Promoter specificity determinants of T7 RNA polymerase

Abstract

The high specificity of T7 RNA polymerase (RNAP) for its promoter sequence is mediated, in part, by a specificity loop (residues 742-773) that projects into the DNA binding cleft (1). Previous work demonstrated a role for the amino acid residue at position 748 (N748) in this loop in discrimination of the base pairs (bp) at positions -10 and -11 (2). A comparison of the sequences of other phage RNAPs and their promoters suggested additional contacts that might be important in promoter recognition. We have found that changing the amino acid residue at position 758 in T7 RNAP results in an enzyme with altered specificity for the bp at position -8. The identification of two amino acid:base pair contacts (i.e., N748 with the bp at -10 and -11, and Q758 with the bp at -8) provides information concerning the disposition of the specificity loop relative to the upstream region of the promoter. The results suggest that substantial rearrangements of the loop (and/or the DNA) are likely to be required to allow these amino acids to interact with their cognate base pairs during promoter recognition.

Figure 1

Promoter structure. (Upper) Alignment of the consensus promoter sequences for T7, T3. K11, and SP6 RNAPs. The sequence of the nontemplate strand is presented; the transcription start site is at +1 (for review, see ref. 3). Positions at which bp are conserved in all phage promoters are shaded; the bases at −8 are enclosed in a box. The solid bar below the sequences denotes the binding region, which is recognized in a double-stranded form; the stippled bar denotes the initiation region, which is thought to be melted open from about −5 to +3 during RNAP binding and initiation (6, 7). (Lower) Summary of promoter recognition contacts (modified from ref. ; drawing courtesy of Dr. Craig T. Martin). The promoter region from −13 to −5 is modeled as B-form DNA. Sugars protected by bound RNAP in hydroxyl radical footprinting experiments are indicated in light gray (16); guanine N7 and phosphate groups identified by chemical modification interference studies are in medium gray (17); base functional groups identified via incorporation of base analogs are in dark gray (7, 21, 22). A dashed line separates the interface between bases in the template and nontemplate strands.

Figure 2

RNA polymerase structure. (Upper) The amino acid sequences of the T7, T3, K11, and SP6 RNAPs in the region of the specificity loop (T7 residues 742–773) are aligned (–34); lowercase letters indicate positions where the sequences differ from that of T7, and dashes indicate a gap that has been inserted into the SP6 sequence to optimize the alignment. The arrowheads along the top indicate travel along the loop from the “fingers” wall of the DNA binding cleft to the tip (residue 756) and return. (Lower) The alpha carbon backbone of T7 RNAP is represented as connected spheres (coordinates are from the Brookhaven Protein Databank). Residues 745–772 in the specificity loop (1) and residues 369–390 in the “thumb” motif (35) are shaded in gray. The positions of residues N748 and Q758, which are involved in recognition of the bp at −11 and −8, are indicated in black. Computer modeling of B-form DNA in the binding cleft was carried out as proposed by Patel et al. (28), placing the specificity loop on top of the DNA and residue N748 near the bp at −11. The base pairs at −8 and −11 are depicted in wire frame.

Figure 3

Altered promoter specificity of T7–Q758K. (A) Plasmid templates that carry a reference T7 promoter (P_T7ref) and a test promoter (P_T7X) were digested with SspI and EcoRV; transcription from each promoter is expected to give rise to runoff products of the lengths indicated (18). (B) Mixtures of three plasmid templates each having a promoter with one of the alternate bp at the test position (P_T7X), in addition to the reference promoter (P_T7ref), were transcribed by wild-type T7 RNAP or Q758K, as noted. The identification of the test promoter set is given by position; thus, for example, the reaction shown in lane 2 utilized a mixture of templates having test promoters with nonconsensus bp at −12. The reactions shown in lanes 1 and 9 utilized a control template (C) having a consensus T7 promoter at the test promoter location. The products were resolved by electrophoresis and visualized by exposure to a PhosphorImager screen. (C) Each reaction contained one plasmid template having a single bp substitution in P_T7X at position −8, as indicated. The control template (lanes 1 and 5) has a consensus promoter (P_T7 −8T) at the test promoter location.

Figure 4

Specificities of mutant RNAPs for the bp at −8. The relative preferences of each mutant RNAP for a promoter having the base indicated in the nontemplate strand are presented (data are from Table 1).