Inhibition of bacterial RNA polymerase by streptolydigin: stabilization of a straight-bridge-helix active-center conformation

Abstract

We define the target, mechanism, and structural basis of inhibition of bacterial RNA polymerase (RNAP) by the tetramic acid antibiotic streptolydigin (Stl). Stl binds to a site adjacent to but not overlapping the RNAP active center and stabilizes an RNAP-active-center conformational state with a straight-bridge helix. The results provide direct support for the proposals that alternative straight-bridge-helix and bent-bridge-helix RNAP-active-center conformations exist and that cycling between straight-bridge-helix and bent-bridge-helix RNAP-active-center conformations is required for RNAP function. The results set bounds on models for RNAP function and suggest strategies for design of novel antibacterial agents.

Fig. 1. Target of Stl

(A,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: 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).

Fig. 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 non-extendable, 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 pre-translocated state (middle; state 0), and the post-translocated 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 pre-translocated state, and the post-translocated state--shown as scans of bands in (C) (in panels) and as normalized integrated peak areas (beneath panels).

Fig. 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, 3F_o-2F_c electron density map [contoured at 1.0σ; phases from density modification, using noncrystallographic symmetry (NCS) averaging and solvent flipping, prior to inclusion of Stl in the model; F_c from NCS averaging and reconstruction; see Supporting Material]; 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 (Fig. 1A; Table 1).

Fig. 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 pre-insertion 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).

Fig. 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 F_o,-F_o difference electron density map (contoured at 3.0σ; calculated using datasets 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; Fig. 1A, Table 1), and the inferred binding subdeterminant and allosteric subdeterminant (magenta ovals).