Comparative Analysis of Structure in RNAs of Related RNA Coliphages

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The studies proposed here come from early work by the principal investigator showing that large reproducible structural features could be seen by electron microscopy in RNA obtained from the RNA coliphages M12 and MS2. These studies suggested that the structures that were seen by electron microscopy in the RNA might represent base pared regions that were unusually stable. The location of these structures within the viral genetic map further suggested that they might play a role in regulating viral gene expression in infected cells. Recently the PI has been examining the structure of related coliphage RNAs by electron microscopy in order to identify conserved structural features; conserved features are expected to have functional significance in infected cells. The conformation of genomic RNAs from the bacteriophages MS2, GA, Qbeta and SP were examined. These bacteriophages represent 4 major viral groups. A large central loop is conserved in all four RNAs and a model was developed at the nucleotide level for 780 nucleotides of this loop in Qbeta. The structure of the loop has been confirmed using biochemical methods. The loop is one of the most stable features (resistant to denaturation) seen by electron microscopy in all of the phage RNAs. The structure and function of this loop will be studied in more detail by genetic analysis. In addition, the PI proposes to develop full models at the nucleotide level for the RNAs of the related coliphages SP, Qbeta and MX1 using electron microscopy, comparative sequence analysis, biochemical probing, and computer modeling. The genetic studies are being done in collaboration with Don Mills, Department of Microbiology and Immunology, SUNY Downstate Medical Center. The Mills lab has developed a genetic system for isolating and growing mutants of Qbeta carrying large deletions by supplying the wild type viral protein in trans. The have shown that deletions in some regions of the viral replicase gene cannot be complemented with viral replicase that is supplied in trans and have called these regions cis-acting RNA elements. The PI believes that these mutants define regions in the genome where the structure of the RNA is important. The conformation of the mutant RNAs will be examined both by electron microscopy and with biochemical methods. In addition, this type of analysis will be extended to the conserved central loop region that is described above. It is hoped that the information obtained by our analysis will prove useful not only in understanding the functional significance of structure in coliphage RNAs, but will also help in understanding the role of RNA structure in other genomic viral RNAs and in cellular messenger RNAs.