The RNA binding site of bacteriophage MS2 coat protein.

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.     

The binding site for coat protein on bacteriophage Qbeta RNA.

The site of interaction of phage Qbeta coat protein with Qbeta RNA was determined by ribonuclease T1 degradation of complexes of coat protein and [32P]-RNA obtained by codialysis of the components from urea into buffer solutions. The degraded complexes were recovered by filtration through nitrocellulose filters, and bound [32P]RNA fragments were extracted and separated by polyacrylamide gel electrophoresis. Fingerprinting and further sequence analysis established that the three main fragments obtained (chain lengths 88, 71 and 27 nucleotides) all consist of sequences extending from the intercistronic region to the beginning of the replicase cistron. These results suggest that in the replication of Qbeta, as in the case of R17, coat protein acts as a translational repressor by binding to the ribosomal initiation site of the replicase cistron.     

Crystal structure of the coat protein from the GA bacteriophage: model of the unassembled dimer.

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.     

Probing sequence-specific RNA recognition by the bacteriophage MS2 coat protein.

We present the results of in vitro binding studies aimed at defining the key recognition elements on the MS2 RNA translational operator (TR) essential for complex formation with coat protein. We have used chemically synthesized operators carrying modified functional groups at defined nucleotide positions, which are essential for recognition by the phage coat protein. These experiments have been complemented with modification-binding interference assays. The results confirm that the complexes which form between TR and RNA-free phage capsids, the X-ray structure of which has recently been reported at 3.0 A, are identical to those which form in solution between TR and a single coat protein dimer. There are also effects on operator affinity which cannot be explained simply by the alteration of direct RNA-protein contacts and may reflect changes in the conformational equilibrium of the unliganded operator. The results also provide support for the approach of using modified oligoribonucleotides to investigate the details of RNA-ligand interactions.     

Hexamer of bacteriophage f2 coat protein as a repressor of bacteriophage RNA polymerase synthesis.

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.     

Cooperative binding of R17 coat protein to RNA.

The binding of the R17 coat protein to synthetic RNAs containing one or two coat protein binding sites was characterized by using nitrocellulose filter and gel-retention assays. RNAs with two available sites bound coat protein in a cooperative manner, resulting in a higher affinity and reduced sensitivity to pH, ionic strength, and temperature when compared with RNAs containing only a single site. The cooperativity can contribute up to -5 kcal/mol to the overall binding affinity with the greatest cooperativity found at low pH, high ionic strength, and high temperatures. Similar solution properties for the encapsidation of the related fr and f2 phage suggest that the cooperativity is due to favorable interactions between the two coat proteins bound to the RNA. This system therefore resembles an intermediate state of phage assembly. No cooperative binding was observed for RNAs containing a single site and a 5' or 3' extension of nonspecific sequence, indicating that R17 coat protein has a very low nonspecific binding affinity. Unexpectedly weak binding was observed for several RNAs due to the presence of alternative conformational states of the RNA.     

Selection of high affinity RNA ligands to the bacteriophage R17 coat protein.

RNA ligands with high affinity for the bacteriophage R17 coat protein were isolated from a pool of random RNA molecules using SELEX. Of the 38 ligands isolated, 36 were found to contain a hairpin very similar to the naturally occurring coat protein binding site in the R17 genome. The common features of these 36 sequences provide a consensus binding site and predict components of a hairpin that promote favorable interaction with the coat protein. These include a tetraloop of primary sequence AUCA and a variable-length stem with a bulged adenosine residue at a specific stem position. The predicted consensus agrees well with the highest-affinity RNA binding site of the R17 coat protein, identified through classical but laborious techniques. These results demonstrate the value of SELEX as a tool for isolating high affinity RNA ligands to a specific target protein, and the further value of those ligands to point the researcher toward natural sequences for that target protein.     

RNA binding site of R17 coat protein

The specific interaction between R17 coat protein and its target of translational repression at the initiation site of the R17 replicase gene was studied by synthesizing variants of the RNA binding site and measuring their affinity to the coat protein by using a nitrocellulose filter binding assay. Substitution of two of the seven single-stranded residues by other nucleotides greatly reduced the Ka, indicating that they are essential for the RNA-protein interaction. In contrast, three other single-stranded residues can be substituted without altering the Ka. When several of the base-paired residues in the binding site are altered in such a way that pairing is maintained, little change in Ka is observed. However, when the base pairs are disrupted, coat protein does not bind. These data suggest that while the hairpin loop structure is essential for protein binding, the base-paired residues do not contact the protein directly. On the basis of these and previous data, a model for the structural requirements of the R17 coat protein binding site is proposed. The model was successfully tested by demonstrating that oligomers with sequences quite different from the replicase initiator were able to bind coat protein.     

The primary structure of the coat protein of the broad-host-range RNA bacteriophage PRR1.

The complete amino acid sequence of the coat protein of RNA bacteriophage PRR1 is presented. After thermolysin digestion, 26 peptides were isolated, covering the complete coat protein chain. Their alignment was established in part using automated Edman degradation on the intact protein, in part with overlapping peptides obtained by enzymic hydrolysis with trypsin, pepsin, subtilisin and Staphylococcus aureus protease, and by chemical cleavage with cyanogen bromide and N-bromosuccinimide. To obtain the final overlaps, a highly hydrophobic, insoluble tryptic peptide was sequenced for seven steps by the currently used manual dansyl-Edman degradation procedure, which was slightly modified for application on insoluble peptides. PRR1 coat protein contains 131 amino acids, corresponding to a molecular weight of 14534. It is highly hydrophobic, and the residues with ionizable side chains are distributed unevenly: acidic residues are absent in the middle third of the sequence, whereas a clustering of basic residues occurs between positions 44 and 62. PRR1 coat protein was compared with the coat proteins of RNA coliphages MS2 and Q beta, and the minimum mutation distance was calculated for both comparisons. It is highly probable that PRR1. Q beta and MS2 share a common ancestor. The basic region present in the three coat proteins is recognized as an essential structural feature of RNA phage coat proteins.     

