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. 2020 Dec 8;59(48):4523-4532.
doi: 10.1021/acs.biochem.0c00571. Epub 2020 Nov 18.

The RNA Polymerase α Subunit Recognizes the DNA Shape of the Upstream Promoter Element

Samuel Lara-Gonzalez  1 , Ana Carolina Dantas Machado  2 , Satyanarayan Rao  2 , Andrew A Napoli  1 , Jens Birktoft  1 , Rosa Di Felice  2   3   4 , Remo Rohs  2   3   5   6 , Catherine L Lawson  1   7
Affiliations

The RNA Polymerase α Subunit Recognizes the DNA Shape of the Upstream Promoter Element

Samuel Lara-Gonzalez et al. Biochemistry. .

Abstract

We demonstrate here that the α subunit C-terminal domain of Escherichia coli RNA polymerase (αCTD) recognizes the upstream promoter (UP) DNA element via its characteristic minor groove shape and electrostatic potential. In two compositionally distinct crystallized assemblies, a pair of αCTD subunits bind in tandem to the UP element consensus A-tract that is 6 bp in length (A6-tract), each with their arginine 265 guanidinium group inserted into the minor groove. The A6-tract minor groove is significantly narrowed in these crystal structures, as well as in computationally predicted structures of free and bound DNA duplexes derived by Monte Carlo and molecular dynamics simulations, respectively. The negative electrostatic potential of free A6-tract DNA is substantially enhanced compared to that of generic DNA. Shortening the A-tract by 1 bp is shown to "knock out" binding of the second αCTD through widening of the minor groove. Furthermore, in computationally derived structures with arginine 265 mutated to alanine in either αCTD, either with or without the "knockout" DNA mutation, contact with the DNA is perturbed, highlighting the importance of arginine 265 in achieving αCTD-DNA binding. These results demonstrate that the importance of the DNA shape in sequence-dependent recognition of DNA by RNA polymerase is comparable to that of certain transcription factors.

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Conflict of interest statement

Conflict of Interest Statement. None declared.

Figures

Figure 1.
Figure 1.
Comparison of two independent crystal structures with αCTDs tandemly bound to the UP-element (A6-tract) DNA. (A) CAD structure, with CAP (cyan) bound to its consensus DNA site, and αCTDs (yellow, green) bound to overlapping sites containing A-tract DNA on either side of CAP. Only αCTD1 (yellow) makes protein–protein contacts with CAP. The two-fold symmetric design was used to simplify crystallographic analysis (the asymmetric unit contains one-half of the full complex). (B) ASD structure, with one σR4 (purple) bound to a consensus −35 element DNA site, and αCTDs (yellow, green) bound to overlapping sites centered about adjacent A-tract DNA. Only αCTD1 (yellow) makes protein–protein contacts with σR41. A second σR4 (pink) forms an asymmetric dimer with DNA-bound σR4 (purple). In (A) and (B), proteins are represented as surfaces; colors are as indicated in the legend at the right. (C) and (D): Corresponding protein–DNA contact footprints in co-crystal structure. Nucleotide bases (letters) and backbone phosphates (open circles) are shaded according to the protein subunit making contact. Intercalation of the minor groove is indicated by shaded rectangles. In (C), thick and thin black rectangles represent positions of major and minor DNA kinks; dotted vertical lines indicate designed breaks in the DNA backbone; arrows indicate the base-pair positions modified in the CAD ‘knockout’ structure. (E) and (F): Superimposed αCTD/A-tract DNA regions (CAD: colors as in the legend, ASD: semi-transparent grey). αCTD residues that participate in αCTD-αCTD contacts (Pro293 of αCTD1, Thr263 and Lys298 of αCTD2) or that intercalate into the DNA minor groove (Arg265, both subunits) are also displayed. The RMSD for 1484 equivalent protein + DNA atom positions in the two structures is 0.7 Å.
Figure 2.
Figure 2.
Minor groove width of the A6-tract is very narrow in CAD and ASD crystal structures, with a consistent minimum of enhanced negative electrostatic potential. (A) Molecular surface of the A6-tract for CAD (left) and ASD (right) is shown color-coded by shape (convex surfaces in green, concave surfaces in dark gray). Darker gray shading corresponds to narrower minor groove. The Arg265 residues from each αCTD that insert into the minor groove are shown in stick model. The isopotential surface at −5kT/e (red mesh) is shown, superimposed on the molecular surfaces. (B) CAD A6-tract region with αCTD Arg265 residue positions. The surface of the base-pair that is mutated in the CAD-KO knockout complexes is highlighted in red. (C) DNA minor groove width profiles from crystal structures and MD trajectories, as a function of nucleotide position: on the horizontal axis, CAD and ASD DNA sequences are merged via overlap in the A6-tract. The underlined position marks the location at which adenine is mutated to cytosine in CAD-KO, CAD_KO_R265Aα1 and CAD_KO_R265Aα1_noCAP. The region corresponding to the A6-tract is highlighted with a black rectangle (the left-most narrow minor groove region is part of the CAP binding site). In the short (3 base-pair) region where the CAD X-ray and ASD X-ray derived curves overlap, the two can be distinguished by continuity of connected points.
Figure 3.
Figure 3.
Shortening of the A-tract impairs binding of the second αCTD. (A) Model map for CAD-KO (2mFo-DFc, gray contour at 2.0 σ) and data vs. model difference map (mFo-DFc, red contour at −3.25 σ) obtained after atomic position refinement with the full (two αCTDs) CAD model. Orientation and color scheme correspond to Figure 1A (right half). (B) Relative αCTD occupancies in CAD, CAD-KO, and ASD crystals obtained by grouped refinement against the measured diffraction datasets (see Methods).
Figure 4.
Figure 4.
Contact between αCTD1 and the DNA minor groove is weakened upon mutation of Arg265 to Ala. (A) DNAproDB polar contact maps for CAD_KO (top) and for CAD_KO_R265Aα1 (bottom), obtained for the most representative structure that covers the final span of the 300 ns MD trajectory (65% cluster population for CAD_KO, 17% cluster population for CAD_KO_R265Aα1, see Table S1); the contact between αCTD1 and the DNA is represented by the HB2 red dot in the minor groove annulus (labeled as ‘mG’). (B) Residue–residue difference distance contact map, obtained with Gromacs; the map is symmetric with respect to the diagonal. A positive value of ΔDistance indicates a residue–residue distance increases as an effect of the R265A protein mutation, in the absence (top) and presence (bottom) of the knock-out DNA mutation. Residue indices: binding helix BH (1–10), binding loop BL (11–15), T6+-tract (16–23), A6+-tract (24–31). The positions of αCTD1 protein residues Arg265 (in BH) and Asn294 (in BL), as well as the positions of the DNA knock-out mutation when present (bottom), are marked by horizontal lines.
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