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 microorganism 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 motifs in both wild-type and mutant E. coli rho proteins and RNA target sites will be examined in catalysis of transcription termination, as will the role of accessory factors. Parallel studies in yeast will be accompanied by analysis of mutations identified in vivo as affecting transcriptional readthrough and both will be correlated with 3'RNA processing, the behavior of pol II, and messenger RNA maturation. 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, crosslinking procedures, competition experiments, and gel shift studies. 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, and their relationships to cellular metabolism. Analyses of interactions between regulatory sequences and the macromolecules or cofactors acting upon them are 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 result from regulatory mechanisms gone awry, in he long run a detailed molecular understanding should contribute to the development of solutions to these health problems.
Showing the most recent 10 out of 26 publications
