Our project is to probe the processes that assemble various proteins and RNA to form a transcriptionally active complex. The protein-protein and RNA-protein interactions will be explored in a system that is uniquely approachable genetically and biochemically, namely E. coli and coliphage. We are focused on the mechanism of transcription termination and its suppression (antitermination). We will study two phage proteins, HK022 Nun termination factor and ?N antitermination factor. Both utilize host proteins, NusA, NusB, NusE (S10) and NusG to support their reactions. Nun and ?N bind ??RNA and nucleate a complex with the four Nus Factors and RNAP. The Nus factors are also active in suppressing transcription termination in ribosomal RNA operons. The project takes advantage of our recent isolation of strains that can support deletions in NusA, NusG, or NusB. We also propose to isolate a strain bearing a transcriptionally-inactive NusE. Informed by new structures of the Nus factors and the phage proteins, we will generate mutant proteins likely to affect their transcriptional activity. We shall interrogate individual domains in order to relate structure and function. The mutants will be tested in vivo and in a variety of in vitro assays, including purified transcription systems, RNA binding tests and structural analyses. The fundamental similarity between this prokaryotic system and eukaryotic transcription systems imply that the results obtained here will have broad biological application.
Regulation of transcription termination is important in both prokaryotic and eukaryotic organisms. This project focuses on how transcription termination in E. coli is essential for chromosome integrity and how it relates to ribosome release.
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