Our laboratory seeks to understand the control of mRNA decay in Bacillus subtilis. For the Gram-positive bacteria, much needs to be learned about the features of an mRNA that determine its half-life, the ribonucleolytic reactions involved in mRNA decay, the genes that encode ribonuc the regulation of their expression. Since the B. subtilis genome does not have sequence homologues for several of the major ribonuclease genes of Escherichia coli, it is altogether uncertain whether models for mRNA decay based on the well-studied E. coli system will pertain to B. subtilis. Small RNA molecules will be used to probe three facets of mRNA turnover in B. subtilis: 1) the entry site for 3' exonucleolytic degradation; 2) endonucleolytic cleavage that initiates decay; and 3) role of the 5' end in determining mRNA half-life. These small RNA molecules are designed such that analysis of their decay will begin to clarify how mRNA turnover is achieved. To identify additional ribonuclease genes (three have been cloned thus far), biochemical experiments are proposed to isolate several proteins predicted to be involved in mRNA decay, i.e., at least one additional 3'-to-5' exoribonuclease, a putative endoribonuclease, and poly(A) polymerase. Once the identities of these proteins are known, the genes encoding them will be disrupted in order to study the effects on mRNA decay. To develop our understanding of ribonuclease function in the Gram-positive bacteria, we will study the function of Bs-RNase III, a narrow-specificity endoribonuc lease that has been shown to be essential in B. subtilis. Genetic means will be employed in an effort to understand the role of Bs-RNase Ill that is critical for viability. The basis for control of Bs-R.Nase III activity in the cell will be investigated, providing the first look at ribonuclease gene regulation in B. subtilis.
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