Promoters and base analogs used in footprinting experiments. (A) RNAP binding to rrnB P1 with an UP element containing a single proximal subsite. The proximal subsite (black rectangle) is a binding site for one αCTD. The position of the other αCTD in this complex is not known. The ς subunit binds to the −10 and −35 hexamers (grey rectangles). The nontemplate strand sequences of the rrnB P1 −10 and −35 hexamers and the two UP elements used in this study (4547, 4549; Estrem et al. 1999) are shown. (B) Structures of an adenine:thymine (A:T) base pair and of the base analogs 7-deaza-7-nitro adenine (A*) and uracil (U). Surfaces facing the major and minor grooves are indicated for the A:T pair. Minor groove N3 position of A (bold) was probed in DMS footprints. Major groove positions N7 of A [modified in 7-deaza-7-nitro adenine to contain a nitro group on C7 (in bold); Min et al. 1996] and the C5 methyl group of T (in bold; not present in U) were probed in interference footprints using templates containing A* or U.

Missing thymine (T) base interference footprint of RNAP binding to rrnB P1 containing UP element 4549. The promoter fragment, with, on average, one missing T per molecule, was labeled in the template strand. RNAP–promoter complexes were isolated, and after strand cleavage at abasic sites, profiles were examined by gel electrophoresis. Gel lanes contain RNAP-bound populations (+) and control samples without RNAP (−). Superimposed profiles from scans of normalized lanes are aligned with the gel. (Purple solid line) RNAP-bound DNA fragments. (Black dotted line) control DNA fragments. UP element proximal subsite and −35 and −10 hexamer template strand sequences are shown; all Ts are in purple. (Circles) Missing Ts interfering with RNAP binding. (Arrows) missing Ts stimulating RNAP binding.

Interference footprints with the base analogs uracil (U; A,B) or 7-deaza-7-nitro adenine (A*; C,D). Promoter fragments (rrnB P1 with UP element 4549), containing, on average, one modified base per molecule, were labeled on the nontemplate strand (A,C) or the template strand (B,D). RNAP–promoter complexes were isolated, and samples were examined by gel electrophoresis after strand cleavage at modified bases. Superimposed profiles from scans of normalized lanes are aligned with gels; RNAP-bound samples (+) are indicated by solid blue lines (uracil; A,B) or green lines (A*; C,D), and control samples without RNAP (−) are black dotted lines. UP element, −35 and −10 hexamer sequences are shown, with all T positions in blue (A,B), and all A positions in green (C,D). Modified bases that affected RNAP binding are indicated: (blue or green circles) interference with binding; (blue or green arrows) stimulation of binding. Symbol sizes correlate with the approximate degree of interference or stimulation observed.

Distamycin experiments. (A) Scans of hydroxyl radical footprints of distamycin bound to a fragment containing rrnB P1 with UP element 4547. Profiles of distamycin-bound fragment (red, 125 nM distamycin; green, 250 nM: blue, 500 nM), are superimposed on profiles from the control fragment (no distamycin; grey lines). RNAP recognition elements (UP element, −35 and −10 hexamers), indicated by rectangles, are aligned with the scans. (B) Effect of pre-bound distamycin on the binding of purified α to a fragment containing rrnB P1 with UP element 4547. α binding was determined by a gel mobility shift assay; the percent of input DNA bound by α (averages with standard deviation from three separate experiments) is shown as a function of distamycin concentration. (C) Effect of pre-bound distamycin on transcription in vitro. Transcription from rrnB P1 with UP element 4547 (filled circles), rrnB P1 with no UP element (open circles), and from the RNA I promoter (filled triangles) was quantified from gel scans, and is expressed as a percentage of transcription from each promoter in the absence of distamycin. Results are the average (with range) from two independent experiments.

