NSF Award
9612367
9630780 Stitt Certain Escherichia coli that contain a mutated transcription termination factor Rho exhibit faster messenger RNA degradation rates and drastically reduce gene expression of infecting T4 bacteriophage. These phenotypes suggest a novel function for Rho in message degradation. An in vivo message degradation system will be used to compare the rate and pattern of degradation of specific mRNAs in wild type and rho(nusD) mutant cell extracts. Experiments will be performed to distinguish between two hypotheses to explain the altered message turnover: increased nuclease activity and increased message susceptibility to nucleases. In particular, Rho will be tested for a proposed role in the protection of RNA from degradation. Two means of overcoming the effects of the rho mutation have been found. First, spontaneous mutants in a new T4 gene, goF, restore the ability of T4 to grow in rho mutant cells. Second, introduction of the plasmid pBR322 into the rho mutant cells also renders them permissive for T4 growth. The rop gene of pBR322 has been found to be responsible for this phenotypic reversal, and the Rop protein is the critical molecule. Expression of either Rop or GoF1 in E. coli lengthens mRNA lifetimes, restoring those in rho(nusD) mutant cells to normal values and lengthening those in rho+ cells. The in vitro message degradation system will be used to determine the effects of Rop and GoFl on message turnover. Affinity chromatography and gel shift techniques will be used to identify interacting molecules in this system. The proposed work will characterize new and previously unsuspected roles for transcription termination factor Rho and pBR322 Rop in the control of mRNA degradation. ***
