The research proposed in this application is a continuation of our general effort to establish how specific features of nucleic acid sequence and structure serve as controlling points during the steps of gene expression. The emphasis is on the synthesis, processing, and function of messenger RNA. A natural set of overlapping polycistronic mRNAs encoding proteins expressed in strikingly disparate yields will be investigated, to determine what roles the post-transcriptional processing events these mRNA transcripts undergo, as well as subsequent controls during translation, play in effecting differential gene expression. Host nuclease activity responsible for the processing of the mRNAs will be characterized, to learn more about enzymes that make specific cleavages in mRNA and about the functions such enzymes have in the cell. Host mutants that fail to carry out these processing events will greatly facilitate addressing these problems, and genetic selections for such mutants will be carried out. Via systematic manipulation of necleotide sequence and direct study of RNA structure, the functional elements of the MRNA processing sites and the determinants of initiation activity in selected translational initiator regions will be identified. Through direct examination of the in vivo transcription process, the effect of sequence-specific DNA binding proteins on the progress of elongating RNA polymerase will be examined. Overall, the unique biological properties of the experimental systems, and their suitability for a combination of in vitro biochemical analysis and in vivo functional studies, ensure substantial contibutions from this work to an improved understanding of these important steps of gene expression.
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