Evgeny A. Nudler
Mechanisms of Transcription Elongation and Its Regulation In Bacteria and Yeast
Transcription, the central step in gene expression and regulation, is carried out by DNA-dependent RNA polymerase (RNAP). Cellular RNAPs are large, multisubunit assemblies. Their complexity presumably reflects an involvement in interactions with numerous regulatory signals and factors that modulate the activity at all stages of transcription. Recent progress in the understanding of transcriptional mechanisms was essentially attributed to the studies on the primary stage of transcription cycle (promoter binding and activation). Although the following steps of transcription (elongation and termination) were shown to be the major target for gene regulation as well, our knowledge about their mechanisms is considerably less compelling.
Our research is focused on structural understanding of the mechanisms of transcription elongation and its regulation in E.coli and yeast systems. To this end we undertook biochemical and structural characterization of the basic intermediates of elongation - the ternary complexes that composed of elongating form of RNAP, DNA template and RNA product. Using the immobilized transcription system - an approach that allows one to generate defined elongation complexes stalled at desired positions on DNA, in combination with footprinting, affinity crosslinking, and protein chemistry methods we have build the new structure-functional model of the elongation complex (see the figure below). This model provide a key to the mechanism of transcription processivity and proofreading. It also has far reaching implications for understanding of regulatory mechanisms of elongation such as termination and pausing. Using this model as a starting point we now pursue two main projects:
1) Structural analysis of transcription elongation complexes from E.coli and yeast. Combining the approaches mentioned above together with an electron microscopy we propose to generate a detailed 3 dimensional topological map of ternary elongation complexes based on the available low resolution structure of E.coli and yeast RNAP II. On this map certain protein regions within RNAP subunits will be assigned to the principal functional domains.
2) Studies on molecular mechanisms that regulate transcription elongation. The thrust of this project is to determine the key intermediates in the pathways leading to termination, pausing or arrest of the elongation by E.coli RNAP and yeast RNAP II and how the elongation factors can modulate these pathways.