RNA polymerase motions during promoter melting

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Science  26 May 2017:
Vol. 356, Issue 6340, pp. 863-866
DOI: 10.1126/science.aam7858
  • Fig. 1 Bacterial RNAP conformations and promoter recognition.

    (A) Model of a promoter melting intermediate with locally underwound –10 element DNA [based on PDB ID 1L9U (26, 27)]. The –10 recognition surface of the σ-subunit is green, and the rest of the RNAP surface is gray. DNA strands are blue (nt) and yellow (t), with forked –10 element red, A–11 and T–7 bound in σ pockets, and –35 element in grey. (Bottom) schematics of fully melted bacterial promoter. Colors are as in (A), and conserved –11 and –7 positions of the nt-strand are highlighted. Numbers indicate positions before the start site (+1) (B) Ligands affecting the equilibrium between oRNAP and cRNAP conformations (9, 10). The mobile β′ clamp and associated σ2 domain regulating RNAP cleft width are highlighted. PDB IDs are 1L9U for oRNAP and 4XLN for cRNAP. (C) Time course of tetramethylrhodamine-labeled (red open circle and solid circle) RNAP (3 nM) fluorescence response to addition of 2 μM –10 element dsDNA. The black trace corresponds to unliganded RNAP, the green trace corresponds to cRNAP preincubated with gp2, and the blue trace corresponds to oRNAP preincubated with Lpm. (Inset) RNAP beacon assay I schematic. (D) Binding of –10 dsDNA to RNAP, cRNAP, or oRNAP measured with the RNAP beacon assay. SEM from at least three independent experiments are shown.

  • Fig. 2 Real-time kinetics of promoter melting and effects of cleft-locking ligands.

    (A) Fluorescence assay II. Cy3 (solid and open gray-filled red ovals) fluorescence enhancement reports on final steps of promoter melting (11). (Inset) –10 element sequence of WT and –11Ab λ PR promoter variants. (B) Time course of Cy3-labeled WT and –11Ab promoters’ response to addition of 100 nM RNAP. (C and D) WT and –11Ab promoter melting in assay II upon addition of 100 nM unliganded RNAP (black), cRNAP (preincubated with Myx; green) or oRNAP (preincubated with Lpm; blue).

  • Fig. 3 Structure of oRNAP in complex with promoter fragment.

    (A) Promoter fragment used for cocrystallization, color coding, and numbering as in Fig. 1A. Boxed parts of the melted construct indicate DNA regions resolved in the structure, with dashed lines corresponding to poorly resolved density. (B) oRNAP shown in gray, with –10 element recognition surface in green. Blue mesh represents 2FoFc map for the DNA displayed at 0.7σ contour level. (Single-letter abbreviations for the amino acid residues are K, Lys; R, Arg.) (C) Exemplary electron density, displayed as in (B), for base pair +14. (D) Downstream duplex trajectories in the intermediates of promoter melting [symbols on the lower right show how RNAP views in (B) and (D) relate]. Above the figure is the kinetic scheme of promoter melting. R, RNAP; P, promoter; RPi1, modeled position of the intermediate preceding melting step; RPi2 (26), duplex DNA position observed in this work; RPo, fully melted promoter DNA (3). (E) Spontaneous DNA unwinding during promoter melting. Schematic model of RPi1–RPi2 transition is shown as the side view. Blue plus signs indicate positively charged surface of the RNAP active site channel (fig. S6).

  • Fig. 4 Schematics of RNAP structural transitions during promoter recognition and melting.

    Promoter DNA is shown in black lines, with –35 promoter element in gray and –10 element in red. Schematic of a sequence-dependent energy landscape of RNAP moving along the DNA in search of a promoter sequence is shown above, adopted from (16). Promoter search is optimized via interplay between oRNAP (blue; lower energy state, fast movement along DNA, scanning for upstream promoter elements, smooth binding energy landscape) and cRNAP (green; higher energy state, slower, precise DNA read-out, base flipping, rough binding energy landscape). –10 element recognition by cRNAP nucleates melting, followed by transition into oRNAP form, allowing DNA loading and unwinding in the cleft; the final closure seals the promoter complex.

Supplementary Materials

  • RNA polymerase motions during promoter melting

    Andrey Feklistov, Brian Bae, Jesse Hauver, Agnieszka Lass-Napiorkowska, Markus Kalesse, Florian Glaus, Karl-Heinz Altmann, Tomasz Heyduk, Robert Landick, Seth A. Darst

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Supplementary Text
    • Figs. S1 to S8
    • Tables S1and S2
    • References

    Images, Video, and Other Media

    Movie S1

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