Linker Regions of the RhaS and RhaR Proteins

Abstract

Substitutions within the interdomain linkers of the AraC/XylS family proteins RhaS and RhaR were tested to determine whether side chain identity or linker structure was required for function. Neither was found crucial, suggesting that the linkers do not play a direct role in activation, but rather simply connect the two domains.In the presence of the sugar L-rhamnose, RhaS and RhaR activate transcription of Escherichia coli genes, whose products are required for the uptake and catabolism of L-rhamnose (6,7,29,30). RhaS and RhaR comprise a regulatory cascade in which L-rhamnose stimulates RhaR to activate transcription of rhaSR (29) and then RhaS activates transcription of the Lrhamnose catabolic operon rhaBAD (7) and the L-rhamnose transport gene rhaT (30). Both RhaS and RhaR are members of the large AraC/XylS family of transcription regulatory proteins, which share sequence similarity in a 100-amino-acid DNA-binding domain (5,10,11,21). AraC/XylS family proteins regulate the expression of genes whose functions include carbon metabolism (3,12,25,28), stress responses (4, 15-18, 22, 32), and pathogenesis (9,14,23,24).RhaS and RhaR each consist of two independently functional domains: an N-terminal domain required for dimerization and response to L-rhamnose (A. Kolin, J. R. Wickstrum, and S. M. Egan, unpublished results) and a C-terminal domain involved in DNA binding and transcription activation (1, 2, 6, 31; J. R. Wickstrum and S. M. Egan, unpublished results). Based on alignment with the AraC dimerization domain and its structure (26) and the MarA protein and its structure (22), we can predict the approximate boundaries of the RhaS and RhaR domains. Such analysis, combined with knowledge of AraC (8), leads to the prediction that there is a flexible linker that connects the two domains of RhaS and RhaR. This linker spans approximately residues 166 to 172 in RhaS and 198 to 207 in RhaR (Fig. 1). Genetic and biochemical studies have shown that many different single and multiple substitutions could be made within the AraC linker without impacting activation (8), suggesting that the AraC arabinose response does not depend on the identity of its linker residues. However, our finding that the ligand responses of RhaS and RhaR differ from that of AraC (A. Kolin and S. M. Egan, unpublished results) left open the possibility that the linker might participate in transmission of the L-rhamnose signal.In this study, we used a genetic approach to analyze the linker regions of RhaS and RhaR. Our previous results indicate that L-rhamnose binds to the N-terminal domains of RhaS and RhaR, while transcription activation involves their C-terminal domains (2, 31; Kolin and Egan, unpublished). Therefore, we sought to determine whether the linker regions of RhaS and/or RhaR were involved in transmitting the L-rhamnose status from the N-to the C-terminal domain or whether, similar to AraC, the linker was required only to flexibly connect the two domains.Single alanine substitutions in the linker region of RhaS and RhaR have, at most,...

Mutagenesis of the Rns regulator of enterotoxigenic Escherichia coli reveals roles for a linker sequence and two helix–turn–helix motifs

“…It is postulated that this area of Rns forms, or is part of, a flexible interdomain linker that is necessary to correctly orientate the N-and C-terminal regions of the protein to enable them to participate in their functional roles. The NACRS sequence of Rns has some features in common with the flexible linkers of the AraC, RhaR and RhaS proteins (Eustance & Schleif, 1996;Kolin et al, 2007). The activity of other AraC-like proteins may also depend on the presence of a central disordered/flexible connecting sequence.…”

“…The RhaS linker was selected because in its native setting, the LENSASR sequence has been suggested to function only to flexibly link the two functional domains of RhaS. Furthermore, a derivative of the related RhaR protein containing the RhaS linker in place of its own linker sequence was functional with only small deficits in its activity (Kolin et al, 2007).…”

Active Role of the Interdomain Linker of AraC

“…Roughly the same was found in studies of the AraC homologs RhaR and RhaS (17) and the AraC-like proteins Rns (20) and VirF (23). Studies on RhaR and RhaS found that some alanine substitutions interfered with normal regulation and that swapping the predicted interdomain linkers from RhaR to RhaS produced measurable but modest effects, and it was concluded from those studies that, overall, the linkers did not play a direct role in activating transcription (17). With Rns it was found that replacing the Rns linker with the RhaS linker results in a protein with WT-like behavior (20).…”

Finely Tuned Regulation of the Aromatic Amine Degradation Pathway in Escherichia coli

“…The C-terminal domain (CTD) and the linker region of FeaR were identified by sequence alignment of five AraC family proteins (FeaR, AraC, MelR, RhaR, and RhaS) using T-coffee (27). The DNA sequence corresponding to the CTD and linker region was amplified by PCR and ligated into pET-21a(ϩ) (Novagen) in frame with sequences encoding a C-terminal hexahistidine tag.…”