These investigations will focus on elucidating the process of mRNA degradation in Escherichia coli. The goal of these studies will be to define the RNA elements responsible for differences in mRNA stability, to better characterize the nature of the rate-determining step in mRNA degradation, and to identify bacterial proteins that influence rates of mRNA decay. Both molecular biological and genetic methods will be employed. Particular attention will be devoted to RNA degradation by RNase E, an endonuclease that appears to control the principal pathway for mRNA decay in E. coli. First, we will analyze in detail the structural features near the 5' ends of E. coli transcripts that determine RNA longevity in vivo by controlling the rate of RNase E cleavage. The cis-acting determinants to be investigated will include 5'-terminal secondary structure, mRNA sequences in the vicinity of the ribosome binding site, and the 5'-proximal elements involved in the autoregulation of rne (RNase E) gene expression. These will be studied by creating defined or random mutations in various genes and analyzing the effect of these mutations on the decay of the resulting RNA transcripts in E. coli. We will also test whether RNase E participates in the rate-determining step in the translational inactivation and decay of mRNAs degraded via the RNase E pathway. Finally, genetic strategies will be employed to identify other E. coli proteins that may influence the activity or specificity of RNase E. The results of these studies should enhance our knowledge of a fundamental aspect of gene regulation that presently is poorly understood. This knowledge should ultimately be of value in maximizing bacterial production of medically useful proteins and in clarifying a biological regulatory mechanism that can play an important role in microbial pathogenesis and mammalian oncogenesis.
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