In vitro selection. (A) Synthesis of rrnB P1 promoter fragments with a randomized upstream region. Oligonucleotides were annealed and extended with T7 DNA polymerase to form a library of double-stranded DNA fragments with different UP element regions (−59 to −38). The top strand oligonucleotide (80 nt) contained (from 5′ to 3′) an EcoRI site (RI), rrnB P1 sequence from −66 to −60, random sequence from −59 to −38, and rrnB P1 sequence from −37 to +1. The bottom strand oligonucleotide (81 nt) contained (from 5′ to 3′) a HindIII site (H3) and rrnB P1 sequence from +50 to −17. Each contained a short additional sequence 5′ to the restriction site to ensure enzyme digestion. The randomized region is indicated by a hatched box, and −10 and −35 hexamers of rrnB P1 by open boxes. (B) Theoretical time course of RNAP binding to promoters containing (UP⁺) or lacking (UP⁻) an UP element. Broken line represents a time at which RNAP-promoter binding reactions were stopped to enrich for UP element-containing fragments.

In vitro transcription of plasmid templates containing rrnB P1 promoters with either the wild-type (WT) UP element (lanes 1–3), no UP element (the SUB sequence, see Fig. 2A, lanes 4–6), or the 4192 selected upstream sequence (lanes 7–9). Plasmids were transcribed with WT RNAP (lanes 1, 4, and 7), αΔ235 RNAP (lanes 2, 5, and 8), or αR265A RNAP (lanes 3, 6, and 9). rrnB P1 transcripts (terminated at rrnB T1 ≈220 nt downstream of the transcription start site) and vector-encoded RNA I transcripts (≈110 nt) are indicated with arrows.

Hydroxyl radical footprints of RNAP or purified α bound to rrnB P1 promoters containing either the 4192 UP element (A) or the rrnB P1 UP element (B). DNA fragments were labeled in the nontemplate strand at position +50. Lanes 1 and 6, A+G sequence markers; lanes 2, 5, 7, and 10, no protein; lanes 3 and 7, purified α; lanes 4 and 9, WT RNAP. The protected regions in the UP element and core promoter are indicated by vertical bars at the right of each panel.

Upstream sequences and relative transcription activities of 31 in vitro-selected promoters used in defining an UP element consensus sequence. (A) Promoters contained wild-type rrnB P1 sequences (solid line; open boxes indicate the −10 and −35 hexamers) and different upstream regions (dotted line). Sequences of the nontemplate strand in the upstream region (−59 to −38) from 31 selected promoters, wild-type rrnB P1, and the rrnB P1 core promoter are shown. Wild-type rrnB P1 contained its natural UP element sequence, and the core rrnB P1 promoter contained an upstream sequence with no UP element function [the SUB sequence with A residues at positions −39 and −40 (6)]. Upstream sequence names are the strain numbers of λ lysogens carrying the promoter–lacZ fusions. Asterisks indicate promoters with single base-pair mutations (probably introduced during PCR amplification) downstream of the transcription start site (between +2 and +17). Sequence variation in this region of rrnB P1 does not affect promoter activity (35). Promoter activities are expressed relative to the activity of the core rrnB P1 promoter (activity = 1; strain RLG3097) and were determined from β-gal measurements in λ lysogens containing promoter–lacZ fusions. Relative activities differed by less than 10% in at least two different experiments. (B) Nucleotide frequencies (percentage of 31 sequences) at each position, −59 to −38, in the set of selected sequences shown in A. (C) Frequency diagram of the data in B. Each nucleotide is represented as a letter proportional in size to its frequency at that position in the selected population. (D) Consensus UP element sequence. One nucleotide is indicated when it is present in more than 55% of the population and two when together they represent more than 95% of the population.

DNase I footprints of RNAP bound to the 4192-rrnB P1 promoter. The DNA fragment was labeled at promoter position +50 in the nontemplate strand. Lanes: 1, A+G sequence markers; 2, no RNAP; 3, wild-type (WT) RNAP; 4, αR265A RNAP. Small arrows indicate upstream region positions protected by WT RNAP, but not by αR265A RNAP. Large arrow indicates a position of enhanced DNase I cleavage in the WT RNAP footprint.

Comparison of upstream sequences with large effects on promoter activity to the UP element consensus sequence (see Discussion). Sequences were aligned by their −35 hexamers where applicable [the spoVG promoter is recognized by a holoenzyme with an alternative σ-subunit (54)]. Sequence numbering refers to the rrnB P1 promoter.