How transcription factors regulate bacterial RNA polymerase activity

• Applicant: Professor Dr. Paul Rösch
• Subject Area: Structural Biology
• Term: from 2015 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 284071855

This project will contribute to the clarification of the mechanisms on an atomic scale of bacterial DNA:RNA transcription. A particular aim is the elucidation of the regulatory processes that lead to transitions between the various steps of transcription and the conformational changes of the contributing proteins during these transitions. Among the long term goals of the project is to lay a foundation for the development of new antimicrobial substances. For these studies we will employ spectroscopic, biochemical and molecular biology methods. The focus is the analysis of interactions of RNA polymerase (RNAP) with Nus factors and related proteins by nuclear magnetic resonance (NMR) spectroscopy in solution. The traditional toolbox of NMR spectroscopy will be enlarged by 13C and 15N specific and unspecific protein isotope labeling schemes combined with perdeuteration. In particular, RNAP will be made accessible to NMR studies by separate expression of the RNAP subunits, 13C labeling of methyl groups of the individual subunits, and reconstitution of the intact multimeric protein. Similarly, the ligand proteins will be rendered observable by 13C labeling of their methyl groups even in complex with RNAP. Thus it will be possible to define rather accurately the interaction surfaces between RNAP and ligand proteins as well as describe the geometry of the complexes. We will supplement these studies by a whole range of biochemical, molecular biology, and in vivo techniques as well as by molecular modeling, molecular docking, and molecular dynamics calculations. Site directed variation of individual amino acids of the respective proteins and studies of protein fragments will play a central role, in parallel with chemical cross-linking experiments. Fluorescence spectroscopy of labeled or intrinsically fluorescent protein variants as well as fluorescence resonance energy transfer (FRET) experiments will allow a more accurate description of the mutual interactions of proteins of the transcription complex. We will include in our studies the transcription factor RfaH, as RfaH shows an unprecedented folding behavior, namely the reversible transition between alpha-helical conformation and beta-strand conformation of a complete domain. This so far unique behavior of RfaH will be studied, in particular we will try to identify those amino acids that facilitate this transition, and we will try to define the processes during transcription that initiate the structural changeover and thus the activation of RfaH. Highly likely, intricate interactions between specific DNA sequences, RNAP, and RfaH are key to this transformation.

• DFG Programme: Research Grants