In this proposal, two hypotheses concerning the regulation of RNA coliphage gene expression by long range RNA-RNA interactions will be examined: 1) Stable long range interactions at the 5' end of Q beta RNA suppress transnational initiation of the viral maturation protein. (2) Two alternate conformations (CH and ACH) of a large structural feature, the central hairpin, serve as a structural switch for the regulations of transnational initiation of viral coat protein. The long range RNA secondary structures, located within coding regions of genomic RNA, have been identified in structural studies on the conformation of genomic and mutant RNAs of coliphage Qbeta by the principal investigator. Their location within the viral genetic map suggest their function in the regulation of transnational initiation. The structural studies have shown that genomic RNA from coliphage Qbeta (4217 nucleotides) can be divided into a series of large structural domains that are well determined, as well as several regions where predicted structures are unusually plastic. The well determined structural domains range in size from 500 to 700 nucleotides and include the initiation region for the translation of viral maturation protein at he 5' end of Qbeta RNA. Variable regions include the initiation region for the translation for the viral coat and viral replicase translation. These sites are located in the lower half of a central hairpin (1500 nucleotides) where the structural rearrangements between the CH and ACH conformations are observed. In the CH conformation, the coat initiation region is free and large amounts of viral coat protein could be synthesized. In the ACH conformation, the coat initiation region is buried in extensive secondary structure. Coat synthesis (and all other viral protein synthesis) would be switched off. The switch may be mediated by viral and/or cellular proteins, possibly by viral replicase. A large structural switch of this type is novel, and has not been proposed previously in any experimental system. The proposed analyses will proceed in several steps. Additional structural studies with native and mutant RNAs are needed to develop reliable structural models at the nucleotide level for the long range interactions both at the 5' end of the RNA and in the region of the CH- ACH switch. On the basis of these studies, mutant RNAs will be created in which the proposed long range interactions will have been disrupted. The effect of these structural changes will be studied both in vitro and in vivo. Several unusual tools will be used in these analysis of potential structural changes in full size genomes by energy dot plot, and a genetic system in which cis-acting structural defects in the RNA can be identified. These unusual methods have been developed by the principal investigator and/or her collaborators. Little is known about the properties of major structural features within coding regions of large mRNAs, and our studies are of direct relevance in understanding the role of RNA structure in human diseases such as AIDS and the common cold.
| Zuker, M; Jacobson, A B (1995) ""Well-determined"" regions in RNA secondary structure prediction: analysis of small subunit ribosomal RNA. Nucleic Acids Res 23:2791-8 |
