Control of Transcription Termination in E.coli

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DESCRIPTION (provided by applicant): Regulation of gene expression at the level of transcription termination is ubiquitous in nature. Nevertheless the precise mechanism of the termination reaction, even in bacteria, is only partially understood. Coliphage HK022 Nun protein binds nascent phage lambda transcript at specific sites (nut) and induces transcription arrest in vitro and termination in vivo. Interactions between Nun and RNA polymerase and host factors NusA, NusB, NusE and NusG are involved in this reaction. Host Mfd protein releases Nun-arrested RNA polymerase in vitro and in vivo. We will explore the possibility that Nun interacts with a host factor analogous to NusA Nun intramolecular contacts as well as interactions between Nun and host proteins will be defined by chemical and genetic approaches. Cysteine-substituted Nun will be derivatized with cross-linkers and reacted with RNA polymerase and Nus factors. The location of the cross-link will be determined by Mass Spectrometry. Cysteine-substituted Nun will also be used to determine the location of Nun in a Nun-RNA polymerase complex by FRET analysis. Nun mutants will be characterized with respect to their interactions with RNA polymerase and NusA. Binding of Nun and the related lambda protein, N, to nut and to the RNaseIII target site in the lambda pL transcript will be studied in vivo and in vitro. The basis of Nun toxicity to E. coli will be investigated and the possibility that Nun recognizes sequences other than nut will be tested.