Both prokaryotic and eukaryotic systems are represented in this study of transcription termination at the ends of genes, and of events associated with producing the mature 3' ends of transcripts in E. coli and S. cerevisiae. The molecular biology of such problems in these microorganisms has the advantage of both sophisticated genetics and biochemical accessibility. The focus will be guided by an additional desire to understand (1) what aspects of protein structure determine the strength and specificity of recognition and binding to nucleic acid, and (2) how RNA sequence and structure (more varied and less well understood than double-stranded DNA) relates to interaction with and recognition by protein. Proposed experiments will use current genetic, biochemical, and chemical approaches in vivo and in vitro. 1. Factors and signals involved in mRNA 3' end formation in E. coli and yeast: Recognition requirements of both wild-type and mutant E. coli rho proteins and RNA target sites for ability to catalyze transcription termination will be studied. Yeast mutat- ions identified in vivo as affecting transcriptional readthrough will be correlated with RNA cleavage and polyadenylation in vitro, and maturation of the RNA transcript. 2. Protein-nucleic acid interactions in transcription. Analysis of the functional interactions between RNA and proteins or macromolecular complexes that act upon it will be undertaken using chemical and enzymatic probes as well as crosslinking procedures. 3. Coupled interactions in transcription. The dynamic interactions that couple RNA binding and ATP hydrolysis to events producing mature 3' ends will be examined as will higher order overall coordination of polymerase termination and the processing events. Analysis of the interaction between regulatory sequences and the macromolecules or cofactors acting upon them is a prerequisite for understanding these processes at the molecular level. The basic principles involved in microbial systems will undoubtedly be applicable to the control of gene expression in higher organisms. Since many diseases are the result of regulatory mechanisms done awry, in the long run a detailed molecular understanding should contribute to the development of solutions to these health problems.
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