The long term goal of our research has been to understand post-transcriptional regulation in Escherichia coli. The general objective is to address current and fundamental questions in translation and mRNA processing/decay. We have focused attention on a transcription unit encoding six genes from the filamentous phage f1 genome that are transcribed in huge amounts but regulated exclusively after transcription to yield proteins made in different amounts. Appropriate regulation of these genes is required to balance phage gene expression and permit the phage to maintain a persistent infection in its host. First, the proposed research will complete studies of controls in the last 2 of the 6 genes. These genes are an in-frame overlapping pair encoding essential DNA replication genes with opposing functions. Present evidence indicates that the genes are regulated at several points, including mRNA stability. We will strengthen evidence that RNA structure inhibits translation of the smaller gene and identify apparently multiple factors that limit internal initiation on the mRNA encoding both genes. Of broader interest, this will clarify how regulation of gene expression in in- frame overlapping gene pairs, numerous in bacterial, phage and plasmid genomes, is achieved. Second, the proposed research will focus heavily on the pathway by which these very abundant phage mRNAs undergo mRNA degradation. The specific rationales are that these mRNAs will reflect a major activity on the decay machinery in infected hosts, and more important, the phage mRNAs illustrate a 5' to 3' wave of decay that is the predominant pathway for decay of bacterial mRNAs. Messenger RNA decay has proven an important parameter that determines levels of gene expression and permits rapid responses to cellular signalling signals, but until recent accelerated progress, has been the slowest of the principal gene regulatory processes to be worked out. Currently, the field is poised to make major progress. As part of this effort, we will define the steps of the f1 mRNA decay pathway, the major aim being to test proposed molecular models for decay. We will identify the cleavages made by major enzymes with functions in endo- and exonucleolytic decay, and as part of planning for in vitro reconstitution studies, define the decay pathways for smaller f1 mRNA substrates. In a new direction, we will follow f1 mRNA decay and degradosome localization within the bacterial cell.
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