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Schwinghammer, Kathrin (2014): Structure and function of human mitochondrial RNA polymerase elongation complex. Dissertation, LMU München: Fakultät für Chemie und Pharmazie



Mitochondria are often described as molecular power stations of the cell as they generate most of the energy that drives cellular processes. Mitochondria are eukaryotic organelles with bacterial origin that contain an extra-nuclear source of genetic information. Although most mitochondrial proteins are encoded in the nucleus, the mitochondrial genome still encodes key components of the oxidative phosphorylation machinery that is the major source for cellular adenosine 5’-triphosphate (ATP). The mitochondrial genome is transcribed by a singlesubunit DNA-dependent RNA polymerase (RNAP) that is distantly related to the RNAP of bacteriophage T7. Unlike its T7 homolog, mitochondrial RNA polymerase (mtRNAP) relies on two transcription factors, TFAM and TFB2M, to initiate transcription. The previously solved structure of free mtRNAP has revealed a unique pentatricopeptide repeat (PPR) domain, a N-terminal domain (NTD) that resembles the promoter-binding domain of T7 RNAP and a C-terminal catalytic domain (CTD) that is highly conserved in T7 RNAP. The CTD adopts the canonical right-hand fold of polymerases of the pol A family, in which its ‘thumb’, ‘palm’ and ‘fingers’ subdomains flank the active center. Since the structure represents an inactive “clenched” conformation with a partially closed active center, only limited functional insights into the mitochondrial transcription cycle have been possible so far. This work reports the first crystal structure of the functional human mtRNAP elongation complex, determined at 2.65 Å resolution. The structure reveals a 9-base pair DNA-RNA hybrid formed between the DNA template and the RNA transcript and one turn of DNA both upstream and downstream of the hybrid. Comparisons with the distantly related T7 RNAP indicate conserved mechanisms for substrate binding and nucleotide incorporation, but also strong mechanistic differences. Whereas T7 RNAP refolds during the transition from initiation to elongation, mtRNAP adopts an intermediary conformation that is capable of elongation without NTD refolding. The intercalating hairpin that melts DNA during mtRNAP and T7 RNAP initiation additionally promotes separation of RNA from DNA during mtRNAP elongation. The structure of the mtRNAP elongation complex (this work) and free mtRNAP (previously published) demonstrate that mtRNAP represents an evolutionary intermediate between singlesubunit and multisubunit polymerases. Furthermore, it illustrates the adaption of a phage-like RNAP to a new role in mitochondrial gene expression.