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

• Published in: Journal of molecular biology 2012-06, Vol.419 (3-4), p.139-157
• Main Authors: Taliaferro, Lanyn P., Keen, Edward F., Sanchez-Alberola, Neus, Wolf, Richard E.
• Format: Article
• Language: English
• Subjects: genetic epistasis; prerecruitment; Rob–micF crystal structure; SoxS; σ70 R4
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2012.03.019

Bacterial transcription activators regulate transcription by making essential protein–protein interactions with RNA polymerase, for example, with region 4 of the σ70 subunit (σ70 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 I and class II promoters. Recently, we showed that interactions between SoxS and σ70 R4 occlude the binding of σ70 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 σ70 R4 at class II promoters and, if so, whether the interactions occlude the binding of σ70 R4 to the −35 hexamer despite the presence of the CTD. We found that although Rob makes fewer interactions with σ70 R4 than SoxS, the two proteins make the same, unusual, position-dependent interactions. Importantly, we found that Rob occludes σ70 R4 from binding the −35 hexamer, just as does SoxS. Thus, the CTD does not substantially alter the way Rob interacts with σ70 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. [Display omitted] ► SoxS makes novel contacts with σ70 during activation of class II promoters. ► Does the CTD of its paralog, Rob, interfere with these contacts? ► In the Rob–micF crystal structure, only one HTH motif binds robbox DNA. ► However, both of Rob's motifs bind robbox DNA in vivo. Thus, Rob's CTD does not interfere. ► Moreover, like SoxS, robbox binding by Rob occludes the binding of σ70 to class II promoters.