Antibiotics trapping transcription initiation intermediates: To melt or to bend, what's first?

Abstract

Promoter DNA melting, culminating in the loading of the single-stranded DNA template into the RNA polymerase active site, is a key step in transcription initiation. Recently, the first transcription inhibitors found to block distinct steps of promoter melting were characterized. Here, the impact of these studies is discussed with respect to the current models of transcription initiation.

Figure 1

(A) Kinetics scheme of RP_o formation on the lacUV5, and λP_R promoters. R: RNAP, P: promoter. Complexes with open DNA marked by blue boxes; closed complexes marked by green ellipses. (B) Structural model of RNAP in complex with dwDNA fragment. The structure of T. thermophilus RNAP is shown as a molecular surface colored in gray. The β lobes (in cyan), β' switch-2 (sw2) (Ser602-Lys621), β' jaw (Arg1266-Gly1328) and β' B-helix-turn-helix (HtH) (Leu469-Pro506) are shown as ribbons, the β-gate loop (Arg243-Pro248) is shown in CPK. Numbering corresponds to T. thermophilus. The β' clamp part of the RNAP surface is colored in red. DNA is shown in red (template) and blue (non-template). The σ subunit is shown as ribbons with the structural domains 2, 3 and 4 in magenta, green and gray, respectively. (C) Influence of the σ_3.2HL deletion on lacUV5 promoter melting. KMnO₄ probing of the open complexes formed between end-labeled lacUV5 promoter and the RNAP carrying either wild type σ⁷⁰ or σ⁷⁰ with deletion of the region 3.2 hairpin loop (Asp513-Leu519). Positions of the template strand thymines reactive to KMnO₄ are indicated. The scan of the gel is shown on the right.

Figure 2

(A) Chemical structures of Myx and Lpm. (B) Overlap of the Lpm and Myx binding sites. Amino acids substitutions conferring resistance to Lpm²⁷ are shown in CPK colored in cyan for β subunit (T. thermophilus Q¹⁰¹⁸, V¹⁰⁸⁷, N¹⁰⁶⁴) and magenta (R⁶¹³) or dark blue (R⁸⁷, P⁵²⁶) for β' subunit, σ_3.2HL is shown as green ribbons and Myx is shown in ball-and-stick and orange. R⁶¹³ is shown in two conformations—as in holoenzyme and as in the RNAP-Myx complex. (C) Model of the mechanism of Lpm and Myx action. RNAP core is shown as a gray ellipse. The σ subunit is shown in green, region 3.2 is shown as a green triangle, and the β' switch-2 is shown as a blue rectangle. The +1 base of the promoter is indicated by a red circle.