Structural basis of Q-dependent antitermination

Abstract

Lambdoid bacteriophage Q protein mediates the switch from middle to late bacteriophage gene expression by enabling RNA polymerase (RNAP) to read through transcription terminators preceding bacteriophage late genes. Q loads onto RNAP engaged in promoter-proximal pausing at a Q binding element (QBE) and adjacent sigma-dependent pause element (SDPE) to yield a Q-loading complex, and Q subsequently translocates with RNAP as a pausing-deficient, termination-deficient Q-loaded complex. Here, we report high-resolution structures of 4 states on the pathway of antitermination by Q from bacteriophage 21 (Q21): Q21, the Q21-QBE complex, the Q21-loading complex, and the Q21-loaded complex. The results show that Q21 forms a torus, a "nozzle," that narrows and extends the RNAP RNA-exit channel, extruding topologically linked single-stranded RNA and preventing the formation of pause and terminator hairpins.

Keywords: RNA polymerase; transcription antitermination; transcription antitermination factor Q; transcription antitermination factor Q21; transcription elongation complex.

Fig. 1.

Biological function of Q. (A) Q-dependent regulatory cassette, consisting of gene for Q (blue) and adjacent transcription unit comprising pR′ promoter (arrow), SDPE (yellow rectangle), terminator (red octagon), and bacteriophage late genes (gray). (B) Steps in assembly and function of a Q-dependent transcription antitermination complex. Promoter −35 and −10 promoter elements, dark gray rectangles; QBE, light gray rectangle; SDPE, yellow rectangle; RNAP core enzyme, large light gray rectangle; σ, brown; Q, blue; DNA nontemplate and template strands, black lines (unwound transcription bubble indicated by raised and lowered line segments); RNA, red line.

Fig. 2.

Crystal structure of Q21. (A) Crystal structure of Q21. Experimental electron density map (green mesh, 2 mFo-DFc map contoured at 1.0 σ) and fitted atomic model (blue). (B) Crystal structure of Q21 (ribbon representation; 2 orthogonal views). (C) Superimposition of Q21 (residues 7 to 156; blue) on ECF σ factor σR4 (PDB 2H27; residues 122 to 190; gray, with HTH motif in red). DALI Z score = 6.4; PDBeFold Z score = 4.8. (D) Superimposition of Q21 (residues 7 to 156; blue) on ECF σ factor σR4 in complex with anti-sigma helix (PDB 1OR7; σR4 residues 124 to 187 and anti-sigma residues 27 to 44; gray, with HTH motif in red and anti-sigma helix in orange). DALI Z score = 6.4; PDBeFold Z score = 5.5.

Fig. 3.

Crystal structure of Q21-QBE complex. (A) Crystal structure of Q21-QBE complex. (A, Left) Molecular assembly in asymmetric unit comprising 2 Q protomers interacting with QBE. (A, Right) Molecular assembly in asymmetric unit comprising 1 Q protomer interacting with QBE. (A, Top and Middle) Experimental electron density map (green mesh, 2 mFo-DFc map contoured at 1.0 σ) and fitted atomic model (red and blue). (A, Bottom) QBE DNA fragment (QBEu, dark green; QBEd, light green). (B) Q21 HT[loop]T motif interacting with DNA (blue and red), superimposed on HTH motif of ECF σ factor σR4 interacting with DNA (PDB 2H27; gray). (C) Interactions of Q21 with DNA major groove. (D) Interactions of Q21 with DNA minor groove. (E) Protein–protein interactions between Qd (B1-B2 loop of HT[loop]H motif; light blue) and Qu (H1 and H4; blue; sidechain oxygen atoms in red).

Fig. 4.

Cryo-EM structure of Q21-loading complex. (A) Nucleic acid scaffold. DNA, red (QBEu, QBEd, and SDPE in dark green, light green, and yellow; noncomplementary region corresponding to unwound transcription bubble indicated by raised and lowered letters); RNA, magenta. (B) Cryo-EM structure of Q21-loading complex (2 view orientations). Qu, dark blue; Qd, light blue; RNAP, gray; σ⁷⁰, brown; DNA and RNA, colored as in A; RNAP active-center Mg²⁺, black sphere. (C) Qu-RNAP interactions. Qu in dark blue, with segments located inside RNAP RNA-exit channel, behind RNAP surfaces, indicated as gray ribbons with black outlines. RNAP β’, β, and α subunits in orange, light yellow, and white. Qu View orientation as in Left subpanel of B. (D) Qd-RNAP interactions. Qu in light blue. Other colors as in C. View orientation as in Left subpanel of B. (E) Conformational changes in Qu upon formation of Q21-loading complex. Qu in Q-QBE complex (Fig. 3 A, Left; red) superimposed on Q21-loading complex (colors as in C). View orientation as in Right subpanel of B. (F) Conformational changes in RNAP FTH on formation of Q21-loading complex. RNAP FTH in transcription initiation complex (PDB 4YLN; orange) superimposed on Q21-loading complex (colors as in C–E). View orientation as in Left subpanel of A. (G) Restriction of RNAP RNA-exit channel by Q torus and proximity of RNA 5′ end to Q torus. RNA nucleotides numbered with RNA 3′ nucleotide as −1. View orientation in Left subpanel is orthogonal to RNA-exit channel; view orientation in Right subpanel is parallel to RNA-exit channel. (H) Summary of protein-RNA interactions by Q torus (blue) and RNAP RNA-exit channel (gray). View orientation as in Left subpanel of G.

Fig. 5.

Cryo-EM structure of Q21-loaded complex. (A) DNA template for preparation of Q21-loading complex. (B) Structure of Q21-loaded complex. View orientations and colors as in Fig. 4B. (C) Threading of RNA 5′ end into and through Q torus (RNA nucleotides numbered with RNA 3′ nucleotide as −1). View orientations and colors as in Fig. 4G. (D) Summary of protein–RNA interactions by Q torus (blue) and RNA-exit channel (gray).

Fig. 6.

Mechanistic conclusions. (A) Transformation of Q21-loading complex to Q21-loaded complex. Black and gray dashed lines, DNA helix axes of upstream dsDNA segments in loading and loaded complexes, respectively; magenta, green, cyan, brown, and gray arrows, structural changes on transformation of loading complex to loaded complex. Other colors as in Figs. 4B and 5B. (B) Antitermination and antipausing by Q21. Top, steric incompatibility of Q torus with pause and terminator RNA hairpins (hairpin stem from PDB 6ASX in magenta, with segments positioned to interpenetrate Q-torus and Q body indicated as transparent ribbons with black outlines; hairpin loop from PDB 1MT4, positioned to interpenetrate Q-torus and Q body, indicated as gray ribbon with black outlines). View orientations and colors as in Figs. 4G and 5C. Bottom, schematic comparison of TECs in absence of Q (Upper row) and TECs in presence of Q (Lower row). Colors as in Figs. 4B and 5B.