Volume 122, Issue 4, Pages (August 2005)

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Volume 122, Issue 4, Pages 541-552 (August 2005) Inhibition of Bacterial RNA Polymerase by Streptolydigin: Stabilization of a Straight- Bridge-Helix Active-Center Conformation  Steven Tuske, Stefan G. Sarafianos, Xinyue Wang, Brian Hudson, Elena Sineva, Jayanta Mukhopadhyay, Jens J. Birktoft, Olivier Leroy, Sajida Ismail, Arthur D. Clark, Chhaya Dharia, Andrew Napoli, Oleg Laptenko, Jookyung Lee, Sergei Borukhov, Richard H. Ebright, Eddy Arnold  Cell  Volume 122, Issue 4, Pages 541-552 (August 2005) DOI: 10.1016/j.cell.2005.07.017 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Target of Stl (A and B) Amino-acid sequence alignments for regions of E. coli RNAP β′ subunit (A) and E. coli β subunit (B) in which single-residue substitutions that confer Stl resistance were obtained (Table 1). Sequences for bacterial RNAP are at top; sequences for human RNAPI, RNAPII, and RNAPIII are at bottom; sites of single-residue substitutions that confer resistance to Stl are boxed (with E. coli and T. thermophilus residue numbers). Species names and SwissProt locus identifiers for the sequences are, in order, the following: E. coli (RPOB_ECOLI, RPOC_ECOLI), Haemophilus influenzae (RPOB_HAEIN, RPOC_HAEIN), Vibrio cholerae (RPOB_VIBCH, RPOC_VIBCH), Pseudomonas aeruginosa (RPOB_PSEAE, RPOC_PSEAE), Treponema pallidum (RPOB_TREPA, RPOC_TREPA), Bordetella pertussis (RPOB_BORPE, RPOC_BORPE), Xylella fastidiosa (RPOB_XYLFA, RPOC_XYLFA), Campylobacter jejuni (RPOB_CAMJE, RPOC_CAMJE), Neisseria meningitidis (RPOB_NEIME, RPOC_NEIMA), Rickettsia prowazekii (RPOB_RICPR, RPOC_RICPR), Chlamydia trachomatis (RPOB_CHLTR, RPOC_CHLTR), Mycoplasma pneumoniae (RPOB_MYCPN, RPOC_MYCPN), Bacillus subtilis (RPOB_BACSU, RPOC_BACSU), Staphylococcus aureus (RPOB_STAAU, BACSU, RPOC_STAAU), Mycobacterium tuberculosis (RPOB_MYCTU, RPOC_MYCTU), Synechocystis sp. PCC 6803 (RPOB_SYNY3, RPOC2_SYNY3), Aquifex aeolicus (RPOB_AQUAE, RPOC_AQUAE), Deinococcus radiodurans (RPOB_DEIRA, RPOC_DEIRA), Thermus thermophilus (RPOB_THETH, RPOC_THETH), Thermus aquaticus (RPOB_THEAQ, RPOC_THEAQ), Homo sapiens RNAPI (RPA2_HUMAN, RPA1_HUMAN), Homo sapiens RNAPII (RPB2_HUMAN, RPB1_HUMAN), and Homo sapiens RNAPIII (RPC2_HUMAN, RPC1_HUMAN). (C) Three-dimensional structure of RNAP showing locations of sites of single-residue substitutions that confer resistance to Stl (high-level resistance in red; moderate-level resistance in pink; Table 1). Two orthogonal views are shown: left, view directly into the NTP-uptake channel, toward the active-center Mg2+ (white sphere); right, view directly into the RNAP-active-center cleft, toward the active-center Mg2+. Atomic coordinates are for T. thermophilus RNAP holoenzyme (Vassylyev et al., 2002; σ subunit and β′ subunit dispensable region omitted for clarity). Cell 2005 122, 541-552DOI: (10.1016/j.cell.2005.07.017) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Effects of Stl on RNAP Translocational State Data are from exonuclease-III DNA-footprinting experiments (Guajardo et al., 1998; Bar-Nahum et al., 2005), analyzing a transcription elongation complex containing a 16 nt RNA product with a nonextendable, 3′-deoxy-3′-amino terminus (TEC16). (A) DNA fragment used in analysis of TEC16. Gray boxes, −35 element, −10 element, and transcription start site (with arrow); red box, halt site (first G in template strand of transcribed region); *, [32P]-phosphate. (B) Predicted structural organization of the backtracked state (top; state −1), the pretranslocated state (middle; state 0), and the posttranslocated state (bottom; state +1) of TEC16, and corresponding predicted 32P-labeled products upon exonuclease-III footprinting (architecture of RNAP [gray], DNA [black], and RNA [red] in transcription elongation complex as in Korzheva et al. [2000]; distance between RNAP active center [blue circle] and exonuclease-III stop point at RNAP trailing edge [arrow] as in Metzger et al. [1989], Wang et al. [1995]). (C) Observed 32P-labeled products upon exonuclease-III footprinting of TEC16, TEC16 in the presence of 20 μM Stl, and TEC16 in the presence of 50 μM complementary incoming NTP (GTP) (10 min reactions). (D) Inferred relative abundances of the backtracked state, the pretranslocated state, and the posttranslocated state—shown as scans of bands in (C) (in panels) and as normalized integrated peak areas (beneath panels). Cell 2005 122, 541-552DOI: (10.1016/j.cell.2005.07.017) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Structural Basis of Inhibition by Stl: Interactions with RNAP (A) Structure of Stl. (B) Structure of RNAP-Stl: electron density for Stl binding region. Gray, 3Fo − 2Fc electron density map [contoured at 1.0 σ; phases from density modification, using noncrystallographic symmetry (NCS) averaging and solvent flipping, prior to inclusion of Stl in the model; Fc from NCS averaging and reconstruction; see Supplemental Data]; cyan, RNAP; green, Stl. (C) Structure of RNAP-Stl: structure of Stl binding region. Cyan, RNAP; cyan dashed line, disordered or poorly ordered RNAP residues; green, Stl; red and pink, substitutions conferring high-level and moderate-level resistance to Stl (Figure 1A; Table 1). Cell 2005 122, 541-552DOI: (10.1016/j.cell.2005.07.017) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Structural Basis of Inhibition by Stl: Modeled Interactions with Transcription Elongation Complex Modeled structure of transcription elongation complex containing Stl. To model positions of the DNA template strand, the DNA nontemplate strand, and the RNA product (blue, cyan, and red nucleic acids), nucleic acids from a structure of the yeast RNAPII transcription elongation complex (Kettenberger et al., 2004; PDB accession 1Y1W) were built into the structure of RNAP-Stl (superimposition based on Cα atoms of residues 482, 822, and 829 of yeast RNAPII RPB1 and Cα atoms of residues 740, 1079, and 1086 of T. thermophilus RNAP β′). To model positions of an NTP at the insertion site (IS), an NTP at the entry site proposed in Westover et al. (2004a) (ES), and an NTP at the preinsertion site proposed in Kettenberger et al. (2004) (PS) (red, gray, and black NTPs), NTPs from structures of yeast RNAPII transcription elongation complexes containing bound NTPs (Westover et al., 2004a; Kettenberger et al., 2004; PDB accessions 19RS, 1R9T and 1Y77) were built into the structure (superimposition as above). Cell 2005 122, 541-552DOI: (10.1016/j.cell.2005.07.017) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Structural Basis of Inhibition by Stl: Stabilization of Straight-Bridge-Helix Active-Center Conformation (A) Bridge-helix conformations in RNAP (red) and RNAP-Stl (blue) and superimposed Fo − Fo difference electron density map (contoured at 3.0 σ; calculated using data sets for RNAP-Stl and RNAP and phases from the refined structure of RNAP-Stl; positive difference density in blue; negative difference density in red). (B) Active-center conformations in RNAP (left) and RNAP-Stl (right). Cyan, RNAP; cyan dashed line, disordered or poorly ordered RNAP residues; white sphere, active-center Mg2+; green, Stl. (C) Active-center conformations in RNAP (left) and RNAP-Stl (right), showing sites of substitutions conferring high-level Stl resistance (red; Figure 1A, Table 1), and the inferred binding subdeterminant and allosteric subdeterminant (magenta ovals). Cell 2005 122, 541-552DOI: (10.1016/j.cell.2005.07.017) Copyright © 2005 Elsevier Inc. Terms and Conditions