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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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Analysis of mtRNAP–nucleic acid contacts by cross-linking experiments (see also Supplementary Fig. 5b and c online)(a) RNA nucleotide –9 cross-links to the specificity loop of mtRNAP. The cross-linked complexes were treated with 2-nitro-5-thiocyano-benzoic acid (NTCB, lanes 2 and 3) or cyanogen bromide (CNBr, lanes 5 and 6). Positions of the cysteine (cys) and methionine (met) residues that produced labeled peptides are indicated in purple and green, correspondingly. Grey numbers indicate methionine residues that did not produce labeled peptides and the expected migration of these peptides.(b) Mapping of the RNA–mtRNAP cross-link at RNA nucleotide –13 with different mtRNAP variants having a single hydroxylamin clevage site (NG) at a defined position. The cross-links were treated with hydroxylamine (NH2OH). The major cross-linked peptides are highlighted in black, minor (less than 10%) cross-linking sites in grey.(c) Mapping of the template strand DNA–mtRNAP cross-link at nucleotide –8. The cross-links were treated with NH2OH as described above.(d) Location of the cross-linked regions in mtRNAP elongation complex.The T7 RNAP specificity loop was built into the mRNAP structure by homology modeling. The structural elements that belong to the identified cross-linked regions and lie within 3–5 Å from the photo cross-linking probe include the modeled specificity loop (yellow, residues 1080–1108), part of the thumb (orange, residues 752–791) and part of the intercalating hairpin (purple, residues 605–623). Cross-linked regions that are not part of a defined structural element are shown in dark grey (e.g. helix G residues 587–571 and helix I residues 570–586).
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Figure 4: Analysis of mtRNAP–nucleic acid contacts by cross-linking experiments (see also Supplementary Fig. 5b and c online)(a) RNA nucleotide –9 cross-links to the specificity loop of mtRNAP. The cross-linked complexes were treated with 2-nitro-5-thiocyano-benzoic acid (NTCB, lanes 2 and 3) or cyanogen bromide (CNBr, lanes 5 and 6). Positions of the cysteine (cys) and methionine (met) residues that produced labeled peptides are indicated in purple and green, correspondingly. Grey numbers indicate methionine residues that did not produce labeled peptides and the expected migration of these peptides.(b) Mapping of the RNA–mtRNAP cross-link at RNA nucleotide –13 with different mtRNAP variants having a single hydroxylamin clevage site (NG) at a defined position. The cross-links were treated with hydroxylamine (NH2OH). The major cross-linked peptides are highlighted in black, minor (less than 10%) cross-linking sites in grey.(c) Mapping of the template strand DNA–mtRNAP cross-link at nucleotide –8. The cross-links were treated with NH2OH as described above.(d) Location of the cross-linked regions in mtRNAP elongation complex.The T7 RNAP specificity loop was built into the mRNAP structure by homology modeling. The structural elements that belong to the identified cross-linked regions and lie within 3–5 Å from the photo cross-linking probe include the modeled specificity loop (yellow, residues 1080–1108), part of the thumb (orange, residues 752–791) and part of the intercalating hairpin (purple, residues 605–623). Cross-linked regions that are not part of a defined structural element are shown in dark grey (e.g. helix G residues 587–571 and helix I residues 570–586).

Mentions: RNA exits over a positively charged surface patch, but shows poor electron density that indicates mobility (Fig. 2d). To investigate whether the weak electron density reflects the RNA exit path, we carried out protein–RNA cross-linking experiments. When we replaced the first RNA base beyond the hybrid by a photo cross-linkable analogue, it was cross-linked to the specificity loop (Figs. 4a, d and Supplementary Fig. 4 online). Thus the mobile specificity loop lines the RNA exit channel, as in the T7 RNAP elongation complex16,17. Exiting RNA at position –13 cross-linked to NTD helices I and G and thus the transcript emerges towards the PPR domain (Figs. 4b, d) that contains conserved RNA recognition motifs21.


