Upstream A-tracts increase bacterial promoter activity through interactions with the RNA polymerase α subunit

Abstract

Upstream A-tracts stimulate transcription from a variety of bacterial promoters, and this has been widely attributed to direct effects of the intrinsic curvature of A-tract-containing DNA. In this work we report experiments that suggest a different mechanism for the effects of upstream A-tracts on transcription. The similarity of A-tract-containing sequences to the adenine-and thymine-rich upstream recognition elements (UP elements) found in some bacterial promoters suggested that A-tracts might increase promoter activity by interacting with the ␣ subunit of RNA polymerase (RNAP). We found that an A-tract-containing sequence placed upstream of the Escherichia coli lac or rrnB P1 promoters stimulated transcription both in vivo and in vitro, and that this stimulation required the C-terminal (DNA-binding) domain of the RNAP ␣ subunit. The A-tract sequence was protected by wild-type RNAP but not by ␣-mutant RNAPs in footprints. The effect of the A-tracts on transcription was not as great as that of the most active UP elements, consistent with the degree of similarity of the A-tract sequence to the UP element consensus. A-tracts functioned best when positioned close to the ؊35 hexamer rather than one helical turn farther upstream, similar to the positioning optimal for UP element function. We conclude that A-tracts function as UP elements, stimulating transcription by providing binding site(s) for the RNAP ␣CTD, and we suggest that these interactions could contribute to the previously described wrapping of promoter DNA around RNAP.Promoters used by the major form of Escherichia coli RNA polymerase (RNAP), E 70 , generally contain two hexamers located about 10 and 35 bp upstream of the start site of transcription, which are recognized by the 70 subunit (1). In addition, sequences upstream of the Ϫ35 hexamer in some promoters in E. coli as well as in other bacterial species increase transcription in the absence of accessory proteins (2-9). These upstream sequences are generally AϩT-rich, and some contain multiple A-tracts in phase with the DNA helical repeat (phased A-tracts). Phased A-tracts inserted upstream of the Ϫ35 hexamer in synthetic hybrid promoters have also been reported to increase transcription (10-14). Because phased A-tracts result in macroscopic DNA curvature [i.e., intrinsic bends whose angle increases with the number of A-tracts (15, 16)], the effects of A-tracts on transcription often have been attributed to direct effects of DNA bending, even though a mechanism for such an effect was not clear (4,5,7,8,(10)(11)(12)(13)(14)(17)(18)(19).Upstream sequences are not as extensively conserved as the Ϫ10, Ϫ35 hexamers in E. coli promoters (1) and were not considered as an RNAP recognition element until recently (9,20). However, an A-tract positioned at about Ϫ40 (21, 22) or AϩT-rich sequences at about Ϫ40 and Ϫ50 (23) were noted in subsets of promoters. Some conservation of alternating A-and T-tracts was also noted in the upstream region of Bacillus subtilis promoters (24). Whereas many promoters co...

Recruitment of σ54-RNA Polymerase to the Pu Promoter of Pseudomonas putida through Integration Host Factor-mediated Positioning Switch of α Subunit Carboxyl-terminal Domain on an UP-like Element

“…Despite this IHF-mediated topological switch, the flexibility of the long unstructured interdomain linker connecting ␣CTD to the rest of ␣ (8,22) (Fig. 1), respectively, could constitute the core subsites (7,10,45). In view of this, we suggested that the distance between the core A-tracts is not consistent with a side-by-side arrangement as for the UP subsites in the canonical UP element (10), and there would be turns of helix between UP-like Pu subsites.…”

Structural origins of adenine-tract bending

“…In some cases, substitution of an entire promoter region by properly curved DNA can activate in vitro transcription (4,5). More recent work indicates that these sequences function as upstream recognition elements (UP elements), the curvatures of which play an unknown role (6). In addition, HIV-1 reverse transcriptase termination of the (ϩ) strand DNA synthesis is thought to occur because of minor groove compression of duplex DNA caused by the A-tracts (7,8).…”

Identification of an upstream regulatory sequence that mediates the transcription of mox genes in Methylobacterium extorquens AM1

“…In some cases these have been shown to bind the a subunit of RNA polymerase (Aiyar et al, 1998), and act as positive transcriptional elements via their DNA-curvature characteristics. Therefore, it is possible that the sequence we have identified is analogous to one of these elements.…”

“…Multiple A-tract sequences act as enhancer elements for promoters in many bacterial systems, including the very highly expressed rrn promoters as well as others (Aiyar et al, 1998;Cheema et al, 1999). In some cases these have been shown to bind the a subunit of RNA polymerase (Aiyar et al, 1998), and act as positive transcriptional elements via their DNA-curvature characteristics.…”

“…Little is known concerning the role of this consensus sequence in M. extorquens AM1. In other organisms, multiple A-or Ttracts function as upstream recognition elements, increasing promoter activity (Cheema et al, 1999) or facilitating DNA curvature for the binding of regulatory proteins (Aiyar et al, 1998). In this study we have investigated the function of this sequence upstream of Mox promoters by site-directed mutagenesis.…”