Methylation protection experiments with rrnB P1 containing UP element 4547. Footprints with purified α subunit (A), wild-type RNAP holoenyzme (B), or RNAP holoenyzme lacking the αCTD (αΔ235 RNAP) (C) bound to a fragment labeled on the nontemplate strand. Preformed complexes were methylated with DMS and samples analyzed by gel electrophoresis after strand cleavage at methylated purines. Superimposed gel scans are shown for protein-bound samples (red solid lines) and control DNAs (no protein; black dotted lines). (D) UP element and −35 hexamer region sequences of the nontemplate strand, with purines in red. Positions where bound protein affects the extent of methylation are indicated by circles (protection) or by an arrow (enhancement). (E) Extent of methylation at individual promoter positions in the presence of wild-type RNAP (red bars) or of RNAP lacking the αCTD (αΔ235 RNAP; hatched grey bars) relative to that of control DNA without RNAP. Average (with range) is shown from two independent experiments.

Summary of footprinting results (protection: DMS and hydroxyl radical; interference: missing base and modified base). Sequence is from rrnB P1 with UP element 4547 (−51 to −6). The −10 and −35 hexamers are boxed. Backbone positions protected by RNAP against hydroxyl radical cleavage (OH·): horizontal black lines above and below the sequences (data from Newlands et al. 1991; Estrem et al. 1999). Modified or missing base interference with RNAP binding (circles) or stimulation of RNAP binding (arrows). (Purple) Missing thymines; (orange) missing purines; (blue) uracil substitution; (green) A* substitution. (Red circles) Protection by RNAP against methylation by DMS; (red arrows) enhancement of methylation by bound RNAP. Sizes of symbols reflect the approximate degree of interference, stimulation, or protection.

Positions of footprint signals and model for αCTD binding to UP element proximal subsite 4547. (A) Positions of footprint protection and interference signals in the proximal subsite and the −35 hexamer. Backbone phosphates are in ribbon form and deoxyribose and bases in stick form. Backbone positions are in green (nontemplate strand) or in blue (template strand), and bases are in magenta (UP element), yellow (−35 hexamer), or white. Positions of footprint signals (for summary, see Fig. 6) are represented as spheres, and include: (1) deoxyribose positions protected in hydroxyl radical footprints (C4‘, C5′ in UP element and downstream edge of −35 hexamer; green on nontemplate strand, blue on template strand; only a few signals are labeled); (2) purines with reduced or enhanced methylation in the presence of bound RNAP in DMS footprints (N3 of A at −41 to −43, magenta; N7 of G at −32, yellow); (3) −35 hexamer positions where uracil substitutions interfered with RNAP binding (shown as C5-methyl groups of thymines at −36, −35, and −31, yellow) or where A* substitution stimulated RNAP binding (shown as N7A at −33, yellow). Positions of enhanced cleavage in DNase I footprints of rrnB P1, in red along the backbone ribbon (−38, nontemplate strand; −35, template strand; Gourse 1988; Ross et al. 1993), are thought to reflect positions of DNA distortion in the RNAP–promoter complex. (B) Secondary structural features of αCTD involved in its (HhH)₂ domain structure (Shao and Grishin 2000). (Green rectangles) α helices in HhH1; (blue rectangles) α helices in HhH2; (white rectangles) hairpin regions. Amino acid residues comprising α helices 1–4 (or nonstandard helix, NSH), are derived from the αCTD solution structure (Jeon et al. 1995; Gaal et al. 1996). (C) Model for αCTD binding to the UP element. DNA is rotated ∼90 degrees around the helix axis from the position in A. The αCTD solution structure (Jeon et al. 1995) is in ribbon form. α helices of the two HhH motifs (Shao and Grishin 2000) are green (HhH1) and blue (HhH2), and hairpin loops connecting the helices are white. Four residues required for binding to the UP element (R265, G296, K298, S299; Gaal et al. 1996), and two residues implicated in interactions with ς (D259, E261; see text) are in stick form. The αCTD is aligned with the DNA such that each HhH motif contacts the backbone of one of the DNA strands, as suggested by features of the RuvA–DNA complex (Ariyoshi et al. 2000). Side chains of R265 and K298 are shown fitting into the minor groove. Figures in A and C were prepared using Insight II software.