MS2 Coat Protein Research Articles

Crystal structure of an RNA bacteriophage coat protein-operator complex.

The RNA bacteriophage MS2 is a convenient model system for the study of protein-RNA interactions. The MS2 coat protein achieves control of two distinct processes--sequence-specific RNA encapsidation and repression of replicase translation--by binding to an RNA stem-loop structure of 19 nucleotides containing the initiation codon of the replicase gene. The binding of a coat protein dimer to this hairpin shuts off synthesis of the viral replicase, switching the viral replication cycle to virion assembly rather than continued replication. The operator fragment alone can trigger self-assembly of the phage capsid at low protein concentrations and a complex of about 90 RNA operator fragments per protein capsid has been described. We report here the crystal structure at 3.0 A resolution of a complex between recombinant MS2 capsids and the 19-nucleotide RNA fragment. It is the first example of a structure at this resolution for a sequence-specific protein-RNA complex apart from the transfer RNA synthetase complexes. The structure shows sequence-specific interactions between conserved residues on the protein and RNA bases essential for binding.

Proteins binding to 5' untranslated region sites: a general mechanism for translational regulation of mRNAs in human and yeast cells.

We demonstrate that a bacteriophage protein and a spliceosomal protein can be converted into eukaryotic translational repressor proteins. mRNAs with binding sites for the bacteriophage MS2 coat protein or the spliceosomal human U1A protein were expressed in human HeLa cells and yeast. The presence of the appropriate binding protein resulted in specific, dose-dependent translational repression when the binding sites were located in the 5' untranslated region (UTR) of the reporter mRNAs. Neither mRNA export from the nucleus to the cytoplasm nor mRNA stability was demonstrably affected by the binding proteins. The data thus reveal a general mechanism for translational regulation: formation of mRNA-protein complexes in the 5' UTR controls translation initiation by steric blockage of a sensitive step in the initiation pathway. Moreover, the findings establish the basis for novel strategies to study RNA-protein interactions in vivo and to clone RNA-binding proteins.

Proteins binding to 5' untranslated region sites: a general mechanism for translational regulation of mRNAs in human and yeast cells.

We demonstrate that a bacteriophage protein and a spliceosomal protein can be converted into eukaryotic translational repressor proteins. mRNAs with binding sites for the bacteriophage MS2 coat protein or the spliceosomal human U1A protein were expressed in human HeLa cells and yeast. The presence of the appropriate binding protein resulted in specific, dose-dependent translational repression when the binding sites were located in the 5' untranslated region (UTR) of the reporter mRNAs. Neither mRNA export from the nucleus to the cytoplasm nor mRNA stability was demonstrably affected by the binding proteins. The data thus reveal a general mechanism for translational regulation: formation of mRNA-protein complexes in the 5' UTR controls translation initiation by steric blockage of a sensitive step in the initiation pathway. Moreover, the findings establish the basis for novel strategies to study RNA-protein interactions in vivo and to clone RNA-binding proteins.

Cellular protein modulates effects of human immunodeficiency virus type 1 Rev.

Replication of human immunodeficiency virus type 1 requires expression of the viral trans activator Rev. Rev binds to a highly structured RNA, the Rev response element, which is present in singly spliced and unspliced genomic viral RNAs. Although Rev helps to transport these transcripts from the nucleus to the cytoplasm, the mechanism(s) involved is not fully understood. Using the yeast two-hybrid system, we isolated a murine protein (YL2) that interacts with the basic domain of Rev, which is essential for the function of Rev in vivo and for the inhibitory splicing activity of Rev in vitro. YL2 has 92% identity to a human 32-kDa protein (p32), which copurifies with alternative splicing factor SF2/ASF. Furthermore, we found that whereas expression of YL2 greatly potentiated the activity of Rev, antisense YL2 transcripts blocked the effects of Rev in mammalian cells. YL2 also increased the activities of Rex on the Rex response element and of hybrid Rev proteins fused to Tat and the coat protein of bacteriophage MS2 on their respective RNAs. Thus, YL2 or p32 is a cellular protein that modulates the function of human immunodeficiency virus type 1 Rev.

Altering the RNA binding specificity of a translational repressor.

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.

Mutations that increase the affinity of a translational repressor for RNA.

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.

