A novel RNA polymerase binding site upstream of the galactose promoter in Escherichia coli exhibits promoter‐like activity

Abstract

RNA polymerase is known to bind and utilize the overlapping promoters P1 and P2 in Escherichia coli galactose operon. We have identified an additional specific site upstream of P2, where RNA polymerase binds in a heparin-resistant manner. Binding of polymerase to this site, termed P3, occurs simultaneous to its binding at P1/P2. We have located this P3 site by DNase I footprinting. A 63 base pair region centered around position 2 100 with respect to galP1 is protected by polymerase. Interestingly, a Pribnow box TATAAT is present within this protected region (2103 to 2108). We have shown that transcription occurs from P3 in vitro. Primer extension analysis provides direct evidence that P3 is transcribed in vivo. The start site of transcription has been mapped at 296 position relative to galP1. b-galactosidase assays with different gal promoter constructs reveal that while P3 alone functions as a weak in vivo promoter, it has a synergistic effect on transcription from the gal operon, since deletion of P3 or specifically mutating its 210 region result in a substantial reduction in the gal promoter activity.Keywords: RNA polymerase; E. coli; promoter; galactose operon; transcription.Gene expression in bacteria involves a series of events, in which the first step is the initiation of transcription. During the initiation process, RNA polymerase holoenzyme (RNPol) recognizes and binds to specific sites on DNA, known as promoters. The initial recognition is followed by the formation of a`closed complex', which, through the steps of isomerization, leads to an`open complex' where a portion of the DNA is melted, and transcription starts [1]. Promoter recognition is a highly specific phenomenon, and the strength of promoters are largely influenced by the hexanucleotide sequences present at the 210 and 235 regions of the coding strand (with the transcription start point designated 11), as well as by the spacing between these regions [2,3]. However, the correspondence of sequences at these sites is not the sole determinant of promoter strength [1,4,5].Control of gene expression can occur at many levels. One of the important points of control that predominates in many bacterial systems is found to occur at the stage of transcription initiation [1]. This control is exercised through the action of negative or positive regulatory proteins [6], as well as through the topology of DNA [7±9]. A large number of promoters that employ these means of regulation have been characterized in E. coli. Thus, the control region consists of the site of interaction of RNA polymerase holoenzyme (promoter) as well as the binding sites for repressor proteins (operator) and for activator proteins. A variety of relative organizations of these sites are found in different operons of the E. coli bacterium.In the galactose operon of E. coli, two overlapping promoters P2 and P1 have been found to be responsible for initiation of transcripts at 25 and at 11, respectively, corresponding to the epimerase-transferase-galactokinase genes [10]. The cyclic AMP r...

A “master” in base unpairing during isomerization of a promoter upon RNA polymerase binding

“…Although the intrinsic amount of P1 RNA synthesis was significantly lower in the template with 2-AP at the ϩ1 position, CRP resurrected this P1 defect to the wild-type level. Unlike 2-AP at ϩ1, cAMP and CRP barely helped the defect of P1 transcription in DNA containing 2-AP ¶ The newly discovered P4 promoter also was named as P3 in the original publication (27). We have changed that to P4 in this report, with the permission of the authors, to avoid confusion.…”

“…1) was generated by PCR for use as templates of transcription by RNA polymerase in vitro. The gal operon has been shown to contain four promotors: P1, P2, P3, and P4 ¶ (27)(28)(29). Of these, P2 was inactivated by substitution mutations (Fig.…”

Sigma70 Promoters in Escherichia coli: Specific Transcription in Dense Regions of Overlapping Promoter-like Signals

“…The lac region presented six promoter-like signals plus the real one, 38,45 the IS50 promoter region has two promoter-like signals near the functional one, 34 and one promoter-like signal is in the region of the gal promoters. 46 Figure 7 shows the positions of the six promoters-like signals found experimentally for the lac promoter region. If we calculated the performance of the RNAP in these experiments, we found a sensitivity of 1, and precision of 0.30, finding an average of 4.3 signals per region.…”

Differential Role of Base Pairs on gal Promoters Strength

“…1a) [18, 19, 21, 23–26]. The DNA sequence of the entire region also contains two additional promoters ( P3 and P4 *), which are observed under specific conditions [16, 27]. [*Footnote: To avoid confusion in nomenclature, we referred to P3 described by Sur et al [27] as P4 to distinguish it from another promoter previously described by Ponnabalam et al as P3 [16]].…”

“…The activated P3 is repressible by CCC binding to the AS . The fourth promoter, P4 , with a functional −10 element (TATAAT) is independent of CCC [27]. Although our investigation of P1 and P2 touches on P3 , P4 was not studied because the P4 promoter is located far upstream of the DNA segment that is not being considered here.…”