods were developed for the rapid purification of plasmid-encoded coat protein, facilitating future biochemical analyses of mutant coat proteins.

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