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)

Analysis of mtRNAP–nucleic acid contacts by cross-linking experiments (see also Supplementary Fig. 5b and c online)(a) RNA nucleotide –9 cross-links to the specificity loop of mtRNAP. The cross-linked complexes were treated with 2-nitro-5-thiocyano-benzoic acid (NTCB, lanes 2 and 3) or cyanogen bromide (CNBr, lanes 5 and 6). Positions of the cysteine (cys) and methionine (met) residues that produced labeled peptides are indicated in purple and green, correspondingly. Grey numbers indicate methionine residues that did not produce labeled peptides and the expected migration of these peptides.(b) Mapping of the RNA–mtRNAP cross-link at RNA nucleotide –13 with different mtRNAP variants having a single hydroxylamin clevage site (NG) at a defined position. The cross-links were treated with hydroxylamine (NH2OH). The major cross-linked peptides are highlighted in black, minor (less than 10%) cross-linking sites in grey.(c) Mapping of the template strand DNA–mtRNAP cross-link at nucleotide –8. The cross-links were treated with NH2OH as described above.(d) Location of the cross-linked regions in mtRNAP elongation complex.The T7 RNAP specificity loop was built into the mRNAP structure by homology modeling. The structural elements that belong to the identified cross-linked regions and lie within 3–5 Å from the photo cross-linking probe include the modeled specificity loop (yellow, residues 1080–1108), part of the thumb (orange, residues 752–791) and part of the intercalating hairpin (purple, residues 605–623). Cross-linked regions that are not part of a defined structural element are shown in dark grey (e.g. helix G residues 587–571 and helix I residues 570–586).
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getmorefigures.php?uid=PMC4321815&req=5

Figure 4: Analysis of mtRNAP–nucleic acid contacts by cross-linking experiments (see also Supplementary Fig. 5b and c online)(a) RNA nucleotide –9 cross-links to the specificity loop of mtRNAP. The cross-linked complexes were treated with 2-nitro-5-thiocyano-benzoic acid (NTCB, lanes 2 and 3) or cyanogen bromide (CNBr, lanes 5 and 6). Positions of the cysteine (cys) and methionine (met) residues that produced labeled peptides are indicated in purple and green, correspondingly. Grey numbers indicate methionine residues that did not produce labeled peptides and the expected migration of these peptides.(b) Mapping of the RNA–mtRNAP cross-link at RNA nucleotide –13 with different mtRNAP variants having a single hydroxylamin clevage site (NG) at a defined position. The cross-links were treated with hydroxylamine (NH2OH). The major cross-linked peptides are highlighted in black, minor (less than 10%) cross-linking sites in grey.(c) Mapping of the template strand DNA–mtRNAP cross-link at nucleotide –8. The cross-links were treated with NH2OH as described above.(d) Location of the cross-linked regions in mtRNAP elongation complex.The T7 RNAP specificity loop was built into the mRNAP structure by homology modeling. The structural elements that belong to the identified cross-linked regions and lie within 3–5 Å from the photo cross-linking probe include the modeled specificity loop (yellow, residues 1080–1108), part of the thumb (orange, residues 752–791) and part of the intercalating hairpin (purple, residues 605–623). Cross-linked regions that are not part of a defined structural element are shown in dark grey (e.g. helix G residues 587–571 and helix I residues 570–586).
Mentions: RNA exits over a positively charged surface patch, but shows poor electron density that indicates mobility (Fig. 2d). To investigate whether the weak electron density reflects the RNA exit path, we carried out protein–RNA cross-linking experiments. When we replaced the first RNA base beyond the hybrid by a photo cross-linkable analogue, it was cross-linked to the specificity loop (Figs. 4a, d and Supplementary Fig. 4 online). Thus the mobile specificity loop lines the RNA exit channel, as in the T7 RNAP elongation complex16,17. Exiting RNA at position –13 cross-linked to NTD helices I and G and thus the transcript emerges towards the PPR domain (Figs. 4b, d) that contains conserved RNA recognition motifs21.

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