Mechanism of bacterial transcription initiation: RNA polymerase - promoter binding, isomerization to initiation-competent open complexes, and initiation of RNA synthesis

Abstract

Initiation of RNA synthesis from DNA templates by RNA polymerase (RNAP) is a multi-step process, in which initial recognition of promoter DNA by RNAP triggers a series of conformational changes in both RNAP and promoter DNA. The bacterial RNAP functions as a molecular isomerization machine, using binding free energy to remodel the initial recognition complex, placing downstream duplex DNA in the active site cleft and then separating the nontemplate and template strands in the region surrounding the start site of RNA synthesis. In this initial unstable "open" complex the template strand appears correctly positioned in the active site. Subsequently, the nontemplate strand is repositioned and a clamp is assembled on duplex DNA downstream of the open region to form the highly stable open complex, RP(o). The transcription initiation factor, σ(70), plays critical roles in promoter recognition and RP(o) formation as well as in early steps of RNA synthesis.

Fig. 1

Model of the E. coli RNAP (σ⁷⁰ α₂ββ′ω) open complex RP_o based on Protein Data Bank IDs 3IYD¹⁰ and 3LU0. (a) View of RP_o illustrating the interactions between promoter DNA [nontemplate strand (NT), black; template (T), dark green] and σ₂, σ₃, and σ₄ (wheat). Linker σ_3.2 is buried in the RNA exit channel. The N-terminal domains of α (bright green, yellow) form a hinge at the bottom of the cleft. σ_NCD is a folded nonconserved domain connecting σ_1.2 and σ₂. ω is shown in light gray. Missing from the figure are σ_1.1 and the flexibly tethered αCTD (not resolved in any holoenzyme structure to date). (b) View down into the active-site channel highlighting mobile regions on the periphery of the cleft and in the cleft. At the upstream entrance to the cleft, β′ clamp helices (black) tightly interact with σ_1.2 and σ₂. The open transcription bubble (–11 to +3 in this model) binds in the cleft, with the template strand start site (+1) next to the active site Mg²⁺ (red sphere) at the bottom. βSI1 (magenta) and β′SI3 (blue) are species-specific sequence insertions (SIs) present in E. coli. The remaining colored regions are highly conserved in bacteria. Along with βSI and β′SI3, β′ jaw (yellow) and β′ clamp (red) appear positioned to clamp on the downstream duplex DNA after the bubble has opened.^– Flexible elements in the cleft that likely bind and stabilize the DNA single strands in RP_o include the bridge helix (visible under the double-stranded–single-stranded boundary of the downstream DNA; pink), rudder (green), fork loop 2 (teal), and switch 2 (light blue). Other mobile elements shown are the β′ upclamp (hot pink; see Supplementary Fig. 1), which is proposed to interact with upstream DNA in forming I₁ (the first kinetically-significant intermediate at the λP_R promoter), and the trigger loop (orange), which is known to be critically involved in the RNA synthesis steps.

Fig. 2

Promoter recognition by amino acids of the α subunit and σ⁷⁰. Orange and blue arrows indicate recognition of promoter regions as double-stranded DNA elements by the α and σ⁷⁰ subunits, respectively. The two red arrows delineate a region of the nontemplate strand DNA recognized by σ⁷⁰ subsequent to strand separation. In the linear representations (not drawn to scale) of both σ⁷⁰ and α, the N-termini are on the right. Only the sequence of the nontemplate strand is shown (5′ end on the left). A typical E. coli promoter does not have all elements shown and exhibits deviations from the consensus sequences shown here for the –10, –35, and UP elements, as well as the consensus spacer length (17 bp).

Fig. 3

Summary of the proposed isomerization steps that form the initiating complex (RP_init) after recruitment of RNAP to form an initial complex at the promoter (RP_c). Formation of the closed complex RP_c triggers a series of subsequent large-scale conformational changes. The RNAP molecular machine places start-site duplex DNA in the active-site cleft in I₁, opens it to form I₂, and stabilizes the open form by assembling a clamp in I₃ and RP_o (model based on Gries et al., Kontur et al.,^, Davis et al., and Craig et al.). Once promoter DNA is open, NTPs can bind, and transcription initiates. I₂ and I₃ are open complexes; current studies are addressing whether they can bind NTPs and initiate transcription.

Fig. 4

Structure of region 2.3 of σ⁷⁰. In the N→C direction are helix 13 (lower helix), a loop, and helix 14 (upper helix). The side chains of K414, K418, Y425, T429, Y430, W433, W434, and Q437 (Lys, green; Tyr, red; Thr, blue; Trp, purple; Gln, pink) stick out towards the viewer from approximately the same face of the protein, where they can interact with promoter DNA. Y430 has been shown to stack with –11A of the –10 region. Y421 sticks out in another direction but may be able to interact with DNA. The structures of the T. aquaticus and T. thermophilus σ_2.3 are very similar.^,