The elongation factor RfaH and the initiation factor sigma bind to the same site on the transcription elongation complex

Abstract

RNA polymerase is a target for numerous regulatory events in all living cells. Recent studies identified a few "hot spots" on the surface of bacterial RNA polymerase that mediate its interactions with diverse accessory proteins. Prominent among these hot spots, the beta' subunit clamp helices serve as a major binding site for the initiation factor sigma and for the elongation factor RfaH. Furthermore, the two proteins interact with the nontemplate DNA strand in transcription complexes and thus may interfere with each other's activity. We show that RfaH does not inhibit transcription initiation but, once recruited to RNA polymerase, abolishes sigma-dependent pausing. We argue that this apparent competition is due to a steric exclusion of sigma by RfaH that is stably bound to the nontemplate DNA and clamp helices, both of which are necessary for the sigma recruitment to the transcription complex. Our findings highlight the key regulatory role played by the clamp helices during both initiation and elongation stages of transcription.

Fig. 1.

RfaH and σ⁷⁰ bind to topologically similar targets on the TEC. (A) In the TEC, core RNAP (gray) is bound to DNA strands (black) that are separated in front of the active site to form a transcription bubble, in which the template DNA strand is paired with the nascent RNA (red) to form an 8- to 9-bp RNA:DNA hybrid, whereas the NT DNA strand is exposed on the TEC surface. RfaH binding to the TEC requires specific interactions of the N-domain (green) with the ops element (dark green) and the hydrophobic tip of the β′ CH. The C-domain (red) is dispensable for effects on RNA chain elongation in vitro and makes no contacts to TEC. Contacts of σ regions 2 and 3 with the β′ CH and the −10-like element in the NT DNA (magenta) were proposed to mediate σ rebinding to the TEC (4), whereas other contacts (made by σ^1.1, σ^3–4 linker, and σ⁴) should be lost upon transition to elongation. (B) Interactions between the β′ CH (with the N-terminal and C-terminal halves colored in purple and cyan, respectively) and RfaH (left, in green) vs. σ (right, in magenta). The two views are related by ≈180° rotation of the CH. The residues whose substitutions eliminate effects of RfaH or σ on elongation are shown as ball-and-stick models. Two residues on the tip of the CH, β′ Ile-290 and Ile-291 (cyan), are engaged in hydrophobic interactions with the N-domain of RfaH; the Tyr-8 residue (orange) is located at the RfaH/β′ interface, whereas Arg-73 (light green) is a part of the DNA binding region of RfaH. In contrast, σ makes many polar contacts to the CH including β′ Arg-278 (purple); σ Leu-402 and Glu-407 residues (yellow) are required for σ-dependent pausing (16). This figure was prepared with PyMol (DeLano Scientific).

Fig. 2.

RfaH reduces σ-dependent pausing downstream from the consensus −10 element. The linear DNA template is shown on top with the transcription start site (+1) and end (224), the ops element, and the extended −10 indicated. The assays were performed as depicted, at least three times for each combination of the conditions tested. A representative 6% denaturing gel is shown below. Positions of opsP, hisP, and σP pause sites were mapped in the presence of chain-terminating NTPs (data not shown). The fraction of RNA at the σP site after a 960-sec incubation (as percentage of total RNA) is presented below each panel; the errors (± SD) were calculated from three independent experiments performed under identical conditions. The same relative effects of the RfaH variants on σ-dependent pausing were observed in different buffer systems, at different Mg²⁺ ion concentrations (2–14 mM), and with different protein preparations. Although it is difficult to interpret variations in the very low pausing efficiencies (which are affected greatly by background subtraction), these differences were also highly reproducible.

Fig. 3.

Contacts with the TEC are critical for RfaH and σ effects during elongation. (A) Analysis of the NT DNA interactions on templates with variant −10 or ops elements shown above each panel. (B) Analysis of the β′ CH determinants on the WT ops–WT −10 template with three different core RNAPs. The assays were performed as in Fig. 2 with protein variants indicated below each panel. To conserve space, only the relevant portions of the gels are shown. The errors were similar to those shown in Fig. 2; the key values (± SD) are listed in the text.

Fig. 4.

RfaH does not compete with σ⁷⁰ during initiation. Core RNAP was preincubated with increasing concentrations of RfaH (full-length or the N-domain alone) before addition of σ⁷⁰, template encoding λP_R promoter, NTP substrates, and a ³²P-labeled DNA oligonucleotide used as a loading control. Samples were analyzed on a 12% denaturing gel; a representative gel is shown. The fraction of halted A26 complex (corrected by using the 45-mer as standard) formed in a single-round assay was quantified relative to that in the absence of RfaH. The assay was repeated five times; the fraction of A26 RNA was between 94% and 102% and independent of RfaH concentration.

Fig. 5.

Contacts to the NT DNA strand. Shown are structural models of RfaH (A) and σ (B) bound to the TEC. The RNAP core is shown in gray with the CH highlighted in cyan. The template and nontemplate strands are shown in red and blue, respectively. The registers (relative to the active site) from −9 to −6 represent the single-stranded NT DNA, whereas those of −10, −11, etc., correspond to the upstream DNA duplex; the numbering does not correspond to positions in the nascent RNA transcript because RfaH- and σ-paused TECs are backtracked, placing the 3′ end of the RNA ahead of the active site. This figure was prepared with Molscript (39).