Transcription in E. coli: Structural Basis of Nus-Factor-Dependent Regulation

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Competition between transcription termination and antitermination constitutes a key mechanism of gene regulation, as exemplified by the paradigmatic processive antitermination during lytic growth of phage λ or during ribosomal RNA (rrn) transcription in Escherichia coli and by the premature termination evoked by phage HK022. These processes depend on an RNA signal element (the N-utilization, nut, site), phage proteins N (λ) or Nun (HK022) and four host N-utilization substances (NusA, NusB, NusE [equivalent to ribosomal protein S10] and NusG), which assemble elaborate ribonudeoprotein complexes on the surface of RNA polymerase (RNAP). Here, I propose to investigate the molecular mechanisms underlying termination/antitermination in the above systems by an integrative approach, building on structural biochemical expertise of my own group (X-ray crystallography) and of the collaborating group of Paul Rösch (Universität Bayreuth, NMR spectroscopy). Specifically, we intend to investigate the mechanisms by which NusB assists the diverse functions of SIO; to elucidate the mode of boxA RNA binding by the NusB-S10 complex; to investigate the functional interplay of NusG with the NusB-S10-boxA RNA complex, with RNA polymerase and with transcription termination factor Rho; and to develop methods to assemble entire transcription termination and antitermination complexes for structural and functional studies. Apart from shedding light onto fundamental regulatory principles of transcription and translation, the outcome of the proposed work will provide novel platforms for the development of antimicrobial substances.