Amino acid residues critical for DNA binding and inducer recognition in CbnR, a LysR-type transcriptional regulator from Cupriavidus necator NH9

Abstract

CbnR, a LysR-type transcriptional regulator from Cupriavidus necator NH9, activates the transcription of chlorocatechol-degradative enzymes. To activate the transcription, CbnR needs to bind not only to the cbnA promoter but also to the inducer. In this study, the transcriptional activity and DNA-binding activity of twenty-five mutants of CbnR were analyzed. Of the 17 mutants of the DNA-binding domain, 11 mutants lost their ability to activate transcription. While most mutants without transcriptional activation did not show DNA-binding activity, Asn17Ala, Gln29Ala, and Pro30Ala retained DNA-binding activity, suggesting that transcriptional activation by CbnR requires more than its binding to promoter DNA. Of the 8 mutants of the regulatory domain, 6 mutants changed their responses to the inducer, when compared with wild-type CbnR. Interestingly, Arg199Ala and Val246Ala induced constitutive expression of the cbnA promoter without the inducer, suggesting that these mutations brought about a conformational change mimicking that induced by the inducer molecule.

Crystal structure of the DNA‐binding domain of the LysR‐type transcriptional regulator CbnR in complex with a DNA fragment of the recognition‐binding site in the promoter region

“…S1). This result is consistent with our earlier mutational analysis, in which Gln29 in CbnR was shown to have no impact on the DNAbinding activity; Gln29Ala retained the cbnA promoter-binding activity [36].…”

“…Interestingly, as described in our earlier report, the affinity for RBS does not directly relate to the transcriptional activation activity of CbnR (or its mutant) [36], suggesting a multistep mechanism of the transcription activation by LTTRs [15,17,30]. In particular, the results of mutational analysis on Gln29 are enigmatic.…”

“…D). Thr33Ala, which lacks the promoter‐binding activity, could not activate the transcription of the cbnA promoter . Similarly, wild‐type CbnR, Thr33Ala, and Thr33Ser all failed to bind to the benA chimera promoter and could not activate its transcription (Fig.…”

“…As shown in Fig. B, this residue is not directly involved in the interaction with DNA, and its mutant proteins exhibit only limited differences by EMSA . However, the inducer was unable to activate transcription of most of the mutant proteins of Gln29.…”

Functional definition of the two effector binding sites, the oligomerization and DNA binding domains of the Bacillus subtilis LysR‐type transcriptional regulator AlsR

“…In agreement, AlsR carrying a S33 similar to BenM revealed BenM like RBS binding. However, similar behavior of AlsR and CbnR was observed for the mutation of the conserved R4, P30 and E40 (Moriuchi et al ., ).…”