RNA polymerases from Bacillus subtilis and Escherichia coli differ in recognition of regulatory signals in vitro

Abstract

Adaptation of bacterial cells to diverse habitats relies on the ability of RNA polymerase to respond to various regulatory signals. Some of these signals are conserved throughout evolution, whereas others are species specific. In this study we present a comprehensive comparative analysis of RNA polymerases from two distantly related bacterial species, Escherichia coli and Bacillus subtilis, using a panel of in vitro transcription assays. We found substantial species-specific differences in the ability of these enzymes to escape from the promoter and to recognize certain types of elongation signals. Both enzymes responded similarly to other pause and termination signals and to the general E. coli elongation factors NusA and GreA. We also demonstrate that, although promoter recognition depends largely on the sigma subunit, promoter discrimination exhibited in species-specific fashion by both RNA polymerases resides in the core enzyme. We hypothesize that differences in signal recognition are due to the changes in contacts made between the beta and beta' subunits and the downstream DNA duplex.

FIG. 1

Promoter selectivity of the E. coli and B. subtilis RNA polymerases. (Top) The consensus E. coli promoter is aligned with λ P_R and T7A1 promoters with the −35 and −10 hexamers shown in boldface, and the transcription start site is indicated with a bent arrow. (Bottom) Elongation complexes were formed for 15 min at 37°C on the linear DNA templates encoding either λ P_R (P_R, pIA226) or T7A1 (A1, pCL102) promoter with holoenzymes composed of the core and ς components indicated below each lane in the presence of limited subset of NTP substrates (see Materials and Methods). The positions of halted RNA transcripts originating from each promoter are indicated on the left. Bands below the A26 and A29 RNAs correspond to abortive products.

FIG. 2

Abortive initiation assays. The pRL550 template shown on the top encodes the T7A1 promoter followed by a mutant initial transcribed sequence that has been shown to impede promoter escape by the E. coli RNAP in vitro (16). The transcription start site is indicated by a bent arrow; the positions of halted A20 RNA and the template end are also shown. Multiple-round abortive initiation assays were carried out with either E_EC · ς⁷⁰ or E_BS · ς⁷⁰, in the absence or presence of the E. coli GreA protein. Aliquots were withdrawn at the times indicated above each lane. After the last aliquot was withdrawn, the sample was incubated with NTPs (at 500 μM each) for 5 min at 37°C to generate the chase sample (the last lane in each panel [i.e., lanes C]). Positions of different RNA species are indicated on the right. The major GreA-sensitive RNA product is shown with a star.

FIG. 3

Recognition of the his pause site. (Top) Linear pCL102 DNA template is drawn to scale with the positions of T7A1 promoter, transcription start and his pause sites, and the his terminator from the attenuator region of the E. coli his biosynthetic operon (33) indicated. The structure of the RNA transcript in the vicinity of the pause site is depicted in the inset. (Bottom) Halted A29 TECs were formed with either E_EC · ς⁷⁰ or E_BS · ς⁷⁰ RNAP and challenged with NTPs (20 μM GTP and 150 μM ATP, CTP, and UTP) and rifampin (Rif) at 50 μg/ml. In the rightmost panel, E_BS · ς⁷⁰ was added to the reaction (to 50 nM) simultaneously with NTPs and rifampin. Aliquots were withdrawn at the times indicated above each lane, followed by the high NTP chase (C lanes as described for Fig. 2. The positions of the halted (A29), paused (P), and terminated (T) transcripts are indicated with arrows.

FIG. 4

Recognition of the ops pause site. (Top) Linear pIA251 DNA template is drawn to scale; the positions of T7A1 promoter, transcription start and ops pause sites, and the his terminator are indicated. The RNA sequence in the vicinity of the pause sites (major at U64, minor at U62) is also shown. (Bottom) Transcription complexes halted at position A29 were incubated with NTPs (20 μM GTP and 150 μM ATP, CTP, and UTP) in the presence of heparin (at 100 μg/ml). Aliquots were withdrawn at times indicated above each lane, followed by the high NTP chase (lanes C) as described in Fig. 2. The positions of the halted (A29), paused (P), and terminated (T) transcripts are indicated.

FIG. 5

Pausing at the U-track signal. The pIA253 template shown on the top encodes the λ P_R promoter followed by a 26-nt C-less cassette and a stretch of eight consecutive U residues (positions 38 to 45). The positions of the transcription start site, the halted A26 RNA, and the major E. coli pause site are indicated with arrows. Transcription complexes halted at position A26 were challenged with NTPs (20 μM UTP and 150 μM ATP, CTP, and GTP) and heparin (at 100 μg/ml); the E. coli NusA or GreA proteins were added where indicated. Aliquots were withdrawn at the times indicated above each lane, followed by the high NTP chase (lanes C) in the last lane of each panel. The positions of the A26, G35, and U44 RNA transcripts are indicated with arrows. The portion of the gel between the U-track and the terminator (T) has been deleted to conserve space.

FIG. 6

Recognition of the pause site in the B. subtilis P RNA transcript. (Top) Linear pIA199 DNA template is drawn to scale with the positions of T7A1 promoter, transcription start and pause sites, and position of the template end indicated; position 17 corresponds to the +1 of P RNA. Structure of the RNA transcript in the vicinity of the pause site (at U73) is also shown. (Bottom) Pause assays were performed with either E_EC · ς⁷⁰ or E_BS · ς⁷⁰. Transcription complexes halted at position G16 were challenged with NTPs (20 μM GTP and 150 μM ATP, CTP, and UTP) and heparin in the absence or presence of the E. coli NusA protein (at 50 nM). Aliquots were withdrawn at the times indicated above each lane; the chase samples (lanes C) were generated as described in Fig. 5. The positions of the pause (P) and runoff (end) transcripts are indicated with arrows.

FIG. 7

Recognition of Rho-independent terminators. The efficiency of termination at several previously characterized terminators (see Materials and Methods for sequences) by B. subtilis (black bars) and E. coli (gray bars) enzymes is depicted graphically. Each value represents an average of four or five independent measurements; the error bars correspond to one standard deviation.