1. Volume 32, Issue 5, Pages 707-717 (December 2008)
Structural Basis for DNA-Hairpin Promoter Recognition by the Bacteriophage N4 Virion RNA Polymerase 
Michael L. Gleghorn, Elena K. Davydova, Lucia B. Rothman-Denes, Katsuhiko S. Murakami 
Molecular Cell 
Volume 32, Issue 5, Pages (December 2008)
DOI: /j.molcel
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2. Figure 1
Promoter DNA Sequences and the Overall Structure of Binary Complex
(A) Sequences and secondary structures of the three vRNAP promoters (WT-P1, WT-P2, and WT-P3) in the N4 genome. The transcription start site (+1) is colored in green, and the direction of transcription is shown by an arrow.
(B) Promoter DNA constructs used for crystallization. The downstream region highlighted by the gray box is disordered in the binary complex structures. The transcription start site (+1) is colored in green.
(C) Overall structure of the N4 mini-vRNAP bound to the P2 promoter (top). The promoter DNA hairpin is depicted by a pink ribbon. The thick bar (bottom) represents the N4 mini-vRNAP primary sequence with amino acid numbering. Domains, subdomains, and structural motifs are labeled and color coded as in the top panel; the color scheme is maintained throughout this paper.
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3. 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.
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4. 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 ∼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 Mg2+ ions; and R666, K670, and Y678 (motif B) for substrate binding. The boxed area is magnified.
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5. Figure 4
Role of R318 in Determining the Site of Transcription Initiation
(A) Effect of mini-vRNAP R318 substitutions on di- and trinucleotide synthesis on P2-4A-8 and P2-4T-8 templates. The percentage of GA synthesis is shown below each lane. The sequences of P2-4A-8 and P2-4T-8 are shown. Inverted repeat sequences that form the hairpin stem are indicated by arrows. The center of the hairpin loop (−11) and transcription start site (+1) with transcripts (GGA, from +1; GA, from +2) in the presence of GTP and ATP are shown.
(B) Phosphodiester bond formation by catalytic autolabeling using templates with increasing number (n) of As (P2-An-CTA) or Ts (P2-Tn-CTA) between the promoter hairpin and CTA and the hydroxybenzaldehyde derivatives of ATP (bATP) or GTP (bGTP). The sequences of the templates are shown at the top (n corresponds to the number of As and Ts between the hairpin and C in the P2-An-CTA and P2-Tn-CTA templates, respectively). (Bottom left) bATP. (Bottom right) bGTP. %, activity. TI, transcription initiation site (+1 corresponds to 11 nucleotides from the center of the hairpin loop).
(C) Selection of the transcription initiation site by wild-type and R318 mutant enzymes on templates containing different combinations of As and Ts between −4 and −1. %GA, transcription initiation at +2.
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6. Figure 5 Conformational Changes
(A) Superimposed ribbon representations of N4 mini-vRNAP alone (black) and mini-vRNAP (color coded as in Figure 1C) in the binary complex. The two structures were aligned by superimposing their palm cores. The movements of the mobile modules from the apo-form structure to their positions in the binary complex are indicated by the arrows (green, a 6.8° rigid body movement including the N-terminal domain, except for the plug and the intercalating β-hairpin, and the C-terminal one-third of the fingers, residues 806–927; orange, a rigid 25.1° body motion of the plug with intercalating β-hairpin).
(B) A 12.8° rigid body movement of the N-terminal two-thirds of fingers (residues 608–805) with a large structural transition of motif B. The structures in the binary complex and the apo-form are colored in light blue/yellow and black, respectively. Motif B rearranges its structure from a loop (apo-form) to a short antiparallel β-hairpin (binary complex) with the largest transition (32.6 Å) at residue N659 (red dashed arrow). The positions of R666 and K670 in motif B that participate in substrate binding are also shown.
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7. Figure 6 The Plug and Motif B Loop Inactivate Mini-vRNAP
(A) The S885-K176 and G6363-G660 distances in the apo-form (left panel) and the binary complex (right panel) are shown. Wild-type, K176C/S885C, and G363C/G660C enzymes were purified in the absence of DTT, preincubated with cystine (−DTT) and then with DTT (+DTT), and analyzed by SDS-PAGE and silver staining (B) for their ability to bind to the promoter hairpin (C), catalyze autolabeling (D), and run off transcription (E). Disulfide bond-containing species is denoted by an asterisk. The percentage of enzyme in each species and their respective activities are shown below each lane.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
The Three Promoter-Recognition Structural Motifs of N4 and T7 RNAPs
(A) The −11 recognition site (gray), specificity loop (cyan), and β-intercalating hairpin (orange) of N4 vRNAP in the binary complex. The promoter hairpin is depicted by a pink ribbon.
(B) The AT-rich recognition loop (gray), specificity loop (cyan), and β-intercalating hairpin (orange) of T7 RNAP in the binary complex (PDB: 2PI5). Template and nontemplate DNA strands are depicted by pink and yellow ribbons, respectively. The orientation of the T7 RNAP binary complex has been aligned with the N4 mini-vRNAP binary complex in (A) by superimposing their palm cores. Amino acid residues involved in promoter recognition are shown as stick and labeled.
(C) Sequential remodeling of the N4 phage genome DNA and vRNAP to achieve phage early gene transcription. Both N4 vRNAP promoters and vRNAP are injected into the host cell in inactive conformations. Host factors—DNA gyrase and EcoSSB—play a role in remodeling the N4 genome DNA to produce three hairpin-form promoters (P1, P2, and P3) that, upon binding to vRNAP, remodel the vRNAP conformation to form the transcription-ready binary complexes. EcoSSB also participates in transcription elongation as a DNA template recycling factor. Structures of N4 mini-vRNAP in the apo-form (left, inactive) and binary complex (right, active) are shown. Color scheme is the same as in Figure 1C.
Molecular Cell  , DOI: ( /j.molcel )
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