Restructuring of an RNA polymerase holoenzyme elongation complex by lambdoid phage Q proteins

Abstract

The structure of an intermediate in the initiation to elongation transition of Escherichia coli RNA polymerase has been visualized through region-specific DNA cleavage by the hydroxyl radical reagent FeBABE. FeBABE was tethered to specific sites of the final sigma(70) subunit and incorporated into two specialized paused elongation complexes that obligatorily retain the final sigma(70) initiation subunit and are targets for modification by lambdoid phage late gene antiterminators. The FeBABE cleavage pattern reveals structures similar to open complex, except for notable changes to region 3 of final sigma(70) that might reflect the presence of stably bound transcript. Binding of the antiterminator protein Q displaces the reactivity of FeBABE conjugated to region 4 of final sigma(70), suggesting that final sigma(70) subunit rearrangement is a step in conversion of RNAP to the antiterminating form.

Figure 1

Open and paused complexes of late promoters of phages λ and 82. Structures were described (39); the 82 sequence is the variant described. Circles and ovals represent, respectively, the assigned positions of σ⁷⁰ regions 2 and 4. Paused complexes are shown in the forward position (38).

Figure 2

Effect of variant σ added in excess in solution on pausing in vitro. (A) Open complex was formed with RNAP containing either wild-type σ⁷⁰ (lanes 1–6) or σ⁷⁰ L402F (lanes 7–12); after washing, excess σ⁷⁰ L402F (lanes 4–6) or wild-type σ⁷⁰ (lanes 10–12) was added, as described. Reactions were sampled at 1, 2, and 5 min for each set of three. (B) As for A, except that the supplement was σ^S. RT, readthrough; T, terminated; P, paused.

Figure 3

Cleavage of DNA in open, paused, and Q-modified complexes by FeBABE conjugates in σ⁷⁰ regions 2 and 4. DNA was labeled at the 3′ end of the nontemplate strand. An A/G ladder is shown for reference; assigned regions of cleavage are designated by adjacent vertical bars.

Figure 4

Cleavage of DNA in phage 82 complexes by a panel of FeBABE conjugates to regions 2, 3, and 4 of σ⁷⁰, and to Q⁸². DNA was labeled at the 3′ end of the nontemplate strand. FeBABE was attached to the N terminus of Q⁸², as described in the text.

Figure 5

Cleavage of DNA in phage λ complexes by a panel of FeBABE conjugates to regions 2, 3, and 4 of σ⁷⁰. DNA was labeled at the 5′ end of the nontemplate strand, and the image was flipped for comparison to Figs. 3 and 4.

Figure 6

Profile of Fe-BABE-Q⁸² cleavage of both DNA strands in paused complexes. Lighter lower traces are of unreacted DNA.

Figure 7

Model of the disposition of σ⁷⁰ regions 2, 3, and 4, Q⁸², and Q^λ in open and paused complexes of phage late gene promoters. Rectangular boxes span regions of reactivity by FeBABE conjugates to σ⁷⁰ residues of the enclosed number, or to Q⁸². Ovals and spheres designate positions of protein regions. Region 4 of σ⁷⁰ in the presence of Q cannot be presumed to bind DNA and is shown displaced. (A) λ; (B) phage 82.