Structural and mechanistic basis of reiterative transcription initiation

Abstract

Reiterative transcription initiation, observed at promoters that contain homopolymeric sequences at the transcription start site, generates RNA products having 5' sequences noncomplementary to the DNA template. Here, using crystallography and cryoelectron microscopy to define structures, protein-DNA photocrosslinking to map positions of RNAP leading and trailing edges relative to DNA, and single-molecule DNA nanomanipulation to assess RNA polymerase (RNAP)-dependent DNA unwinding, we show that RNA extension in reiterative transcription initiation 1) occurs without DNA scrunching; 2) involves a short, 2- to 3-bp, RNA-DNA hybrid; and 3) generates RNA that exits RNAP through the portal by which scrunched nontemplate-strand DNA exits RNAP in standard transcription initiation. The results establish that, whereas RNA extension in standard transcription initiation proceeds through a scrunching mechanism, RNA extension in reiterative transcription initiation proceeds through a slippage mechanism, with slipping of RNA relative to DNA within a short RNA-DNA hybrid, and with extrusion of RNA from RNAP through an alternative RNA exit.

Keywords: DNA scrunching; RNA polymerase; reiterative transcription initiation; transcription; transcriptional slippage.

Fig. 1.

Crystal structures of RPrtc,4 and RPrtc,5: RNA extension through RNA slipping without DNA scrunching. (A–D) Crystal structures of transcription initiation complexes engaged in standard transcription initiation and reiterative transcription initiation. Left, experimental electron density (mFo-DFc; contoured at 2.0σ in A and 1.5σ in B–D) and atomic model, showing interactions of RNAP and σ with transcription-bubble nontemplate strand, transcription-bubble template strand, and downstream dsDNA (RNAP β subunit and β′ nonconserved domain omitted for clarity). Right, nucleic-acid scaffold. RNAP, gray; RNAP active-center catalytic Mg²⁺(I) ion, violet; σ, yellow; σ finger, asterisk in Left subpanel and yellow-brown in Right subpanel; σR3–σR4 linker in RNA exit channel, brown; −10 element of DNA nontemplate strand, dark blue; discriminator element of DNA nontemplate strand, light blue; rest of DNA nontemplate strand, pink; DNA template strand, red; RNA product, magenta. Cyan rectangles in B indicate disordered regions containing scrunched nucleotides. Cyan rectangles in C and D indicate ordered scrunched nucleotides. Bulged-out nucleotides in B–D, Right, indicate bulged-out scrunched nucleotides. Violet rectangles indicate RNA–DNA hybrids. Raised template-strand nucleotides in C and D indicate non–base-paired nucleotides. (A) RPo (PDB ID: 4G7H; ref. 34). (B) RPitc containing 5-nt RNA product generated by in crystallo standard transcription initiation (RPitc,5). (C) RPrtc containing 5-nt RNA product generated by in crystallo reiterative transcription initiation on nucleic-acid scaffold having a template-strand G₊₁G₊₂G₊₃ homopolymeric sequence (RPrtc,5 [G₊₁G₊₂G₊₃]). (D) RPrtc containing 4-nt RNA product generated by in crystallo reiterative transcription initiation on nucleic-acid scaffold having a template-strand C₊₁C₊₂C₊₃ homopolymeric sequence (RPrtc,4 [C₊₁C₊₂C₊₃]).

Fig. 2.

