These investigations will focus on elucidating the process of mRNA degradation in Escherichia coli. The goal of these studies will be to identify and characterize RNA elements responsible for differences in mRNA stability, to define the features of a key bacterial ribonuclease that determine its specificity and function, and to elucidate important aspects of the mechanism by which this ribonuclease acts. Molecular biological, biochemical, and genetic methods will be employed. Particular attention will be devoted to RNA degradation by RNase E, the ribonuclease thought to govern the principal pathway for mRNA decay in E. coli. First, we will examine the molecular characteristics that are important for the ability of this endonuclease to sense the 5' phosphorylation state of its RNA substrates and to cleave those that are monophosphorylated at an accelerated rate. In addition, we will investigate the initial degradative events that govern mRNA longevity in E. coli. Finally, we will study the mechanism by which RNase E autoregulates its synthesis in E. coli. 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. ? ?
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