A Chimeric Human Immunodeficiency Virus Type 1 TAR Region Which Mediates High Level Trans-activation in Both Rodent and Human Cells

Trans-activation of the HIV-1 LTR by the Tat protein functions by a novel mechanism which involves the direct interaction of the Tat protein and cellular factors with nascently transcribed viral RNA encoding the Tat responsive element (TAR). Rodent cells do not efficiently support HIV-1 Tat activity because of a deficiency of human-specific factor(s). Human chromosome 12 appears to encode one of these Tat cofactors. We have designed chimeric TAR sequences which contain the heterologous RNA sequence derived from the bacteriophage R17 genome which binds to the bacteriophage MS2 coat protein. These chimeric TAR constructs were co-transfected into rodent and human cells with a plasmid encoding a chimeric Tat protein which contains the RNA binding domain of the MS2 coat protein. TAR constructs which contain the MS2 coat protein binding region inserted into the three nucleotide "bulge" region support a high level of trans-activation by Tat-MS2 coat protein chimeras in both human and rodent cells. This result suggests that the human-specific Tat cofactor(s) may act to allow Tat to interact effectively in a ribonucleoprotein complex which includes Tat, cellular factors, and TAR RNA.

Folding of the MS2 Coat Protein in Escherichia coli is Modulated by Translational Pauses Resulting from mRNA Secondary Structure and Codon Usage: A Hypothesis

Possible translational pauses within the coat protein of the RNA bacteriophage MS2 were located on the basis of a distribution plot of rare codons and RNA secondary structure. It appeared that the position of certain codon pauses corresponds with the size of some nascent polypeptide intermediates, which have been isolated from MS2-infected cells. Other accumulated polypeptide intermediates seemed to be related to RNA regions, where double-stranded secondary structures occur, which probably impede the movement of ribosomes during chain elongation. We assume that a discontinuous translation rate is designed to allow optimal folding of this (and other) polypeptide(s).

Multiple presentation of foreign peptides on the surface of an RNA-free spherical bacteriophage capsid.

We have produced a plasmid expression vector for the coat protein of RNA bacteriophage MS2. The vector has been modified to introduce a unique KpnI restriction site within the coat protein gene at a site corresponding to the most radially distant feature of the bacteriophage capsid, namely the top of the N-terminal beta-hairpin (between residues 15 and 16). Insertion of DNA oligonucleotides at this site allows the production of chimeric MS2 coat proteins having foreign peptide sequences expressed as the central part of the hairpin. We have produced chimeras with a number of different peptide sequences (up to 24 amino acids in length) chosen because of their known antigenic properties. The chimeric coat proteins self-assemble into largely RNA-free phage-like capsids in Escherichia coli and can be easily disassembled and reassembled in vitro. Such peptide-presenting particles may have a number of biotechnological applications, including use as a cost-effective, synthetic vaccine. We have tested the antigenicity of one such construct in vivo in mice and have shown that these particles are immunogenic and that antibody titres against the inserted peptide epitope can be obtained.

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.

Encapsidation of heterologous RNAs by bacteriophage MS2 coat protein.

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.

Bacteriophage and spliceosomal proteins function as position-dependent cis/trans repressors of mRNA translation in vitro.

The translational regulation of ferritin expression currently represents the only well characterized example for eukaryotic translational control by high affinity interactions between a specific cytoplasmic protein, iron regulatory factor [IRF], and an mRNA-binding site, the iron-responsive element [IRE], located in the 5' untranslated region [UTR] of ferritin mRNAs. To elucidate whether IRE/IRF may represent the first physiological example of a more general mechanism for mRNA-specific translational control, high affinity RNA-binding sites for the bacteriophage MS2 coat protein or the spliceosomal protein U1A were introduced into the 5' UTR of capped chloramphenicol acetyltransferase [CAT] transcripts. In the absence of these RNA-binding proteins, CAT mRNA was efficiently translated. Addition of purified MS2 coat protein or U1A caused a specific, dose-dependent repression of CAT biosynthesis in rabbit reticulocyte and wheat germ in vitro translation systems. The translational blockage imposed by the RNA/protein complex was reversible and did not alter the stability of the repressed mRNAs. Translational repression caused by binding of U1A or MS2 proteins to their target mRNAs is shown to be position-dependent in vitro. Thus, mRNA/protein complexes without an a priori role in eukaryotic mRNA translation function as translational effectors with characteristics resembling those of IRE/IRF.

Control of translational repression by protein-protein interactions.

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.

Trans-activation by HIV-1 Tat via a heterologous RNA binding protein

The HIV-1 trans-activator Tat increases levels of viral gene expression and replication. The target for Tat is an RNA stem-loop called TAR, located at the 5' end of all viral transcripts. To study the mechanism of action and map functional domains of Tat, we fused Tat to the coat protein of bacteriophage MS2, an RNA binding protein. TAR in the HIV-1 LTR was replaced by the operator, the RNA target of the coat protein. The hybrid Tat-coat protein trans-activated HIV-1 LTRs containing either TAR or operator sequences. Mutations in the operator that weaken binding of the coat protein in vitro led to decreased levels of trans-activation in vivo. Deletions in Tat within the hybrid Tat-coat protein identified activation and RNA binding domains of Tat. These experiments suggest that trans-activation by Tat can occur independently of TAR RNA and DNA binding proteins and that Tat exerts its effects on HIV-1 transcription by directly interacting with the TAR RNA stem-loop.