Specific RNA binding by Q beta coat protein

The interaction between the bacteriophage Q beta coat protein and its specific binding site on Q beta genomic RNA was characterized by using a nitrocellulose filter binding assay. Q beta coat protein bound to a synthetic 29-nucleotide RNA hairpin with an association constant of 400 microM-1 at 4 degrees C, 0.2 M ionic strength, pH 6.0. Complex formation had a broad pH optimum centered around pH 6.0 and was favored by both enthalpy and entropy. The salt dependence of Ka revealed that four to five ion pairs may be formed in the complex although approximately 80% of the free energy of complex formation is contributed by nonelectrostatic interactions. Truncation experiments revealed that coat protein binding required only the presence of a hairpin with an eight base pair stem and a three-base loop. Analysis of the binding properties of hairpin variants showed that the sequence of the stem was not important for coat protein recognition and only one of the three loop residues was essential. A bulged adenosine present in the coat protein binding site was not required for coat protein binding. Q beta coat protein binding specific is therefore primarily achieved by the structure and not by the sequence of the operator.     

Template activity of complexes formed between bacteriophage f2 RNA and coat protein.

Formation of complexes between f2 RNA polymerase cistron was partially inhibited, some RNA and coat protein was studied using salt conditions which are optimum for phage protein synthesis. In this ionic environment, coat protein precipitation can be prevented by sulfhydryl group-protecting agents. Complexes formed at different protein-RNA input molar ratios were isolated and tested for template activity in an in vitro protein synthesizing system. Simultaneously, the number of protein molecules bound per RNA strand in such complexes was measured by the membrane (Millipore) filtration technique. Under conditions in which translation of the RNA strands were complexed with six molecules of coat protein, whereas some remained unbound. Strong inhibition of the translation of the RNA polymerase cistron was observed when each of the RNA strands present in the mixture was associated with six molecules of coat protein.     

Specific RNA binding by amino-terminal peptides of alfalfa mosaic virus coat protein.

Specific RNA-protein interactions and ribonucleoprotein complexes are essential for many biological processes, but our understanding of how ribonucleoprotein particles form and accomplish their biological functions is rudimentary. This paper describes the interaction of alfalfa mosaic virus (A1MV) coat protein or peptides with viral RNA. A1MV coat protein is necessary both for virus particle formation and for the initiation of replication of the three genomic RNAs. We have examined protein determinants required for specific RNA binding and analyzed potential structural changes elicited by complex formation. The results indicate that the amino-terminus of the viral coat protein, which lacks primary sequence homology with recognized RNA binding motifs, is both necessary and sufficient for binding to RNA. Circular dichroism spectra and electrophoretic mobility shift experiments suggest that the RNA conformation is altered when amino-terminal coat protein peptides bind to the viral RNA. The peptide--RNA interaction is functionally significant because the peptides will substitute for A1MV coat protein in initiating RNA replication. The apparent conformational change that accompanies RNA--peptide complex formation may generate a structure which, unlike the viral RNA alone, can be recognized by the viral replicase.     

Complementation of RNA binding site mutations in MS2 coat protein heterodimers.

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.     

Mechanism of translational coupling between coat protein and replicase genes of RNA bacteriophage MS2.

We have analyzed the molecular mechanism that makes translation of the MS2 replicase cistron dependent on the translation of the upstream coat cistron. Deletion mapping on cloned cDNA of the phage shows that the ribosomal binding site of the replicase cistron is masked by a long distance basepairing to an internal coat cistron region. Removal of the internal coat cistron region leads to uncoupled replicase synthesis. Our results confirm the model as originally proposed by Min Jou et al. (1). Activation of the replicase start is sensitive to the frequency of upstream translation, but never reaches the level of uncoupled replicase synthesis.     

Dynamics of fd coat protein in the bacteriophage

The dynamics of the coat protein in fd bacteriophage are described with solid-state 15N and 2H NMR experiments. The virus particles and the coat protein subunits are immobile on the time scales of the 15N chemical shift anisotropy (10(3) Hz) and 2H quadrupole (10(6) Hz) interactions. Previously we have shown that the Trp-26 side chain is immobile, that the two Tyr and three Phe side chains undergo only rapid twofold jump motions about their C beta-C gamma bond axis [Gall, C. M., Cross, T. A., DiVerdi, J. A., & Opella, S. J. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 101-105], and that most of the backbone peptide linkages are highly constrained but do undergo rapid small amplitude motions [Cross, T. A., & Opella, S. J. (1982) J. Mol. Biol. 159, 543-549] in the coat protein subunits in the virus particles. In this paper, we demonstrate that the four N-terminal residues of the coat protein subunits are highly mobile, since both backbone and side-chain sites of these residues undergo large amplitude motions that are rapid on the time scales of the solid-state NMR experiments. In addition, the dynamics of the methyl-containing aliphatic residues Ala, Leu, Val, Thr, and Met are analyzed. Large amplitude jump motions are observed in nearly all of these side chains even though, with the exception of the N-terminal residue Ala-1, their backbone peptide linkages are highly constrained. The established information about the dynamics of the structural form of fd coat protein in the virus particle is summarized qualitatively.(ABSTRACT TRUNCATED AT 250 WORDS)