Transcription Activation by Escherichia coli Rob at Class II Promoters: Protein-Protein Interactions between Rob's N-Terminal Domain and the σ70 Subunit of RNA Polymerase

Bacterial transcription activators regulate transcription by making essential protein-protein interactions with RNA polymerase, for example, with region 4 of the sigma(70) subunit (sigma(70) R4). Rob, SoxS, and MarA comprise a closely related subset of members of the AraC/XylS family of transcription factors that activate transcription of both class land class II promoters. Recently, we showed that interactions between SoxS and sigma(70) R4 occlude the binding of sigma(70) R4 to the -35 promoter element of class II promoters. Although Rob shares many similarities with SoxS, it contains a C-terminal domain (CTD) that the other paralogs do not. Thus, a goal of this study was to determine whether Rob makes protein-protein interactions with sigma(70) R4 at class II promoters and, if so, whether the interactions occlude the binding of sigma(70) R4 to the -35 hexamer despite the presence of the CTD. We found that although Rob makes fewer interactions with sigma(70) R4 than SoxS, the two proteins make the same, unusual, position-dependent interactions. Importantly, we found that Rob occludes sigma(70) R4 from binding the -35 hexamer, just as does SoxS. Thus, the CTD does not substantially alter the way Rob interacts with sigma(70) R4 at class II promoters. Moreover, in contrast to inferences drawn from the co-crystal structure of Rob bound to robbox DNA, which showed that only one of Rob's dual helix-turn-helix (HTH) DNA binding motifs binds a recognition element of the promoter's robbox, we determined that the two HTH motifs each bind a recognition element in vivo. (C) 2012 Elsevier Ltd. All rights reserved.