Although the presence of polyadenylate sequences at the 3' -ends of eukaryotic mRNAs has ben known for almost two decades, very little is understood about their physiological function. The more recent discovery that polyadenylate sequences are also present at the termini of bacterial mRNAs provides the opportunity to subject the polyadenylation process to combined biochemical and genetic analysis, which should greatly facilitate the elucidation of its function and the characterization of its components. The proposed research aims to elucidate the mechanism of polyadenylation by identifying the enzymes and nucleic acid sequences that play an essential role in mRNA polyadenylation and by reconstituting these components to yield an in vitro system capable of polyadenylating specific mRNAs. One major focus is to examine the effects of mutations affecting ribonucleases and poly (A) polymerases on the degree and pattern of polyadenylation of a specific mRNA. Another line of investigation will attempt to reconstitute a cell-free polyadenylation system in order to define the protein components required from optimal polyadenylation as well as factors that may modulate the polyadenylation process. A third focus is the analysis of the essential sequence elements at the polyadenylation sites of specific Escherichia coli mRNAs by cloning and sequencing complementary DNAs and by examining the effects of site-directed mutagenesis.
A final aim i s the characterization of the poly (A) polymerases of Escherichia coli, of which there are two major isozymes, and the study of their genes, one of which has already been cloned. It is hop[ed that the proposed application of combined biochemical and molecular genetic approaches to the study of mRNA polyadenylation in E. coli will provide insights into the biological function of this process, which as thus far resisted elucidation in more complex organisms.
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