Translational repression by bacteriophage MS2 coat protein expressed from a plasmid. A system for genetic analysis of a protein-RNA interaction.

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.

Use of synthetic oligoribonucleotides to probe RNA-protein interactions in the MS2 translational operator complex

Synthetic oligoribonucleotides have been used to probe the interaction of MS2 coat protein with the translational operator of the MS2 replicase gene. We have investigated the possible formation of a transient covalent bond between the single-stranded uridine residue, at position -5, and a cysteine side-chain on the coat protein, by the incorporation of a chemically modified residue (5-BrU) at this position. This chemically synthesised operator variant has a binding constant of between 10 and 50 times greater than that of the wild type and is therefore comparable with the tight binding variant having a cytidine substituted at the -5 position. Dissociation kinetics show that the complex with the 5-BrU operator is more stable than the -5C variant; a result which is consistent with the formation of a Michael adduct at the -5 position. In addition, a number of other chemical variants of the operator have been analysed. These include operators incorporating deoxyadenine residues at each of the important single-stranded adenine sites. Recently the Michael adduct proposal has been challenged on the basis of mutagenesis of the coat protein cysteine residues. These results are discussed in the light of our data in support of Michael adduct formation.

Translationsl repression by bacteriophage MS2 coat protein does not require cysteine residues

Previous studies implicated cysteine residues in the translational repressor (i.e. RNA binding) activity of the coat protein of bacteriophage MS2. It has been proposed that a protein sulfhydryl forms a transient covalent bond with an essential pyrimidine in the translational operator by a Michael addition reaction. We have utilized codon-directed mutagenesis methods to determine the importance of each of the two coat protein cysteines for repressor function in vivo. The results indicate that cys46 can be replaced by a variety of amino acids without loss of repressor function. Cys101, on the other hand, is more sensitive to substitution. Most position 101 substitutions inactivate the repressor, but one (arginine) results in normal repressor activity. Although the possibility of a transient covalent contact between cys101 and RNA is not categorically ruled out, construction of double mutants demonstrates that cysteines are not absolutely required for translational repression by coat protein.

Missense misreading of asparagine codons as a function of codon identity and context.

During asparagine starvation the frequency of lysine for asparagine substitutions increases to levels that enable one to isolate and sequence mistranslated protein. We have used site-directed mutagenesis to construct a series of derivatives of the gene encoding the coat protein of the bacteriophage MS2. The mutant set constructed has either AAU or AAC as codon three in the gene with each possible adjoining 3' base. Lysine incorporation in coat protein encoded by these genes shows that AAU is misread from 4- to 9-fold more frequently than AAC with any 3' context. Although in some cases context effects of approximately 2-fold were noted, there seems to be no simple hypothesis to explain them.

Mistranslation during phenylalanine starvation.

Starvation for phenylalanine led to leucine misincorporation frequencies of 0.1 and 0.6 at UUC codons in the argI transcript of Escherichia coli, but no detectable misincorporation at a UUU codon. Under similar starvation conditions the relative synthesis of full sized MS2 coat protein, encoded by the RNA virus or a DNA copy, is greatly reduced, preventing analysis of the protein. This reduction in amount is unaffected by a rpsL mutation.

Effect of polyamines on translation fidelity in vivo.

Polyamines, when added to cell-free protein-synthesizing systems, have been shown either to reduce mistranslation or to increase it depending upon the composition of the reaction mixture. To study this question under conditions as natural as possible we investigated the effects of polyamines on the fidelity of protein synthesis in intact Escherichia coli bacterial cells, using strains which were auxotrophic for polyamine synthesis. Error was measured in two ways: the incorporation of [3H]histidine into coat protein of bacteriophage MS2, the gene of which does not code for histidine, and the synthesis of a basic variant of MS2 coat protein in which a lysine erroneously replaces an asparagine, causing a change in isoelectric point. We found that when cell cultures were supplemented with polyamines there was no effect on the first type of error and the second type decreased twofold. The measured errors occurred at the level of translation because their frequency increased in the presence of streptomycin and decreased in cells bearing a streptomycin-resistance mutation known to lower the occurrence of translational misreading. The average erroneous incorporation per mol coat protein in the presence of polyamines was 1.43 +/- 0.59 mmol histidine and 25-34 mmol lysine/asparagine substitution. The reason for the different effect of polyamines on the two types of error is not known but could be related to the difference between their corresponding frequencies or to codon-specific effects.