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

Upstream A-tracts stimulate transcription from a variety of bacterial promo ters, and this has been widely attributed to direct effects of the intrinsi c curvature of A-tract-containing DNA. In this work we report experiments t hat suggest a different mechanism for the effects of upstream A-tracts on t ranscription. The similarity of A-tract containing sequences to the adenine - and thymine-rich upstream recognition elements (UP elements) found in som e bacterial promoters suggested that A-tracts might increase promoter activ ity by interacting with the alpha subunit of RNA polymerase (RNAP). We foun d that an A-tract-containing sequence placed upstream of the Escherichia co li lac or rrnB P1 promoters stimulated transcription bath in vivo and in vi tro, and that this stimulation required the C-terminal (DNA-binding) domain of the RNAP alpha subunit. The A-tract sequence was protected by wild-type RNAP but not by alpha-mutant RNAPs in footprints. The effect of the A-trac ts 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 U P element consensus. A-tracts functioned best when positioned close to the -35 hexamer rather than one helical turn farther upstream, similar to the p ositioning optimal for UP element function. We conclude that A-tracts funct ion as UP elements, stimulating transcription by providing binding site(s) for the RNAP alpha CTD, and we suggest that these interactions could contri bute to the previously described wrapping of promoter DNA around RNAP.