The strong efficiency of the Escherichia coli gapA P1 promoter depends on a complex combination of functional determinants

Abstract

The Escherichia coli multi-promoter region of the gapA gene ensures a high level of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) production under various growth conditions. In the exponential phase of growth, gapA mRNAs are mainly initiated at the highly efficient gapA P1 promoter. In the present study, by using site-directed mutagenesis and chemical probing of the RPo (open complex) formed by Esigma70 (holoenzyme associated with sigma70) RNAP (RNA polymerase) at promoter gapA P1, we show that this promoter is an extended -10 promoter that needs a -35 sequence for activity. The -35 sequence compensates for the presence of a suboptimal -10 hexamer. A tract of thymine residues in the spacer region, which is responsible for a DNA distortion, is also required for efficient activity. We present the first chemical probing of an RPo formed at a promoter needing both a -10 extension and a -35 sequence. It reveals a complex array of RNAP-DNA interactions. In agreement with the fact that residue A-11 in the non-template strand is flipped out in a protein pocket in previously studied RPos, the corresponding A residue in gapA P1 promoter is protected in RPo and is essential for activity. However, in contrast with some of the previous findings on RPos formed at other promoters, the -12 A:T pair is opened. Strong contacts with RNAP occur both with the -35 sequence and the TG extension, so that the sigma4 and sigma2 domains may simultaneously contact the promoter DNA. RNAP-DNA interactions were also detected immediately downstream of the -35 hexamer and in a more distal upstream segment, reflecting a wrapping of RNAP by the core and upstream promoter DNA. Altogether, the data reveal that promoter gapA P1 is a very efficient promoter sharing common properties with both extended -10 and non-extended -10 promoters.

Dual temporal transcription activation mechanisms control cesT expression in enteropathogenic Escherichia coli

“…Our finding that a 218T/G substitution in cesTp reduced promoter activity fourfold compared with wild-type cesTp (Fig. 3) is in agreement with studies with the gapA1 promoter which showed that a 218T/G substitution reduced promoter activity threefold (Thouvenot et al, 2004). It has been demonstrated that there is a preference for T at position 218 in E. coli promoters (Singh et al, 2011) and that this position may contribute to a structural conformation favourable for Es 70 interactions (Murakami et al, 2002).…”

Redefining Escherichia coli σ 70 Promoter Elements: −15 Motif as a Complement of the −10 Motif

“…Specifically, it was observed earlier (32) that promoters containing Ϫ15 TG Ϫ14 are able to tolerate weaker Ϫ10 elements. Furthermore, both gapAP1 and Pminor promoters have weak Ϫ10 elements, and the presence of a strong Ϫ15 element is required for promoter function (21,49). On the other hand, mutations that improve the Ϫ10 element were shown to abolish the requirement for a strong Ϫ15 element, therefore demonstrating the direct role of the Ϫ15 element in complementing weak Ϫ10 element strength.…”

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

“…1C). Our previous work demonstrated that P minor and P min belong to a newly identified promoter class, Ϫ 35/TGn, that is characterized by a requirement for both an excellent Ϫ35 element and an extended Ϫ10 TGn sequence to compensate for a poor Ϫ10 element (1,37,38). To investigate whether the effect of region 1.1 with P minor reflects a general characteristic of Ϫ35/TGn promoters, we compared the formation of active transcription complexes at P min with that at P min16 , a derivative with perfect Ϫ35 and Ϫ10 elements, and at P min7 , a derivative with a good Ϫ35 and a perfect TGn and Ϫ10 element (Fig.…”

“…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).…”