A mutant spacer sequence between -35 and -10 elements makes the P _lac promoter hyperactive and cAMP receptor protein-independent

Abstract

To determine whether the spacer region between the ؊35 and ؊10 elements plays any sequence-specific role, we randomized the GC-rich sequence ( ؊20 CCGGCTCG ؊13 ) within the spacer region of the cAMP-dependent lac promoter and selected an activatorindependent mutant, which showed extraordinarily high intrinsic activity. The hyperactive promoter is obtained by incorporation of a specific 10-bp-long AT-rich DNA sequence within the spacer, referred to as the ؊15 sequence, which must be juxtaposed to the upstream end of the ؊10 sequence for the hyperactivity. The transcription enhancement functions only in the presence of a ؊35 element. The spacer sequence enhanced both RNA polymerase binding and open complex formation. Isolated in the lac promoter, it also enhanced transcription when placed at two other unrelated promoters. Sequence analysis shows a low GC content and an abundance of stereochemically flexible TG:CA and TA:TA dimeric steps in the ؊18͞؊9 region and a strong correlation between the presence of flexible dimeric steps in this region and the intrinsic strength of the promoter.A promoter is a stretch of DNA sequence that encodes information for RNA polymerase binding and initiation of transcription. Genetic and statistical analysis of promoters in Escherichia coli defined two kinds of E 70 RNA polymerase holoenzyme-dependent promoters (1). Both kinds require a 6-bp Ϫ10 sequence (consensus 5Ј-TATAAT-3Ј) located Ϸ7 bp 5Ј to the transcription start site. Functionally, the Ϫ10 element participates in RNA polymerase binding by interacting with the region 2.3-2.4 of 70 (2-10) and is part of an Ϸ15-nt putative single-stranded region in the open complex (4,8,(11)(12)(13)(14)(15). The first kind of promoters (Ϫ35 promoters) is more common and characterized by the presence of the Ϫ10 element as well as a 6-bp (consensus sequence 5Ј-TTGACA-3Ј) in the Ϫ35 position (16). The Ϫ35 element also helps RNA polymerase binding through interaction with region 4.2 of 70 (2,5,17). It is believed that the spacer region between Ϫ35 and Ϫ10 (optimal length 17 bp) does not have any specific sequence requirement and simply facilitates the spatial alignment of the Ϫ10 and Ϫ35 elements in binding to the 2.4 and 4.2 regions of the factor (7, 17, 18). The second kind, called ''extended Ϫ10'' promoters, contains an extra 2-bp 5Ј-TG-3Ј sequence located 1 bp 5Ј to the Ϫ10 element, (consensus sequence Ϫ15 TGNTATAAT Ϫ7 ) (19). The DNA sequence immediately upstream of an extended Ϫ10 element may enhance the activity of the extended Ϫ10 promoter (20, 21). The TG element also binds to RNA polymerase by contacting the 3.0 (formerly 2.5) segment of 70 (22). The Ϫ35 promoters may be improved by the presence of the extended Ϫ10 sequence and vice versa.Many naturally occurring promoters are more or less inefficient because of the presence of non-consensus sequence elements or suboptimal spacer lengths and are resurrected by extra regulatory factors (23). The extra factor may be a DNA sequence, e.g., an UP element (24-26). The UP element, located imme...

Transcriptional Analysis of the grlRA Virulence Operon from Citrobacter rodentium

“…However, this promoter also possesses an extended Ϫ10 (TGn) motif, and its spacer is extremely AT rich (AT/GC ratio of 8:1). The TGn motif probably compensates for a poor Ϫ35 region, while the AT richness contributes to the promoter strength (5,30,33). As for grlRA, a number of other bacterial operons possess internal promoters for supplementary expression of downstream genes (35,58,59).…”

The promoter spacer influences transcription initiation via σ 70 region 1.1 of Escherichia coli RNA polymerase

“…In this way, region 1.1 may help polymerase discriminate against nonpromoter DNA and disfavor promoter sequences that are specific for alternate sigma factors, which lack region 1.1 (57). It is also worth noting that, unlike the promoter sequence elements, the spacer structure may be responsive to conditions (temperature) (41), DNA modifications (methylation), or transcription factors (CRP, MerR) (40,58), and thereby provide an opportunity to regulate gene expression by modulating the formation of RPo.…”

“…In particular, a GC-rich spacer can result in reduced promoter activity compared with that seen with an AT-rich spacer (39,40). Spacers with runs of Ts (46) or As (47) have been shown to dramatically increase the overall activity of a promoter.…”

“…Although the length of a promoter spacer dictates the distance between the promoter elements and is an important factor in promoter usage, the spacer is not known to make sequence-specific contact with RNAP (reviewed in refs. 1 and 4) and exactly how the spacer affects the interaction of RNAP with a promoter is not fully understood (39)(40)(41). The spacer in P minor is the preferred length for a 70 -dependent promoter, eliminating the possibility that simply its length makes P minor unusual.…”

“…Previously, it has been shown that a spacer can influence promoter activity (38,(40)(41)(42)(43)(44)(45). In particular, a GC-rich spacer can result in reduced promoter activity compared with that seen with an AT-rich spacer (39,40).…”