Crystal structures of RPrtc,4 and RPrtc,5: short RNA–DNA hybrid. (A) RNA–DNA base pairing in crystal structures of transcription initiation complexes engaged in standard transcription initiation (RPitc,5) and reiterative transcription initiation (RPrtc,5 [G₊₁G₊₂G₊₃] and RPrtc,4 [C₊₁C₊₂C₊₃]). Left, template-strand DNA bases (red) and corresponding RNA bases (magenta) in view orientation parallel to RNA–DNA hybrid helix axis. Right, template-strand DNA bases (red) and corresponding RNA bases (magenta) in view orientation perpendicular to RNA–DNA hybrid helix axis. Positions are numbered relative to the RNAP active-center P site. Dashed lines indicate Watson-Crick H-bonds. Violet rectangles indicate RNA–DNA hybrids. At positions P-4, P-3, and P-2 of RPrtc,5 [G₊₁G₊₂G₊₃], and at positions P-3 and P-2 of RPrtc,4 [C₊₁C₊₂C₊₃], template-strand DNA bases are displaced relative to their locations in RPitc,5, and no base pairing occurs. (B) Superimposition of DNA template strand and RNA of RPrtc,5 [G₊₁G₊₂G₊₃] (red spheres, DNA phosphates; magenta spheres, RNA phosphates; violet sphere, RNAP active-center catalytic Mg²⁺ ion) on DNA template strand and RNA of RPitc,5 (gray spheres, DNA and RNA phosphates). Left, view orientation parallel to RNA–DNA hybrid helix axis; Right, view orientation perpendicular to RNA–DNA hybrid helix axis. Distances in cyan, displacement of template-strand DNA nucleotides at positions P-4 and P-3 of RPrtc,5 [G₊₁G₊₂G₊₃] relative to their locations in RPitc,5.

Fig. 3.

Cryo-EM structure of RPrtc,≥11: RNA extension through RNA slipping without DNA scrunching. (A) Overall structure (β′ nonconserved region omitted for clarity; two orthogonal view orientations). Dark blue brackets indicate the standard RNA exit and alternative RNA exit. Cyan rectangles indicate scrunched nucleotides. Violet rectangles indicate RNA–DNA hybrids. Other symbols and colors in panels A to D are as in Fig. 1. (B) Left, cryo-EM density and atomic model, showing interactions of RNAP and σ with transcription-bubble nontemplate strand, transcription-bubble template strand, and downstream dsDNA. Right, nucleic-acid scaffold. Yellow-brown, σ finger (note displacement of σ-finger tip); magenta dots, RNA outside RNAP active-center cleft (nucleotides rN ≥ 11). (C) Superimposition of DNA in RPrtc,≥11 [C₊₁C₊₂C₊₃] (pink and red) on DNA in RPo (black; PDB ID: 512D; ref. 30) (two view orientations). (D) Close-up of cryo-EM density and atomic model for RNA (nucleotides rN1 to rN11 numbered in white).

Fig. 4.

Mapping of RNAP leading-edge and trailing-edge positions in RPrtc by use of protein–DNA photocrosslinking: RNA extension without DNA scrunching. (A) RNAP trailing-edge and leading-edge positions in transcription initiation complexes at the N25 promoter (WT) and derivatives of the N25 promoter containing template-strand G₊₁G₊₂G₊₃ and C₊₁C₊₂C₊₃ homopolymeric sequences (G₊₁G₊₂G₊₃ and C₊₁C₊₂C₊₃). First bracketed subpanel, protein–DNA photocrosslinking data for RPo; second bracketed subpanel, protein–DNA photocrosslinking data for transcription initiation complexes engaged in standard transcription initiation (RPitc) and reiterative transcription initiation (RPrtc). Promoter sequences are shown with positions numbered relative to the transcription start site and with positions of the −10 element, the discriminator element, and the homopolymeric sequence highlighted in blue, light blue, and red. Dark and light olive-green bars indicate strong and weak RNAP trailing-edge crosslinks, and dark and light forest green bars indicate strong and weak RNAP leading-edge crosslinks. Bottom, observed modal TE-LE distances and differences in modal TE-LE distance relative to modal TE-LE distance in RPo at WT N25 promoter [Δ(TE-LE distance)]. (B) Mechanistic interpretation of data in A. Three states are shown: RPo, RPitc [specifically, RPitc having a 5-nt RNA product in a posttranslocated state (RPitc,5 post), corresponding to the major crosslink in A], and RPrtc. Gray, RNAP; yellow, σ; yellow-brown, σ finger (note displacement of σ-finger tip in RPrtc); brown, σ region-3/region-4 linker; light green, trailing-edge Bpa and crosslinking site for trailing-edge Bpa; dark green, leading-edge Bpa and crosslinking site for leading-edge Bpa; black boxes with blue fill, −10 element nucleotides; black boxes with light blue fill, discriminator-element nucleotides; black boxes with red fill, template-strand homopolymeric-sequence nucleotides; other black boxes, other DNA nucleotides (nontemplate-strand nucleotides above template-strand nucleotides); magenta boxes, RNA nucleotides; violet rectangles, RNA–DNA hybrids; P and A, RNAP active-center product and addition sites. Raised template-strand nucleotides and black x's indicate non–base-paired nucleotides. Scrunching of nontemplate and template DNA strands is indicated by bulged-out nucleotides. Initial product formation in both standard transcription initiation and reiterative transcription initiation involves one step of DNA scrunching. RNA extension in standard transcription initiation involves additional DNA scrunching, but RNA extension in reiterative transcription does not.

