Region 1.2 of the RNA polymerase sigma subunit controls recognition of the -10 promoter element

Abstract

Recognition of the -10 promoter consensus element by region 2 of the bacterial RNA polymerase sigma subunit is a key step in transcription initiation. sigma also functions as an elongation factor, inducing transcription pausing by interacting with transcribed DNA non-template strand sequences that are similar to the -10 element sequence. Here, we show that the region 1.2 of Escherichia coli sigma70, whose function was heretofore unknown, is strictly required for efficient recognition of the non-template strand of -10-like pause-inducing DNA sequence by sigma region 2, and for sigma-dependent promoter-proximal pausing. Recognition of the fork-junction promoter DNA by RNA polymerase holoenzyme also requires sigma region 1.2 and thus resembles the pause-inducing sequence recognition. Our results, together with available structural data, support a model where sigma region 1.2 acts as a core RNA polymerase-dependent allosteric switch that modulates non-template DNA strand recognition by sigma region 2 during transcription initiation and elongation.

Figure 1

Mapping of the σ-DNA crosslink in paused TEC. (A) The lacUV5 pause-inducing sequence and the −10 promoter element consensus sequence (Keilty and Rosenberg, 1987). Matches to consensus are indicated in bold typeface. The +5G to A substitution is indicated (+5mut). An asterisk marks a position (+6T), which when substituted to A abolishes pausing (Brodolin et al, 2004). Transcription start sites (+1) are shown by arrows. (B) An autoradiogram of a denaturing gel showing ³²P-labeled RNA products produced during run-off transcription from the lacUV5 promoter fragments containing either WT or mutant (+5mut) pause-inducing sequence (PS). (C) Formaldehyde crosslinking of the +17 paused complexes formed at the WT or mutant (+5mut) ³²P-labeled lacUV5 promoter DNA. Crosslinked σ⁷⁰-DNA and β′-DNA complexes are labeled as σ and β′. (D) Mapping of the crosslinking site in the σ⁷⁰ subunit crosslinked to ³²P-labeled lacUV5 DNA in TEC16 by Met-specific chemical cleavage (σ-DNA). The panel on the right (σ_m) shows Met-specific cleavage of the σ⁷⁰ subunit ³²P-labeled at the C terminus. Continuous lines with arrows between panels σ_m and σ-DNA connect bands corresponding to the identical cleavage products. Positions of σ⁷⁰ Met residues, which when cleaved give rise to observed cleavage products, are indicated on the right side of the figure. Suffixes C or N refer to C- or N-terminal cleavage product, respectively. (E) The diagram shows the map of the σ⁷⁰ subunit; the positions of Met cleavage sites are indicated by arrows. ³²P-labeled peptides produced by cleavage at indicated σ⁷⁰ Met residues are shown beneath as lines. σ⁷⁰ region containing the crosslink site to DNA is indicated by a black bar.

Figure 2

σ-exchange assay using immobilized +16 paused complexes. (A) A scheme demonstrating the principle of ‘σ-exchange' experiments on the lacUV5 promoter-proximal pause. The pausing site (PS) is marked by an open rectangle. Promoter open complex (RPo) and paused elongation complex (TEC16) are shown. An exchange reaction between σ originated from the promoter complex (σ1 in gray) and exogenously added σ (σ2 in black) is illustrated. (B) ³²P-labeled RNA transcripts produced by σ⁷⁰-containing (lanes 1–4 and 9–11) and σ⁷⁰-less (lanes 5–8) TEC16 immobilized on Ni²⁺-NTA beads. Transcription complexes were chased by the addition of NTPs and 0.5, 1, or 5-min incubation either before (lanes 2–4) or after heparin wash (lanes 6–11). Lanes 9–11: σ⁷⁰-less complexes were supplemented with exogenous σ⁷⁰ (500 nM final concentration) before chase. (C) Crosslinking of the σ⁷⁰ subunit to ³²P-labeled lacUV5 DNA in immobilized TEC16 either before (lane 1) or after (lanes 2–5) heparin wash. Complexes were supplemented with exogenous σ⁷⁰ (lanes 3–5). NTPs were added before (lane 5) and after (lane 4) the addition of σ⁷⁰. Crosslinked σ⁷⁰–DNA complexes resolved on SDS–PAGE are shown (labeled as σ-crosslink).

Figure 3

σ⁷⁰ fragments used and their binding to RNAP core. (A) A scheme of σ⁷⁰; the universally conserved regions are shown in white and are numbered. The structural domains of σ (σ1.1, σ2, σ3, and σ4; Campbell et al, 2002) are indicated beneath. σ fragments used in this work are shown as simple lines below the σ⁷⁰ scheme. Contact sites with the −10 and −35 promoter elements and the β′ coiled-coil (β′c–c) are indicated. (B, D) Inhibition of σ⁷⁰-dependent abortive transcription initiation reaction from the lacUV5 promoter by σ⁷⁰ fragments. Concentrations of σ⁷⁰ and its fragments are indicated at the top of the panel. Reactions contained either 50 nM (panel B) or 10 nM (panel D) of RNAP core. ³²P-labeled abortive ApUpU RNA product is marked. (C) Quantification of experimental data presented in panel B. The amount of ³²P-labeled RNA synthesized in the presence of σ fragments was normalized to the amount synthesized without fragments added (black bar at the left). Gray bars correspond to lanes 2, 5, 8, and 11 of panel B. Light gray bars correspond to lanes 3, 6, 9, and 12. White bars correspond to lanes 4, 7, 10, and 13. Mean values and s.d. from two independent experiments are shown. (E) Quantification of experimental data presented in panel D. The amount of ³²P-labeled abortive product synthesized in the presence of indicated concentrations of σ fragments is shown.

