Asymmetric contributions to RNA binding by the Thr(45) residues of the MS2 coat protein dimer.
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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. (+info)
In vivo oligo(A) insertions in phage MS2: role of Escherichia coli poly(A) polymerase.
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Previously we introduced an RNase III site into the genome of RNA phage MS2 by extending a hairpin with a perfect 18 bp long stem. One way in which the phage escaped from being killed by RNase III cleavage was to incorporate uncoded A residues on either side of the stem. This oligo(A) stretch interrupts the perfect stem that forms the RNase III site and thus confers resistance. In this paper we have analyzed the origin of these uncoded adenosines. The data strongly suggest that they are added by the host enzyme poly(A) polymerase. Apparently the 3'-OH created by RNase III cleavage becomes a substrate for poly(A) polymerase. Subsequently, MS2 replicase makes one contiguous copy from the two parts of the genome RNA. The evolutionary conversion from RNase III sensitivity to resistance provides a large spectrum of solutions that could be an important tool to understand what essentially constitutes an RNase III site in vivo. (+info)
RNA aptamers for the MS2 bacteriophage coat protein and the wild-type RNA operator have similar solution behaviour.
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We have probed the effects of altering buffer conditions on the behaviour of two aptamer RNAs for the bacterio-phage MS2 coat protein using site-specific substitution of 2'-deoxy-2-aminopurine nucleotides at key adenosine positions. These have been compared to the wild-type operator stem-loop oligonucleotide, which is the natural target for the coat protein. The fluorescence emission spectra show a position and oligonucleotide sequence dependence which appears to reflect local conformational changes. These are largely similar between the differing oligonucleotides and deviations can be explained by the individual features of each sequence. Recognition by coat protein is enhanced, unaffected or decreased depending on the site of substitution, consistent with the known protein-RNA contacts seen in crystal structures of the complexes. These data suggest that the detailed conformational dynamics of aptamers and wild-type RNA ligands for the same protein target are remarkably similar. (+info)
Forced retroevolution of an RNA bacteriophage.
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The operator hairpin ahead of the replicase gene in RNA bacteriophage MS2 contains overlapping signals for binding the coat protein and ribosomes. Coat protein binding inhibits further translation of the gene and forms the first step in capsid formation. The hairpin sequence was partially randomized to assess the importance of this structure element for the bacteriophage and to monitor alternative solutions that would evolve on the passaging of mutant phages. The evolutionary reconstruction of the operator failed in the majority of mutants. Instead, a poor imitation developed containing only some of the recognition signals for the coat protein. Three mutants were of particular interest in that they contained double nonsense codons in the lysis reading frame that runs through the operator hairpin. The simultaneous reversion of two stop codons into sense codons has a very low probability of occurring. Therefore the phage solved the problem by deleting the nonsense signals and, in fact, the complete operator, except for the initiation codon of the replicase gene. Several revertants were isolated with activities ranging from 1% to 20% of wild type. The operator, long thought to be a critical regulator, now appears to be a dispensable element. In addition, the results indicate how RNA viruses can be forced to step back to an attenuated form. (+info)
General effect of Sam68 on Rev/Rex regulated expression of complex retroviruses.
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We have previously demonstrated that overexpression of Sam68 functionally substitutes for, as well as synergizes with, HIV-1 Rev in RRE-mediated gene expression and virus replication. In addition, C-terminal deletion mutants of Sam68 exhibit a transdominant negative phenotype in HIV replication. We now report that Sam68 also enhances the activities of Rev-like proteins of other complex retroviruses (e.g. HTLV-1 and EIAV) on their respective RNA targets. Furthermore, we demonstrate that Sam68 can function alone as well as synergize with Rev-MS2 and/or Rex-MS2 chimeric proteins on expression mediated by the corresponding RRE-MS2 fusion RNA element. Additionally, dominant negative mutants of Sam68 also repressed the synergistic activation of Sam68 with Rex, E-Rev, and/or Rev-MS2/Rex-MS2 on their corresponding RNA targets. Thus, Sam68 may play an important role in the post-transcriptional regulation of all complex retroviruses. Oncogene (2000) 19, 4071 - 4074 (+info)
RNA analysis by ion-pair reversed-phase high performance liquid chromatography.
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Ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC) is presented as a new, superior method for the analysis of RNA. IP RP HPLC provides a fast and reliable alternative to classical methods of RNA analysis, including separation of different RNA species, quantification and purification. RNA is stable under the analysis conditions used; degradation of RNA during the analyses was not observed. The versatility of IP RP HPLC for RNA analysis is demonstrated. Components of an RNA ladder, ranging in size from 155 to 1770 nt, were resolved. RNA transcripts of up to 5219 nt were analyzed, their integrity determined and they were quantified and purified. Purification of mRNA from total RNA is described, separating mouse rRNA from poly(A)(+) mRNA. IP RP HPLC is also suitable for the separation and purification of DIG-labeled from unlabeled RNA. RNA purified by IP RP HPLC exhibits improved stability. (+info)
Evaluation of methylphosphonates as analogs for detecting phosphate contacts in RNA-protein complexes.
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The well-studied interaction between the MS2 coat protein and its cognate hairpin was used to test the utility of the methylphosphonate linkage as a phosphate analog. A nitrocellulose filter binding assay was used to measure the change in binding affinity upon introduction of a single methylphosphonate stereoisomer at 13 different positions in the RNA hairpin. Comparing these data to the available crystal structure of the complex shows that all phosphates that are in proximity to the protein show a weaker binding affinity when substituted with a phosphorothioate and control positions show no change. However, in two cases, a methylphosphonate isomer either increased or decreased the binding affinity where no interaction can be detected in the crystal structure. It is possible that methylphosphonate substitutions at these positions affect the structure or flexibility of the hairpin. The utility of the methylphosphonate substitution is compared to phosphate ethylation and phosphorothioate substitution experiments previously performed on the same system. (+info)
Peptide display on live MS2 phage: restrictions at the RNA genome level.
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The potential of the RNA phage MS2 to accommodate extra amino acids in its major coat protein has been examined. Accordingly, a pentapeptide was encoded in the genome as an N-terminal extension. In the MS2 crystal structure, this part of the coat protein forms a loop that extends from the outer surface of the icosahedral virion. At the RNA level, the insert forms a large loop at the top of an existing hairpin. This study shows that it is possible to maintain inserts in the coat protein of live phages. However, not all inserts were genetically stable. Some suffer deletions, while others underwent adaptation by base substitutions. Whether or not an insert is stable appears to be determined by the choice of the nucleic acid sequence used to encode the extra peptide. This effect was not caused by differential translation, because coat-protein synthesis was equal in wild-type and mutants. We conclude that the stability of the insert depends on the structure of the large RNA hairpin loop, as demonstrated by the fact that a single substitution can convert an unstable loop into a stable one. (+info)