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Structural and biochemical investigation of bacteriophage N4-encoded RNA polymerases.

Lenneman BR, Rothman-Denes LB - Biomolecules (2015)

Bottom Line: These encode the components of new transcriptional machinery (N4 RNAPII and cofactors) responsible for the synthesis of middle RNAs.Both N4 RNAPs belong to the T7-like "single-subunit" family of polymerases.Herein, we describe their mechanisms of promoter recognition, regulation, and roles in the phage life cycle.

View Article: PubMed Central - PubMed

Affiliation: Committee on Genetics, Genomics, and Systems Biology, The University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA. blenneman@uchicago.edu.

ABSTRACT
Bacteriophage N4 regulates the temporal expression of its genome through the activity of three distinct RNA polymerases (RNAP). Expression of the early genes is carried out by a phage-encoded, virion-encapsidated RNAP (vRNAP) that is injected into the host at the onset of infection and transcribes the early genes. These encode the components of new transcriptional machinery (N4 RNAPII and cofactors) responsible for the synthesis of middle RNAs. Both N4 RNAPs belong to the T7-like "single-subunit" family of polymerases. Herein, we describe their mechanisms of promoter recognition, regulation, and roles in the phage life cycle.

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Related in: MedlinePlus

Comparison of the mini-vRNAP apo (Accession number 2PO4) (a) and BC (Accession number 3C3L) (b) structures highlighting rearrangements near the enzyme active site. Both structures show “cupped right hand” architecture (thumb, palm, fingers) characteristic of this family of RNAPs. Upon promoter binding, large scale structural rearrangements of the plug and β-IH motif occur, allowing the rearrangement of the motif B loop into the O helix to allow single-stranded DNA access to the active site. Bottom bar represents the mini-vRNAP primary sequence with amino acid numbering indicated in parentheses. Domains and structural motifs are labeled and colored as in the crystal structures, with template strand DNA represented as a purple ribbon.
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biomolecules-05-00647-f003: Comparison of the mini-vRNAP apo (Accession number 2PO4) (a) and BC (Accession number 3C3L) (b) structures highlighting rearrangements near the enzyme active site. Both structures show “cupped right hand” architecture (thumb, palm, fingers) characteristic of this family of RNAPs. Upon promoter binding, large scale structural rearrangements of the plug and β-IH motif occur, allowing the rearrangement of the motif B loop into the O helix to allow single-stranded DNA access to the active site. Bottom bar represents the mini-vRNAP primary sequence with amino acid numbering indicated in parentheses. Domains and structural motifs are labeled and colored as in the crystal structures, with template strand DNA represented as a purple ribbon.

Mentions: Despite the low level of sequence similarity between mini-vRNAP and T7-like polymerases, they share a common architecture. The “cupped right hand” architecture shared by all related DNA and RNA polymerases is evident in the crystal structure of the apo form of mini-vRNAP, solved at 2.0 Å resolution [36]. A comparison with the crystal structure of T7 RNAP shows that the active sites of T7 RNAP and mini-vRNAP superimpose, reinforcing results of mutational analyses [34,36,37]. This comparison also identified three structural motifs in mini-vRNAP shown to be required for promoter recognition in T7 RNAP: the AT-rich recognition loop, β-intercalating hairpin (β-IH), and specificity loop [38,39]. Surprisingly, the crystal structure of apo mini-vRNAP reveals two structural motifs, the “plug” and the “motif B loop,” that block the pathway of the DNA to the active site, suggesting that the structure represents an inactive conformation (Figure 3a) [36].


Structural and biochemical investigation of bacteriophage N4-encoded RNA polymerases.

Lenneman BR, Rothman-Denes LB - Biomolecules (2015)

Comparison of the mini-vRNAP apo (Accession number 2PO4) (a) and BC (Accession number 3C3L) (b) structures highlighting rearrangements near the enzyme active site. Both structures show “cupped right hand” architecture (thumb, palm, fingers) characteristic of this family of RNAPs. Upon promoter binding, large scale structural rearrangements of the plug and β-IH motif occur, allowing the rearrangement of the motif B loop into the O helix to allow single-stranded DNA access to the active site. Bottom bar represents the mini-vRNAP primary sequence with amino acid numbering indicated in parentheses. Domains and structural motifs are labeled and colored as in the crystal structures, with template strand DNA represented as a purple ribbon.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496689&req=5

biomolecules-05-00647-f003: Comparison of the mini-vRNAP apo (Accession number 2PO4) (a) and BC (Accession number 3C3L) (b) structures highlighting rearrangements near the enzyme active site. Both structures show “cupped right hand” architecture (thumb, palm, fingers) characteristic of this family of RNAPs. Upon promoter binding, large scale structural rearrangements of the plug and β-IH motif occur, allowing the rearrangement of the motif B loop into the O helix to allow single-stranded DNA access to the active site. Bottom bar represents the mini-vRNAP primary sequence with amino acid numbering indicated in parentheses. Domains and structural motifs are labeled and colored as in the crystal structures, with template strand DNA represented as a purple ribbon.
Mentions: Despite the low level of sequence similarity between mini-vRNAP and T7-like polymerases, they share a common architecture. The “cupped right hand” architecture shared by all related DNA and RNA polymerases is evident in the crystal structure of the apo form of mini-vRNAP, solved at 2.0 Å resolution [36]. A comparison with the crystal structure of T7 RNAP shows that the active sites of T7 RNAP and mini-vRNAP superimpose, reinforcing results of mutational analyses [34,36,37]. This comparison also identified three structural motifs in mini-vRNAP shown to be required for promoter recognition in T7 RNAP: the AT-rich recognition loop, β-intercalating hairpin (β-IH), and specificity loop [38,39]. Surprisingly, the crystal structure of apo mini-vRNAP reveals two structural motifs, the “plug” and the “motif B loop,” that block the pathway of the DNA to the active site, suggesting that the structure represents an inactive conformation (Figure 3a) [36].

Bottom Line: These encode the components of new transcriptional machinery (N4 RNAPII and cofactors) responsible for the synthesis of middle RNAs.Both N4 RNAPs belong to the T7-like "single-subunit" family of polymerases.Herein, we describe their mechanisms of promoter recognition, regulation, and roles in the phage life cycle.

View Article: PubMed Central - PubMed

Affiliation: Committee on Genetics, Genomics, and Systems Biology, The University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA. blenneman@uchicago.edu.

ABSTRACT
Bacteriophage N4 regulates the temporal expression of its genome through the activity of three distinct RNA polymerases (RNAP). Expression of the early genes is carried out by a phage-encoded, virion-encapsidated RNAP (vRNAP) that is injected into the host at the onset of infection and transcribes the early genes. These encode the components of new transcriptional machinery (N4 RNAPII and cofactors) responsible for the synthesis of middle RNAs. Both N4 RNAPs belong to the T7-like "single-subunit" family of polymerases. Herein, we describe their mechanisms of promoter recognition, regulation, and roles in the phage life cycle.

Show MeSH
Related in: MedlinePlus