Structural transitions in the transcription elongation complexes of bacterial RNA polymerase during σ-dependent pausing

Abstract

A transcription initiation factor, the σ(70) subunit of Escherichia coli RNA polymerase (RNAP) induces transcription pausing through the binding to a promoter-like pause-inducing sequence in the DNA template during transcription elongation. Here, we investigated the mechanism of σ-dependent pausing using reconstituted transcription elongation complexes which allowed highly efficient and precisely controlled pause formation. We demonstrated that, following engagement of the σ subunit to the pause site, RNAP continues RNA synthesis leading to formation of stressed elongation complexes, in which the nascent RNA remains resistant to Gre-induced cleavage while the transcription bubble and RNAP footprint on the DNA template extend in downstream direction, likely accompanied by DNA scrunching. The stressed complexes can then either break σ-mediated contacts and continue elongation or isomerize to a backtracked conformation. Suppressing of the RNAP backtracking decreases pausing and increases productive elongation. On the contrary, core RNAP mutations that impair RNAP interactions with the downstream part of the DNA template stimulate pausing, presumably by destabilizing the stressed complexes. We propose that interplay between DNA scrunching and RNAP backtracking may have an essential role in transcription pausing and its regulation in various systems.

Figure 1.

σ⁷⁰-dependent pausing in reconstituted TECs. (A) Synthetic nucleic acid scaffolds. Left panel, the structure of the ‘Cons’ scaffold used for analysis of pausing is shown in comparison with the initially transcribed region of the lacUV5 promoter. The −10 promoter element of lacUV5 is boxed, the transcription start point (+1) and the pause positions (+17/18) are shown above the sequence. The −10 and TG-like elements of the pause-inducing sequence are shadowed. The pause positions observed on the scaffold template are shown with arrowheads, the major position is shadowed. RNA nucleotides that are added to the starting 20-mer RNA during transcription at the pause site are gray. Right panel, sequences of different scaffold variants; nucleotides substituted in comparison with the ‘Cons’ scaffold are underlined italics. (B) Kinetics of σ⁷⁰-dependent pausing on different scaffold variants; transcription was performed at 100 µM NTPs. Positions of the starting 20-mer RNA, paused 25, 26-mer RNAs and the run-off (RO) transcript are indicated. The plot shows averages and standard deviations from three to four independent measurements. (C) Kinetics of RNAP pausing on the ‘Cons’ scaffold at 3 µM NTP concentration. (D) σ⁷⁰-dependent pausing on the ‘Cons’ scaffold at 1 mM NTPs analyzed either in the absence or in the presence of the GreB protein.

Figure 2.

RNA cleavage in TECs stalled at different positions at the pause site in the presence (top) or in absence (bottom) of the σ⁷⁰ subunit. The 25-mer TECs were obtained either by run-off RNA synthesis in the presence of σ⁷⁰, or by walking of the 23-mer complex in the absence σ⁷⁰.

Figure 3.

Analysis of DNA melting in TECs during σ⁷⁰-dependent pausing by KMnO₄ probing. (A) Analysis of the cleavage products by gel-electrophoresis. The scanned cleavage profiles in the 20-mer and 24-mer TECs obtained either in the absence or in the presence of the σ⁷⁰ subunit are shown on the left (for the non-template strand) and on the right (for the template strand) of the gel. For each strand, the relative signal amplitudes are on the same scale. (B) Schematic representation of the footprinting results in 20-mer and 24-mer TECs. The central part of the ‘Cons’ scaffold template is shown. Positions of modified thymines in σ-less and σ⁷⁰-containing TECs are shown with open and closed arrowheads, respectively; the sizes of the arrowheads correspond to the observed modification efficiencies.

Figure 4.

Analysis of the TEC conformation during σ⁷⁰-dependent pausing by ExoIII footprinting. (A) ExoIII footprinting on the non-template (up) and template (bottom) DNA strands in TECs obtained either in the absence or in the presence of the σ⁷⁰ subunit. ‘M’ is an A+G cleavage marker. (B) ExoIII footprinting profiles of the front TEC border on the non-template DNA strand in σ⁷⁰-containing complexes [the data for the 30” point from (A)]. The profiles are normalized by the signal intensities at position +20 A. (C) The summary of the footprinting results for σ-less (up) and σ⁷⁰-containing (bottom) complexes. Complementary interactions that change during transcription at the pause site are shown with gray lines. The downstream-most positions of the ExoIII stops in different TECs on the template and non-template DNA strands are indicated below and above the scaffolds, respectively.

Figure 5.

Effects of mutations in E. coli core RNAP on transcription pausing. The reaction was performed on the ‘Cons’ scaffold template at different NTP concentrations for 1′ at 37°C. The plot shows averages and standard deviations from two to three independent experiments. Location of the mutations on the T. thermophilus TEC structure [2PPB, (48)] is shown in the left bottom part of the figure. The template and non-template DNA strands are black and gray, respectively; RNA is red. Trigger loop (TL), bridge helix (BH) and F-loop (FL) in the RNAP active center are shown in green, magenta and red, respectively. The β′ coiled-coil (β′CC) region of the clamp domain is shown in yellow, SW2 is blue (with R339 residue shown as a CPK model), the ΔJaw deletion is dark green, the region of Ω216 insertion is brown; the site of the ΔSI3 deletion in the TL is indicated with an arrow.

Figure 6.

The effect of antisense oligonucleotides on σ⁷⁰-dependent pausing. (A) DNA oligonucleotides used in the study are shown below the ‘Cons’ scaffold. The −10-like element is boxed; RNA nucleotides 21 through 25 are shown in gray. (B) Transcription pausing by wild-type E. coli RNAP in the presence of oligonucleotides. The reaction was performed in the presence of 100 µM NTPs for 1′ at 25°C. (C) Kinetics of the pausing in the presence of the oligonucleotide antiRNA15 and GreB measured at 1 mM NTPs. The plots show the efficiencies of pausing at positions 25 and 26.

Figure 7.

RNAP pausing through DNA scrunching and backtracking. See the text for details.