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Ringel, Eva Rieke (2011): Molecular basis of RNA polymerase III transcription repression by Maf1 & Structure of human mitochondrial RNA polymerase. Dissertation, LMU München: Fakultät für Chemie und Pharmazie



Topic I Molecular basis of RNA polymerase III transcription repression by Maf1 RNA polymerase III (RNAP III) is a conserved 17-subunit enzyme that transcribes genes encoding short untranslated RNAs such as transfer RNAs (tRNAs) and 5S ribosomal RNA (rRNA). These genes are essential and involved in fundamental processes like protein biogenesis; hence RNAP III activity needs to be tightly regulated. RNAP III is repressed upon stress and this is regulated by Maf1, a protein conserved from yeast to humans. Many stress pathways were shown to converge on Maf1 and result in its phosphorylation, followed by its nuclear import and eventual repression of RNAP III activity. However, the molecular mechanisms of this repression activity were not known at the beginning of these studies. This work establishes the mechanism of RNAP III specific transcription repression by Maf1. The crystal structure of Maf1 was solved. It has a globular fold with surface accessible NLS sequences, which sheds new light on already published results and explains how stress-induced phopshorylation leads to import of Maf1 into the nucleus. Additionally, cryo EM studies and competition assays show that Maf1 binds RNAP III at its clamp domain and thereby induces structural rearrangements of RNAP III, which inhibits the interaction with Brf1, a subunit of the transcription initiation factor TFIIIB. This specifically impairs recruitment of RNAP III to its promoters and implies that Maf1 is a repressor of transcription initiation. Competition and transcription assays show that Maf1 also binds RNAP III that is engaged in transcription, leaving RNAP III activity intact but preventing re-initiation. Topic II Structure of human mitochondrial RNA polymerase The nuclear-encoded human mitochondrial RNAP (mitoRNAP) transcribes the mitochondrial genome, which encodes rRNA, tRNAs and mRNAs. MitoRNAP is a single subunit (ss) polymerase, related to T7 bacteriophage and chloroplast polymerases. All share a conserved C-terminal core, whereas the N-terminal parts of mitoRNAP do not show any homology to other ss RNAPs. Unlike phage RNAPs, which are self-sufficient, human mitoRNAP needs two essential transcription factors for initiation, TFAM and TFB2M. Both of these factors are likely to control the major steps of transcription initiation, promoter binding and melting. Thus human mitoRNAP has evolved a different mechanism for transcription initiation and exhibits a unique transcription system. Structural studies thus far concentrated on the nuclear enzymes or phage RNAPs, whereas the structure of mitochondrial RNA polymerase remained unknown. The structural organization of human mitoRNAP and the molecular mechanisms of promoter recognition, binding and melting were subject of interest in these studies. In this work the crystal structure of human mitoRNAP was solved at 2.4 Å resolution and reveals a T7-like C-terminal catalytic domain, a N-terminal domain that remotely resembles the T7 promoter-binding domain (PBD), a novel pentatricopeptide repeat (PPR) domain, and a flexible N-terminal extension. MitoRNAP specific adaptions in the N-terminus include the sequestering of one of the key promoter binding elements in T7 RNAP, the AT-rich recognition loop, by the PPR domain. This sequestration and repositioning of the N-terminal domain explain the need for the additional initiation factor TFAM. The highly conserved active site within the C-terminal core was observed to bind a sulphate ion, a well known phosphate mimic, and thereby suggests conserved substrate binding and selection mechanisms between ss RNAPs. However, conformational changes of the active site were observed due to a movement of the adjacent fingers subdomain. The structure reveals a clenching of the active site by a repositioned fingers subdomain and an alternative position of the intercalating -hairpin. This explains why the conserved transcription factor TFB2M is required for promoter melting and initiation. A model of the mitochondrial initiation complex was build to further explore the initiation mechanism, and to rationalize the available biochemical and genetic data. The structure of mitoRNAP shows how this enzyme uses mechanisms for transcription initiation that differ from those used by phage and cellular RNAPs, and which may have enabled regulation of mitochondrial gene transcription and adaptation of mitochondrial function to changes in the environment.