Fig. 5.

Measurement of transcription-bubble size in RPrtc by use of single-molecule DNA nanomanipulation: RNA extension without DNA scrunching. (A, B) Experimental approach (7, 47, 56). (A) Apparatus. (B) End-to-end extension (l) of a mechanically stretched, positively supercoiled (Top) or negatively supercoiled (Bottom) DNA molecule is monitored. Unwinding of n turns of DNA by RNAP results in compensatory gain of n positive supercoils or loss of n negative supercoils and movement of the bead by n*56 nm. (C) Single-molecule time traces for RPo and RPitc at the N25 promoter (WT; Left), and for RPo and RPrtc at derivatives of the N25 promoter containing template-strand G₊₁G₊₂G₊₃ and C₊₁C₊₂C₊₃ homopolymeric sequences (G₊₁G₊₂G₊₃ and C₊₁C₊₂C₊₃; Middle and Right). Upper subpanels, positively supercoiled DNA; lower subpanels, negatively supercoiled DNA. Green points, raw data (30 frames/s); red points, averaged data (1 s window); horizontal black lines, unbound and RPo states; dashed horizontal black lines, RPitc and RPrtc states (with the difference in Δlobs between RPo and RPitc being substantially greater than the difference in Δl_obs between RPo and RPrtc). (D) Single-molecule transition-amplitude histograms for RPo and RPitc at the N25 promoter (WT; Left), and for RPo and RPrtc at derivatives of the N25 promoter containing template-strand G₊₁G₊₂G₊₃ and C₊₁C₊₂C₊₃ homopolymeric sequences (G₊₁G₊₂G₊₃ and C₊₁C₊₂C₊₃; Middle and Right). Upper subpanels, positively supercoiled DNA; Lower subpanels, negatively supercoiled DNA. Vertical dashed lines, means; Δlobs,pos, transition amplitudes with positively supercoiled DNA; Δlobs,neg, transition amplitudes with negatively supercoiled DNA. (E) Differences in Δl_obs,pos and DNA unwinding relative to those in RPo at WT N25 promoter (means ±2 SEM).

Fig. 6.

Mechanisms of standard transcription initiation and reiterative transcription initiation. Standard transcription initiation (Left column) and reiterative transcription initiation (first four panels of Left column followed by panels of Right column). Cyan, reactions present only in reiterative transcription: i.e., cycles of RNA extension and slippage. Other colors and symbols are as in Fig. 4. Scrunching is indicated by bulged-out nucleotides (∼8 to 10 scrunched bp prior to promoter escape in the standard transcription initiation pathway; 1 scrunched bp in the reiterative transcription initiation pathway). Scrunched nucleotides of nontemplate and template DNA strands during initial transcription are accommodated as bulges within the unwound transcription bubble.