The characterization of two major polyA polymerases of E. coli and the identification of their genes together with the definition of the sites of polyadenylation and the recent identification of a third polyA polymerase, have set the stage for the detailed study of the function and regulation of bacterial mRNA polyadenylation. The general aim of the proposed research is to elucidate the physiological function of bacterial mRNA polyadenylation through a combination of biochemical approaches. Specifically, the proposed experiments will focus on the following aspects. 1. Purification of the third polyA polymerase and identification of its gene. This should fill the last gap in the understanding of the major E. coli polyA polymerases and their genes and pave the way for the study of polyA polymerase function. 2. Genetic studies of the relative roles of the E. coli polymerases. Strains with deletions of the genes for the three polyA polymerases, either singly or in combination, will be constructed in order to examine the physiological effects of the loss of these enzymes. Examination of the growth phenotypes of such strains and their parent will reveal whether a particular polyA polymerase is essential for growth under specific conditions and whether the loss of all three enzymes is lethal. Comparison of cDNA libraries from strains lacking one of the polyA polymerases and their parent will reveal possible selectivity of the three polyA polymerases for different classes of mRNA. 3. Effect of polyadenylation on mRNA function. The contribution of polyA tracts to the efficiency of mRNA translation will be examined in a reconstituted in vitro translation system. Special attention will be focussed on the role of ribosomal protein S1 as a potential polyA binding protein. The role of mRNA polyadenylation as a modulator of mRNA degradation will be examined by comparing the half-lives of specific mRNAs in E. coli strains with lesions in the polyA polymerases. The multiplicity of polyA polymerases in E. coli suggests a fail-safe mechanism to protect a critical biological function. It is anticipated that these studies will significantly enhance the understanding of bacterial gene expression by elucidating the mechanism and function of mRNA polyadenylation, a process which has only recently been recognized to be an important aspect of mRNA metabolism in all organisms.
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