Transcription initiation: molecular mechanisms of TFE

The information that determines the basic layout, development and behaviour of living organisms is mostly contained within their genes. The retrieval of this genetic information is termed transcription and is facilitated by the enzyme RNA Polymerase (RNAP). In order to access the right information in a timely manner this enzyme interacts with additional proteins (transcription factors). In some cases transcription factors are malfunctioning because they are altered (mutated) / resulting in the wrong information being read and applied at the wrong time and this causes diseases. We need to understand how the factors interact with the enzyme in order to understand the cause of such diseases and ultimately how to treat them. My research proposal focuses on one of the transcription factors, TFE. We have a reasonable knowledge of what TFE achieves during transcription but we do not know how. In essence, we do not understand the mechanisms underlying TFE function. The project we are proposing will address these questions by testing combinations of mutant variants of this factor and RNAP in experimental setups that characterise transcription. We will use biophysical techniques to examine how the structure of RNAP changes due to the influence of TFE. In summary, the research proposal is focused on the structure and function of the basal TFE factor and the RNA Polymerase enzyme.

Important regulatory events in the cell take place at the level of gene expression, which relies on the orchestrated interplay between transcription factors and RNA polymerase (RNAP). The archaeal transcription machinery is a genuine model system for the eukaryotic RNAPII apparatus because both share a complement of basal factors required for promoter-directed transcription (TBP, TFB and TFE). Whereas TBP and TFB are well characterised, the function of TFE remains unclear. Previous studies have indicated that TFE increases the stability of initiation- but destabilises transcription elongation-complexes. TFE consists of two domains (winged helix and Zinc-ribbon) and we do not currently understand how the two domains interact with RNAP and nucleic acids and how this affects transcription. We will apply a range of biochemical and biophysical strategies to characterise the molecular mechanisms of TFE. Our biochemical/molecular biological repertoire includes in vitro transcription assays that measure the activity of RNAP, gel retardation assays for the detection of protein-nucleic acid complex formation, size exclusion chromatography for protein-protein interactions and chemical cross-linking experiments that are suitable to investigate more transient molecular interactions. In addition we want to develop a biophysical fluorescence-based system (FRET) for the detection of conformational changes within RNAP in order to establish whether and how TFE induces RNAP subunit/domain rearrangements during the transcription initiation process. This system will also be able to follow the stepwise assembly of the initiation complex (DNA-TBP-TFB-RNAP-TFE) and its disassembly following the escape of RNAP from the promoter into the elongation phase of transcription.