Steps toward translocation-independent RNA polymerase inactivation by terminator ATPase ρ

Abstract

Factor-dependent transcription termination mechanisms are poorly understood. We determined a series of cryo-electron microscopy structures portraying the hexameric adenosine triphosphatase (ATPase) ρ on a pathway to terminating NusA/NusG-modified elongation complexes. An open ρ ring contacts NusA, NusG, and multiple regions of RNA polymerase, trapping and locally unwinding proximal upstream DNA. NusA wedges into the ρ ring, initially sequestering RNA. Upon deflection of distal upstream DNA over the RNA polymerase zinc-binding domain, NusA rotates underneath one capping ρ subunit, which subsequently captures RNA. After detachment of NusG and clamp opening, RNA polymerase loses its grip on the RNA:DNA hybrid and is inactivated. Our structural and functional analyses suggest that ρ, and other termination factors across life, may use analogous strategies to allosterically trap transcription complexes in a moribund state.

Fig. 1.. Engagement.

(A) Semi-transparent surface/cartoon representations of the engagement complex, highlighting contact sites of ρ subunits. Rotation symbols in this and the following figures indicate views relative to (A), upper left. (B) Post-translocated state of the nucleic acids at the active site; tDNA, template DNA; ntDNA, non-template DNA; +1, template nucleotide pairing with the next incoming NTP. (C-H) Close-up views of ρ/EC contacts. Elements discussed in the text, magenta. (I) ρ-cutaway view; ρ-contacting RNAP elements around the RNA exit, magenta.

Fig. 2.. Effects of ρ PBS/SBS ligands and NusG^NTD.

(A) Effects of optimal (green) and poor (red) PBS/SBS ligands on ρ termination; here and in other figures, positions of proximal (pink) and distal (magenta) terminated RNAs and the read-through transcript (RT; purple) are indicated with a colored bar. PBS (dN₁₅) ligands were present at 5 μM, SBS (rN₁₂) ligands at 500 nM. A fraction of RT versus the sum of all RNA products is shown at the bottom. Values represent means ± SD of three independent experiments. (B) Close-up view on NusG^NTD in the engagement complex. Elements discussed in the text, magenta. (C) Modulation of ρ effects by the indicated NusG (“G”) variants. The center panel shows lane profiles from the gel on the left; the Y-axis signals were normalized based on the total signal in that lane. The right panel shows a distribution of ρ-terminated RNAs between the proximal and distal regions. Values represent means ± SD of three independent experiments. * P<0.01; ** P<0.001; *** P<0.0001 [unpaired Student’s t-test].

Fig. 3.. Priming.

(A) Surface views of the engagement (I), primed (II) and RNA capture (III) complexes, illustrating rotation of NusA underneath ρ₆ (I to II) and shift of ρ₆ from ρ₅ to ρ₁ (II to III). (B) Semi-transparent surface/cartoon representations of the primed complex, highlighting contact sites of ρ subunits and distal uDNA on top of β’^ZBD. (C) Overlay of selected elements of the primed complex (solid surfaces) and engagement complex (semi-transparent surfaces; ρ, magenta), highlighting movements of NusA and ρ, and handover of NusA^NTD from α₁^CTD to α₂^CTD.

Fig. 4.. NusA interactions.

(A) Comparison of β^FT-NusA^NTD interactions in the primed and engagement complexes, after superposition of NusA^NTDs. (B) ρ₅/ρ₆/NusA^KH1 interaction network in the primed complex. (C) Correlation of accommodation of distal uDNA on the β’^ZBD and NusA rotation underneath ρ₆ in the primed complex. (D) NusA (“A”) effects on termination by WT RNAP, or RNAP variants lacking α^CTDs or ω; dashed lines indicate spliced images. The RNA fractions are means ± SD of three independent experiments. ns, not significant; * P<0.1; ** P<0.001; *** P<0.0001.

Fig. 5.. RNA capture.

(A) Surface view of the RNA capture complex (nucleic acids as cartoon) with superimposed ρ₆ from the primed complex. Arrow, movement of ρ₆ during the transition from the primed to the RNA capture state. (B) Close-up views on ρ₆^PBS with bound RNA. Angled arrows, direction of intervening RNA region that might ascend 5’-to-3’ through the open ρ ring and return on the outside. Inset, details of RNA binding at ρ₆^PBS. 5’-portion of the RNA and selected ρ₆^PBS residues as sticks colored by atom type. In this and the following figures: Carbon RNA, red; carbon ρ residues, magenta; oxygen, light red, nitrogen blue; phosphorus, orange. (C) Quantification of β-gal activity derived from a reporter construct (scheme) in cells with ρ^WT or ρ^Y80c, in the presence of the indicated plasmid-encoded β’ variants. Values represent means ± SEM of at least nine independent experiments.

Fig. 6.. Inhibition.

(A) Comparison of selected elements of the inhibited complex (regular colors) with the β’ clamp of the RNA capture complex (magenta), illustrating partial clamp opening (arrow). (B) tDNA is post-translocated in complexes I-III, but β’ lid moves and the +1 nucleotide is rotated out of the templating position in complex IV. Templating nt, cyan; BH, bridge helix; Mg1, catalytic magnesium ion. (C) Effects of deleting β’ jaw, lid, or SI3, alone or in the presence of NusA or NusG. Reactions were run on the same gel; dashed lines indicate positions where intervening lanes were removed. (D) Comparison of selected elements of the moribund complex (regular colors) with the β’ clamp of the RNA capture complex (magenta), illustrating dramatic clamp opening (arrow).

Fig. 7.. Inactivation.

(A,B) Side-by-side comparison of selected elements in the inhibited complex (top) and in the moribund complex (bottom), highlighting movement of the β’ clamp helices (CH, magenta) and nucleic acid-guiding loops (lid/rudder/switch 2, magenta) (A), as well as repositioning of the hybrid and displacement of the RNA 3’-end from the active site (arrow) (B). (C) Pause-resistant β^V550A substitution decreases ρ termination. Reactions were run on the same gel, and a dashed line indicates the splice position. (D) Effects of NTP concentration at λ tR1.

Fig. 8.. Model for an EC-dependent ρ-mediated termination pathway.

Trafficking and termination/hybrid unwinding correspond to hypothetical steps (behind semi-transparent gray boxes) preceding and following the stages resolved by cryoEM in this work. Legend on the lower right and bottom. Coloring as in structural figures except: DNA, upstream to downstream progressively lighter brown; hybrid, orange.