Escherichia coli RNA polymerase recognition of a sigma70-dependent promoter requiring a -35 DNA element and an extended -10 TGn motif

Abstract

Escherichia coli sigma70-dependent promoters have typically been characterized as either -10/-35 promoters, which have good matches to both the canonical -10 and the -35 sequences or as extended -10 promoters (TGn/-10 promoters), which have the TGn motif and an excellent match to the -10 consensus sequence. We report here an investigation of a promoter, P(minor), that has a nearly perfect match to the -35 sequence and has the TGn motif. However, P(minor) contains an extremely poor sigma70 -10 element. We demonstrate that P(minor) is active both in vivo and in vitro and that mutations in either the -35 or the TGn motif eliminate its activity. Mutation of the TGn motif can be compensated for by mutations that make the -10 element more canonical, thus converting the -35/TGn promoter to a -35/-10 promoter. Potassium permanganate footprinting on the nontemplate and template strands indicates that when polymerase is in a stable (open) complex with P(minor), the DNA is single stranded from positions -11 to +4. We also demonstrate that transcription from P(minor) incorporates nontemplated ribonucleoside triphosphates at the 5' end of the P(minor) transcript, which results in an anomalous assignment for the start site when primer extension analysis is used. P(minor) represents one of the few -35/TGn promoters that have been characterized and serves as a model for investigating functional differences between these promoters and the better-characterized -10/-35 and extended -10 promoters used by E. coli RNA polymerase.

FIG. 1.

Promoter constructs. Sequences between EcoRI (GAATTC) and SalI (GTCGAC) cloning sites (enclosed in boxes) in pFW11 are shown. (A) P_null (pFW11-null), P₁ (pFW11-P1), and P₂ (pFW11-P2) are as described elsewhere (52). A 67-bp P_minor fragment is from pDKT90 (29). Promoter elements (−35, TGn, and −10) and the +1 start site are noted in red above the sequence. Note that the +1A starts of P₁ and P₂ are located within the SalI site, whereas the P_minor +1 is 8 nt upstream. Arrows indicate 5′ end of the P_minor fragment before the EcoRI site in the P_-63, P_-44, P_-35, and P_-29 clones. (B) The P_-35 construct, used as wild-type P_minor promoter in the present study, is shown. Derivative promoters with the indicated changes are listed below P_minor. The dotted line indicates that the sequence is identical to P_minor.

FIG. 2.

The P_minor transcriptional start is identified. In vitro transcription reactions were assembled by adding 1.95 μl of a solution containing reconstituted polymerase (0.2 pmol core plus 0.5 pmol of σ⁷⁰) in protein buffer I to 0.02 pmol of linear DNA in 2.05 μl of DNA buffer I. (A) Transcription was initiated by adding 1 μl of NTP mix to the protein-DNA mix containing P_minor DNA. As indicated, each NTP was added at the following concentrations: 1 mM UTP and 0.25 mM each ATP and GTP. The specific activity of [γ-³²P]GTP or [γ-³²P]ATP (where indicated by the asterisk) was 7 × 10⁵ dpm/pmol. Assignments of labeled RNA products are shown. The black arrow signifies 5-nt products, consistent with the migration of the pppACN₃ marker (not shown) kindly provided by N. Nossal. (B) Denaturing acrylamide gel showing the products of primer extension assays next to a P_minor DNA sequencing ladder. The unlabeled P_minor, P_+1C, and P₂ transcripts were generated by multiple-round transcription assays, which were initiated by the addition of 1 μl of NTP mix II. (C) Single-round transcription was initiated by adding 1 μl of NTP mix I with heparin. Transcripts arising from the P_minor, P_+1C, and P₂ promoters are indicated.

FIG. 3.

Minimal P_minor promoter defined. (A) Graph showing the β-galactosidase assay activity (in Miller units) determined in vivo for each promoter. (B) A denaturing acrylamide gel showing the products of multiple-round in vitro transcription reactions is overlaid with a graph of the quantitation data. Transcription reactions were assembled as described in Fig. 2C. Multiple-round transcription was initiated by adding 1 μl of NTP mix I (without heparin). The amounts of RNA were determined by densitometry and are shown relative to P₂, which is set at 100. The values represent the average of three or more transcriptions.

FIG. 4.

TTGAAA functions as −35 element and is required for P_minor transcription. A denaturing acrylamide gel showing the products of single-round in vitro transcription reactions is overlaid with a graph of the quantitation data. Transcription reactions were assembled and carried out as described in Fig. 2C. The amounts of RNA were determined by densitometry and are shown relative to P_minor, which is set at 100. The values represent the average of three or more transcriptions.

FIG. 5.

Potassium permanganate footprints of P_minor (A) and P_{−14A−13A−12T} (B). Reactions were assembled by adding 1.95 μl of a solution containing reconstituted polymerase (Eσ⁷⁰; 0.4 pmol core plus 1.0 pmol of σ⁷⁰) or core alone (0.4 pmol) in protein buffer I to 0.2 pmol of the indicated DNA in 2.05 μl of DNA buffer I. Eσ⁷⁰-dependent bands are marked by arrows and are numbered relative to the P_minor transcriptional +1 site. Nontemplate strand results for P_minor are consistent with the findings of Vuthoori et al. (50), but bands −6 and −4 are referred to as −2 and +1 in that study. Traces for the lanes are shown.

FIG. 6.

P_minor requires TGn motif to compensate for a weak −10 element. A denaturing acrylamide gel showing the products of single-round in vitro transcription reactions is overlaid with a graph of the quantitation data. Transcription reactions were assembled and carried out as described in Fig. 2C. The amounts of RNA transcript were determined by densitometry and are shown relative to P_minor, which is set at 100. The values represent the average of three or more transcriptions.