The decay rate of a particular mRNA can greatly influence the amount of protein produced from that message and can thereby play an important role in the overall regulation of gene expression. The goal of this project is to elucidate the mechanisms by which cells degrade messenger RNA molecules and are able to regulate this decay process. The proposed experiments will determine the mechanism by which the E. coli chloramphenicol acetyltransferase (cat) message is degraded and how the stability of this message is regulated in response to culture growth conditions. Three cleavage sites have been detected within the cat message that may be involved in initiating the decay process. These cleavage sites will be mapped, and the importance of these cleavages to the decay process will be determined by deletion and site-directed mutagenesis and by gene fusion analyses. An in vitro message degradation assay will be used to study the decay process and to eventually purify the ribonucleases involved in these degradative events. Changes in the stability of the cat message in response to non-steady state growth conditions and medium composition will also be measured and compared to other messages such as ompA, bla, lpp, lac, and trxA. The results of these experiments will define the important components of the cat message decay pathway, which should be broadly applicable to other messages in E. coli, and will be lend insight into how message stability is regulated in response to culture growth conditions.
