Differences in the mechanism of the allosteric I-rhamnose responses of the AraC/XyIS family transcription activators RhaS and RhaR

• Published in: Molecular microbiology Vol. 68; no. 2; p. 448
• Main Authors: Kolin, Ana, Balasubramaniam, Vinitha, Skredenske, Jeff M, Wickstrum, Jason R, Egan, Susan M
• Format: Journal Article
• Language: English
• Published: Oxford Blackwell Publishing Ltd 01.04.2008
• Subjects: Binding sites; Deoxyribonucleic acid; DNA; Mutation; Proteins
• ISSN: 0950-382X; 1365-2958

Proteins in the largest subset of AraC/Xy1S family transcription activators, including RhaS and RhaR, have C-terminal domains (CTDs) that mediate DNA-binding and transcription activation, and N-terminal domains (NTDs) that mediate dimerization and effector binding. The mechanism of the allosteric effector response in this family has been identified only for AraC. Here, we investigated the mechanism by which RhaS and RhaR respond to their effector, 1-rhamnose. Unlike AraC, N-terminal truncations suggested that RhaS and RhaR do not use an N-terminal arm to inhibit activity in the absence of effector. We used random mutagenesis to isolate RhaS and RhaR variants with enhanced activation in the absence of 1-rhamnose. NTD substitutions largely clustered around the predicted 1-rhamnose-binding pockets, suggesting that they mimic the structural outcome of effector binding to the wild-type proteins. RhaS-CTD substitutions clustered in the first HTH motif, and suggested that 1-rhamnose induces improved DNA binding. In contrast, RhaR-CTD substitutions clustered at a single residue in the second HTH motif, at a position consistent with improved RNAP contacts. We propose separate allosteric mechanisms for the two proteins: Without 1-rhamnose, RhaS does not effectively bind DNA while RhaR does not effectively contact RNAP. Upon 1-rhamnose binding, both proteins undergo structural changes that enable transcription activation. [PUBLICATION ABSTRACT]