The long term goal of this proposal is to obtain a complete understanding of the regulation of transcription termination in a procaryotic cell. This laboratory has focused on elucidating the mechanism by which certain transcriptional activator proteins act to suppress the ability of the transcription machinery to recognize and respond to genetic signals for termination and release of mRNA. The model system used in this study is phage lambda whose gene N product has been hypothesized to act on RNA polymerase at specific cis-acting loci to allow the formation of a termination-resistant transcription apparatus. The modification of the transcription apparatus by N protein involves a number of cellular proteins of which some act to positively effect termination and others act as components of the ribosome in the cell. The proposed experiments, combining genetic, biochemical and immunochemical approaches, will attempt to identify all cellular components involved in N-dependent antitermination process, determine whether and which of these factors are components of the normal and N-modified elogating transcription apparatus, and determine the order with which they assemble and the respective recognition signals which allow their assembly. The domains of the interacting proteins and respective genetic signals will be identified through the analysis of suppressor mutations, gene fusions an construction of specifically designed chimeras of the transcription factors, and by employing chemically synthesized peptides and oligonucleotides. The sites of contacts in the interacting domains of the respective proteins and the genetic signals will be determined by chemical and UV cross-linking studies, and by deliberate alterations of these entities through site-directed mutagenesis. The hypothesis that the N protein, as a stable subunit of the elongating transcription apparatus, competes with termination signals and factors for interaction with the termination domain of RNA polymerase will be tested. In addition to providing a molecular mechanism of termination suppression, these studies should provide fundamental information on the chemical nature of the elongating transcription apparatus and the physiological parameters and the genetic signals which govern transcription elongation in the procaryotic cell.
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