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Structure of human mitochondrial RNA polymerase elongation complex.

Schwinghammer K, Cheung AC, Morozov YI, Agaronyan K, Temiakov D, Cramer P - Nat. Struct. Mol. Biol. (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Gene Center, Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany.

ABSTRACT
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.

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Active center and nucleic acid strand separation observed in the crystal structure(a) Conservation of active centers in mtRNAP (color code as in Figs. 1 and 2) and T7 RNAP (PDB 3E2E15, light blue). Structures were superimposed based on their palm subdomains and selected residues were depicted as stick models.(b) Downstream DNA strand separation.(c) RNA separation from DNA at the upstream end of the hybrid and thumb–hybrid interactions.(d) Primer extension assays showed that a thumb subdomain plays a key role in elongation complex stability. Elongation complexes of WT (lanes 1 and 2) and Δthumb (lanes 3 and 4) mtRNAP variants were halted 18 nucleotides (nts) downstream of the light-strand promoter (LSP) by omitting cytidine triphosphate (CTP)31 (see also Supplementary Fig. 5a online).
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Figure 3: Active center and nucleic acid strand separation observed in the crystal structure(a) Conservation of active centers in mtRNAP (color code as in Figs. 1 and 2) and T7 RNAP (PDB 3E2E15, light blue). Structures were superimposed based on their palm subdomains and selected residues were depicted as stick models.(b) Downstream DNA strand separation.(c) RNA separation from DNA at the upstream end of the hybrid and thumb–hybrid interactions.(d) Primer extension assays showed that a thumb subdomain plays a key role in elongation complex stability. Elongation complexes of WT (lanes 1 and 2) and Δthumb (lanes 3 and 4) mtRNAP variants were halted 18 nucleotides (nts) downstream of the light-strand promoter (LSP) by omitting cytidine triphosphate (CTP)31 (see also Supplementary Fig. 5a online).

Mentions: The active site closely resembles that of T7 RNAP and harbors the RNA 3′-end at its catalytic residue D1151 (refs. 6–8) (Fig. 3a). Comparison with phage RNAP structures that contain the nucleoside triphosphate (NTP) substrate9,10 supports a conserved mechanism of substrate binding, selection, and catalysis. The location and relative arrangement of amino acid residues in the active center that bind catalytic metal ions and the NTP substrate are conserved in both enzymes. The trajectory of several side chains differs, but this was likely due to the absence of metal ions and NTP in our structure.


Structure of human mitochondrial RNA polymerase elongation complex.

Schwinghammer K, Cheung AC, Morozov YI, Agaronyan K, Temiakov D, Cramer P - Nat. Struct. Mol. Biol. (2013)

Active center and nucleic acid strand separation observed in the crystal structure(a) Conservation of active centers in mtRNAP (color code as in Figs. 1 and 2) and T7 RNAP (PDB 3E2E15, light blue). Structures were superimposed based on their palm subdomains and selected residues were depicted as stick models.(b) Downstream DNA strand separation.(c) RNA separation from DNA at the upstream end of the hybrid and thumb–hybrid interactions.(d) Primer extension assays showed that a thumb subdomain plays a key role in elongation complex stability. Elongation complexes of WT (lanes 1 and 2) and Δthumb (lanes 3 and 4) mtRNAP variants were halted 18 nucleotides (nts) downstream of the light-strand promoter (LSP) by omitting cytidine triphosphate (CTP)31 (see also Supplementary Fig. 5a online).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321815&req=5

Figure 3: Active center and nucleic acid strand separation observed in the crystal structure(a) Conservation of active centers in mtRNAP (color code as in Figs. 1 and 2) and T7 RNAP (PDB 3E2E15, light blue). Structures were superimposed based on their palm subdomains and selected residues were depicted as stick models.(b) Downstream DNA strand separation.(c) RNA separation from DNA at the upstream end of the hybrid and thumb–hybrid interactions.(d) Primer extension assays showed that a thumb subdomain plays a key role in elongation complex stability. Elongation complexes of WT (lanes 1 and 2) and Δthumb (lanes 3 and 4) mtRNAP variants were halted 18 nucleotides (nts) downstream of the light-strand promoter (LSP) by omitting cytidine triphosphate (CTP)31 (see also Supplementary Fig. 5a online).
Mentions: The active site closely resembles that of T7 RNAP and harbors the RNA 3′-end at its catalytic residue D1151 (refs. 6–8) (Fig. 3a). Comparison with phage RNAP structures that contain the nucleoside triphosphate (NTP) substrate9,10 supports a conserved mechanism of substrate binding, selection, and catalysis. The location and relative arrangement of amino acid residues in the active center that bind catalytic metal ions and the NTP substrate are conserved in both enzymes. The trajectory of several side chains differs, but this was likely due to the absence of metal ions and NTP in our structure.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Gene Center, Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus