Structure and function of human mitochondrial RNA polymerase elongation complex

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

Mechanism of Transcription Anti-termination in Human Mitochondria.

In human mitochondria, transcription termination events at a G-quadruplex region near the replication origin are thought to drive replication of mtDNA by generation of an RNA primer. This process is suppressed by a key regulator of mtDNA-the transcription factor TEFM. We determined the structure of an anti-termination complex in which TEFM is bound to transcribing mtRNAP. The structure reveals interactions of the dimeric pseudonuclease core of TEFM with mobile structural elements in mtRNAP and the nucleic acid components of the elongation complex (EC). Binding of TEFM to the DNA forms a downstream sliding clamp, providing high processivity to the EC. TEFM also binds near the RNA exit channel to prevent formation of the RNA G-quadruplex structure required for termination and thus synthesis of the replication primer. Our data provide insights into target specificity of TEFM and mechanisms by which it regulates the switch between transcription and replication of mtDNA

Mechanism of Transcription Anti-termination in Human Mitochondria

In human mitochondria, transcription termination events at a G-quadruplex region near the replication origin are thought to drive replication of mtDNA by generation of an RNA primer. This process is suppressed by a key regulator of mtDNA—the transcription factor TEFM. We determined the structure of an anti-termination complex in which TEFM is bound to transcribing mtRNAP. The structure reveals interactions of the dimeric pseudonuclease core of TEFM with mobile structural elements in mtRNAP and the nucleic acid components of the elongation complex (EC). Binding of TEFM to the DNA forms a downstream “sliding clamp,” providing high processivity to the EC. TEFM also binds near the RNA exit channel to prevent formation of the RNA G-quadruplex structure required for termination and thus synthesis of the replication primer. Our data provide insights into target specificity of TEFM and mechanisms by which it regulates the switch between transcription and replication of mtDNA

Empirische Forschung in der Deutschdidaktik. Band 2: Erhebungs- und Auswertungsverfahren

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Structure of human mitochondrial RNA polymerase elongation complex

Here we report the crystal structure of the human mitochondrial RNA polymerase (mtRNAP) transcription elongation complex, determined at 2.65-Å resolution. The structure reveals a 9-bp 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 RNA polymerase (RNAP) from bacteriophage T7 indicates 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 refolding. The intercalating hairpin that melts DNA during T7 RNAP initiation separates RNA from DNA during mtRNAP elongation. Newly synthesized RNA exits toward the pentatricopeptide repeat (PPR) domain, a unique feature of mtRNAP with conserved RNA-recognition motifs