Our laboratory seeks to understand the control of messenger RNA decay in the gram-positive bacterium, Bacillus subtilis. While much is known about the mechanism and regulation of mRNA synthesis (transcription) and translation of mRNA into protein, little is known about the intermediate step in gene expression-degradation of mRNA. Experiments in this proposal focus on 2 aspects of mRNA decay: the elements (e.g., sequences, structures) of an mRNA that determine its half-life, and the ribonuclease activities that are required for initiation and completion of mRNA decay. The availability of B. subtilis strains that are deficient in 1 or more 3'-to-5' exoribonucleases will make it possible to examine the role of individual ribonucleases in mRNA turnover. The decay of 3 small mRNAs, whose characteristics have been studied to some extent, will be analyzed in detail and will serve as models for the study of mRNA decay generally. Experiments are proposed to clarify: 1) what is the initiation site for decay? 2) which ribonuclease(s) participates in initiation of decay? 3) how does the decay mechanism deal with stable secondary structure? and 4) how do the various 3'-to-5' exoribonucleases bind and degrade mRNA? The role of the 4 known B. subtilis 3'-to-5' exoribonucleases -- PNPase, RNase R, RNase PH, and YhaM-will be assessed in the turnover of model mRNAs, as well as newly-identified mRNAs that are stabilized in a PNPase-deficient mutant strain. The likely participation of 2 B. subtilis endoribonucleases -- RNase J1 and RNase J2 -in mRNA decay will be assessed. An in vitro system will be established that will be useful in probing the characteristics of purified ribonucleases, which will be overexpressed and isolated from E. coli. Relevance: Messenger RNA (mRNA) is the template molecule upon which proteins are synthesized. Bacteria rely on rapid mRNA decay to adapt to changing environments, and the details of this process will be studied in detail in the model microorgansim, Bacillus subtilis. Elucidating the mechanism of mRNA in this bacterium could lead to the design of new antibiotics that inhibit the mRNA decay process and thereby prevent successful bacterial colonization of human tissues. ? ? ?
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