Initial events in bacterial transcription initiation

Abstract

Transcription initiation is a highly regulated step of gene expression. Here, we discuss the series of large conformational changes set in motion by initial specific binding of bacterial RNA polymerase (RNAP) to promoter DNA and their relevance for regulation. Bending and wrapping of the upstream duplex facilitates bending of the downstream duplex into the active site cleft, nucleating opening of 13 bp in the cleft. The rate-determining opening step, driven by binding free energy, forms an unstable open complex, probably with the template strand in the active site. At some promoters, this initial open complex is greatly stabilized by rearrangements of the discriminator region between the -10 element and +1 base of the nontemplate strand and of mobile in-cleft and downstream elements of RNAP. The rate of open complex formation is regulated by effects on the rapidly-reversible steps preceding DNA opening, while open complex lifetime is regulated by effects on the stabilization of the initial open complex. Intrinsic DNA opening-closing appears less regulated. This noncovalent mechanism and its regulation exhibit many analogies to mechanisms of enzyme catalysis.

Keywords: RNA polymerase; kinetics; mechanism; promoter; transcription regulation.

Figure 1

Sequence-specific interactions between σ⁷⁰ RNAP and regions of the promoter. Schematic representations of the subunits of RNAP core, σ⁷⁰, and promoter DNA. RNAP: α₂: cyan; β and β': gray; ω: black. σ regions: as shown. Promoter: UP element: cyan; −35 element: blue; extended −10: red; −10 element: yellow; discriminator: orange; transcription start site: green; DNA downstream of the transcription start site: gray. Linker regions in α and σ subunits are shown as springs. Nontemplate strand sequences of a “consensus” and λP_R, T7A1 and rrnB P1 promoters are shown below; missing bases are indicated by dashes.

Figure 2

Structural representation of key functional regions of Eσ⁷⁰ holoenzyme. Structure created from PDB 4LK1, looking down the cleft in (A) and rotated 90° into the page and 90° counterclockwise in (B). Colors of regions of σ are the same as in Figure 1. Additionally shown: β' jaw (β' 1149–1990), brown; β' SI3 (β' insert 6; β' 943–1130), green; β SI1 (β insert 4; β 225–343), pink; active site Mg²⁺, red ball; αNTD (α 1–234), cyan.

Figure 3

Schematic representation of proposed intermediates in open complex formation and dissociation at λP_R promoter. Closed complexes like RP_C, I_1,E (early), or I_1,L (late) can be significant members of the rapidly equilibrating I₁ ensemble. The αCTDs are shown in cyan; other colors are the same as in Figure 1.

Figure 4

Mechanism of transcription initiation: resolvable kinetic steps. Minimal mechanisms of open complex formation and dissociation, showing by color coding the steps that contribute to the observed rate constants (k_obs, k_d) for promoters like λP_R that form a stable open complex RP_O. For the common experimental situation of excess RNAP, the rate constant for open complex formation (k_obs) is determined by the front half of the mechanism (boxed in purple), including the equilibrium constant K₁ for formation of the ensemble of closed (I₁) intermediates, the forward rate constant k₂ of the isomerization step that includes DNA opening, and the excess RNAP concentration. Likewise, k_d is determined by the late steps of the mechanism (boxed in green), including the rate constant k_-2 for DNA closing and the equilibrium constant K₃ for stabilization of the initial open complex I₂ to form longer-lived I₃ and/or RP_O complexes [65,66].

Figure 5

Reaction progress diagrams for open complex formation by (A) FL and (B) UT−47 λP_R, interpreted using Mechanism 2. Free energies of the rapidly-equilibrating closed intermediates I_1,E and I_1,L, the rate- determining transition state (I_1,L-I₂)^‡, and the initial (I₂) and stable (RP_O) open complexes are calculated relative to promoter DNA (P) for [R] = 30 nM from experimentally-determined values of K₁, k₂, k_-2 and k_d for FL λP_R [24,60] and UT-47 λP_R [75] at 37 °C (see text for details). Free energies of FL and UT-47 λP_R are set at the same value. For purposes of illustration, for both variants, we estimate that k_open = 1 s⁻¹ and that (I_1,L-I₂)^‡ decomposition frequencies are 10³ s⁻¹ in each direction, and assume that k_-2 for UT-47 λP_R is the same as that determined for FL λP_R [24].