PDBsum entry 3c46

Transferase/DNA

PDB id

3c46

Contents

			Protein chains

					1095 a.a.

			DNA/RNA

			Ligands

					2HP ×2

			Waters ×967

References listed in PDB file

Key reference

Title

Structural basis for DNA-Hairpin promoter recognition by the bacteriophage n4 virion RNA polymerase.

Authors

M.L.Gleghorn, E.K.Davydova, L.B.Rothman-Denes, K.S.Murakami.

Ref.

Mol Cell, 2008, 32, 707-717. [DOI no: 10.1016/j.molcel.2008.11.010]

PubMed id

19061645

Note: In the PDB file this reference is annotated as "TO BE PUBLISHED". The citation details given above have been manually determined.

Abstract

Coliphage N4 virion-encapsidated RNA polymerase (vRNAP) is a member of the phage T7-like single-subunit RNA polymerase (RNAP) family. Its central domain (mini-vRNAP) contains all RNAP functions of the full-length vRNAP, which recognizes a 5 to 7 base pair stem and 3 nucleotide loop hairpin DNA promoter. Here, we report the X-ray crystal structures of mini-vRNAP bound to promoters. Mini-vRNAP uses four structural motifs to recognize DNA sequences at the hairpin loop and stem and to unwind DNA. Despite their low sequence similarity, three out of four motifs are shared with T7 RNAP that recognizes a double-stranded DNA promoter. The binary complex structure and results of engineered disulfide linkage experiments reveal that the plug and motif B loop, which block the access of template DNA to the active site in the apo-form mini-vRNAP, undergo a large-scale conformational change upon promoter binding, explaining the restricted promoter specificity that is critical for N4 phage early transcription.



	Figure 2. Figure 2. The Interaction between the Promoter Hairpin and the N4 vRNAP (A) P2 promoter DNA structure in the binary complex. The hairpin-stem promoter consists of a double-stranded stem (−5 to −9 and −17 to −13) and a 3 nt loop (−10 to −12). Template DNA contains bases from −4 to +2 with the transcription start site at +1. (B) Promoter hairpin-loop recognition. R119 and K114 interact with −11G (N7 and 6-keto) and −10G (N7), respectively. W129 participates in a stacking interaction with −11G. The fingers residues K849 and K850 form salt bridges with the phosphate backbone at −12 and −13, respectively. Hydrogen bonds and salt bridges are depicted by red and green dashed lines, respectively. Color code of the structure motifs is indicated. (C) Promoter recognition by the specificity loop (cyan) and the β-intercalating hairpin (orange) in the P2_7a binary complex. D901 and R904 of the specificity loop recognize bases −9/−10 and −8, respectively, from the major grove. R902 interacts with the phosphate backbone at −7 and −6. Residues K267 and K268 in the β-intercalating hairpin face the DNA stem to separate the last 2 bp of the 7 bp stem, yielding a 5 bp stem, and direct the template DNA toward the active site. (D) Difference of −11 and R119/W129 interactions between P1 (blue) and P2 (pink) promoters in the binary complexes. Only residues R119 and W129 in the P2 binary complex are shown. The bifurcate hydrogen bonds between −11G (P2 promoter) and R119 are depicted by dashed red lines.		Figure 3. Figure 3. Positioning of the Transcription Start Site at the vRNAP Active Site A view of the P2_7a promoter-binary complex active center. Thumb and plug are removed to see the active center. Residue R318 in the N-terminal domain has a cation-π interaction with base −2 and salt bridges with the phosphate backbone (depicted by yellow and green dashed lines, respectively) that induce a DNA kink between bases −2 and −1. The +3 base is rotated by vert, similar 90°, presenting only DNA bases from −1 to +2 to the active site. Amino acid residues essential for activity at the active site are shown: R424 (T/DxxGR motif) for substrate binding; D559 (motif A) and D951 (motif C) for chelating the catalytically essential Mg^2+ ions; and R666, K670, and Y678 (motif B) for substrate binding. The boxed area is magnified.

PROCHECK

	Headers