Figure 4

Binding of the exogenously added σ⁷⁰ fragments to paused TEC16. (A) Crosslinking of TEC16 complexes to ³²P-labeled lacUV5 DNA before (lane 1) and after (lanes 2–7) heparin wash. Heparin-washed σ⁷⁰-less complexes were supplemented with 0.5 μM of wild-type σ⁷⁰ (FL) or fragments: 5 μM of σ_2a, 3 μM of σ₂₋₃, 1.5 μM of σ₁₋₂, and 2.5 μM of σ_2.4 (lanes 3–7). Crosslinked complexes were resolved by SDS–PAGE and revealed by autoradiography. Crosslinked subunits are indicated. (B) Crosslinking of TEC16 complexes washed with heparin (lanes 2–9) and supplemented with 1.5 μM of σ_2b or σ₁₋₂ (lanes 3–6, 8, and 9). NTPs were added before (lanes 7–9) or after (lanes 5 and 6) the addition of σ⁷⁰ fragments. Lane 1: TEC16 before heparin wash. Reaction products were analyzed as in panel A. (C) ³²P-labeled RNA transcripts produced upon addition of NTPs to immobilized TEC16. Complexes were chased by the addition of NTPs for 0.5, 1, and 5 min. Lanes 5–19: complexes were supplemented with wild-type σ⁷⁰ (FL) or indicated σ⁷⁰ fragments before addition of NTPs. (D) Quantification of the results of experiment shown in panel C. The amount of +17 RNA was calculated as percentage of the initial amount of the starting 16-mer RNA present before the addition of NTPs. Mean values and s.d. from two independent experiments are shown.

Figure 5

Probing of the interactions between σ⁷⁰ or its fragments and the-10 oligonucleotide using UV crosslinking assay. (A) Crosslinking of the −10 non-template oligonucleotide with the wild-type σ⁷⁰ (FL) or σ⁷⁰ fragments (1–2, 2b, and 2a) present at the indicated concentrations in the absence or in the presence of 20 nM of RNAP core. Crosslinked complexes were resolved by 8% SDS–PAGE. (B) Quantification of data from panel A obtained in the presence of 0.1 μM σ⁷⁰ or its fragments ‘Stimulation fold' is a ratio of crosslinking signals observed in presence and in the absence of RNAP core. Mean values and standard deviations from three independent experiments are shown. (C) Crosslinking of the non-template (NT) (lanes 1–4) and control template (T) (lanes 5–12) −10 element oligonucleotides with RNAP holoenzymes (lanes 1–8) containing either wild-type σ⁷⁰ (FL) or indicated σ⁷⁰ fragments. Lanes 9–12: crosslinking of σ⁷⁰ and σ⁷⁰ fragments without core RNAP. The samples contained 0.5 μM (lanes 1–4) or 5 μM of σ (lanes 5–12).

Figure 6

Interaction of fork-junction templates with holoenzymes containing the wild-type σ⁷⁰ subunit or σ fragments. (A) A scheme of the fork-junction templates. The positions of −10 elements are marked by black rectangle and corresponding DNA sequences are shown. Arrow indicates substitution of the −8G to C. (B) Crosslinking of the core RNAP or σ⁷⁰ containing holoenzyme to fork DNA. Templates contained at position −8 either G (−8G) or C (−8C). Crosslinked complexes were resolved by SDS–PAGE and revealed by autoradiography. (C) Complex formation between RNAP and forks bearing −10 element (‘−10') or anticonsensus −10 sequence (‘anti-10') was detected by nitrocellulose filtration method as described in Materials and methods. Binding is shown as percentage of total DNA in the sample. Mean values and s.d. from two independent experiments are shown.

Figure 7

Modeling of the σ_2b fragment interactions with RNAP core on the crystallographic structures of T. aquaticus and T. thermophilus RNAP. (A) The structure of T. thermophilus RNAP (PDB accession number 1IW7) fitted with the T. aquaticus RNAP structure in the complex with fork-junction DNA (PDB accession number 1L9Z). RNAP is shown as a molecular surface and DNA is shown as a CPK structure with the non-template strand in orange and the template strand in green. The large non-conserved domain present in Thermus β′ is omitted for clarity. The σ_2b fragment is shown as ribbons with region 1.2 amino acids 94–101 shown as a red molecular surface. RNAP core subunits are colored as follows: β′ in rose and β in cyan; the β′ coiled-coil in violet. The β lobe that interacts with σ region 1.1 and forms the upper jaw is shown in green. (B) Modeling of σ crosslink in TEC16 on the T. aquaticus RNAP and fork-junction DNA complex structure. Color codes are as in panel A. The position corresponding to crosslinked +5G is shown in blue. (C) Interactions between σ region 1.2 (red) and 2.1–2.2 (green) and the β′ coiled-coil (magenta) in the T. thermophilus RNAP holoenzyme structure. Two projections are shown. The molecular modeling and figures were acquired using MOLMOL package (Koradi et al, 1996).