Structural basis for operator and antirepressor recognition by Myxococcus xanthus CarA repressor

Blue light induces carotenogenesis in Myxococcus xanthus. The carB operon encodes all but one of the structural genes involved, and its expression is regulated by the CarA-CarS repressor-antirepressor pair. In the dark, CarA-operator binding represses carB. CarS, produced on illumination, interacts physically with CarA to dismantle the CarA-operator complex and activate carB. Both operator and CarS bind to the autonomously folded N-terminal domain of CarA, CarA(Nter), which in excess represses carB. Here, we report the NMR structure of CarA(Nter), and map residues that interact with operator and CarS by NMR chemical shift perturbations, and in vivo and in vitro analyses of site-directed mutants. We show CarA(Nter) adopts the winged-helix topology of MerR-family DNA-binding domains, and conserves the majority of the helix-turn-helix and wing contacts with DNA. Tellingly, helix alpha2 in CarA, a key element in operator DNA recognition, is also critical for interaction with CarS, implying that the CarA-CarS protein-protein and the CarA-operator protein-DNA interfaces overlap. Thus, binding of CarA to operator and to antirepressor are mutually exclusive, and CarA may discern structural features in the acidic CarS protein that resemble operator DNA. Repressor inactivation by occluding the DNA-binding region may be a recurrent mechanism of action for acidic antirepressors.     

The N terminus of Myxococcus xanthus CarA repressor is an autonomously folding domain that mediates physical and functional interactions with both operator DNA and antirepressor protein

Expression of the Myxococcus xanthus carB operon, which encodes the majority of the enzymes involved in light-induced carotenogenesis, is down-regulated in the dark by the CarA repressor binding to its bipartite operator. CarS, produced on illumination, relieves repression of carB by physically interacting with CarA to dis-mantle CarA-DNA complexes. Here, we demonstrate that the N- and C-terminal portions of CarA are organized as distinct structural and functional domains. Specifically, we show that the 78 N-terminal residues of CarA, CarA(Nter), form a monomeric, highly helical, autonomously folding unit with significant structural stability. Significantly, CarA(Nter) houses both the operator and CarS binding specificity determinants of CarA. CarA(Nter) binds operator with a lower affinity than whole CarA, and the CarA(Nter)-CarS complex has a 1:1 stoichiometry. In vitro, sufficiently high concentrations of CarA(Nter) block M. xanthus RNA polymerase-promoter binding, and this is relieved by CarS. In vivo, substitution of the gene carA by that for CarA(Nter) results in constitutive expression of carB just as in a carA-deleted background. However, re-engineering the latter strain to overexpress CarA(Nter) restores repression of carB. Thus, the 78-residue N-terminal portion of CarA is an autonomously folded, dual function domain that orchestrates specific DNA-protein and protein-protein interactions and, when overexpressed, can be functionally competent in vivo.     

Light-induced carotenogenesis in Myxococcus xanthus: evidence that CarS acts as an anti-repressor of CarA

In the bacterium Myxococcus xanthus, carotenoids are produced in response to illumination, as a result of expression of the crt carotenoid biosynthesis genes. The majority of crt genes are clustered in the crtEBDC operon, which is repressed in the dark by CarA. Genetic data suggest that, in the light, CarS is synthesized and achieves activation of the crtEBDC operon by removing the repressive action of CarA. As CarS contains no known DNA-binding motif, the relief of CarA-mediated repression was postulated to result from a direct interaction between these two proteins. Use of the yeast two-hybrid system demonstrated direct interaction between CarA and CarS. The two-hybrid system also implied that CarA and, possibly, CarS are capable of homodimerization. Direct evidence for CarS anti-repressor action was provided in vitro. A glutathione S-transferase (GST)-CarA protein fusion was shown to bind specifically to a palindromic operator sequence within the crtEBDC promoter. CarA was prevented from binding to its operator, and prebound CarA was removed by the addition of purified CarS. CarS is therefore an anti-repressor.     

1H, 13C and 15N backbone and side chain resonance assignments of a Myxococcus xanthus anti-repressor with no known sequence homologues

The CarS antirepressor activates a photo-inducible promoter in Myxococcus xanthus by physically interacting with the CarA repressor and eliminating the latter’s binding to operator DNA. Interestingly, interactions with both CarS and operator are crucially dependent on the DNA recognition helix of the CarA winged-helix DNA-binding domain. The CarA–CarS and the CarA-operator interfaces therefore overlap, and CarS may have structural features that mimic operator DNA. CarS has no known sequence homologues and its Gly and Pro contents are unusually high. Here, we report ¹H, ¹³C and ¹⁵N backbone and side chain assignments of CarS1, an 86-residue truncated yet fully functional variant of CarS. Secondary structural elements inferred from these data differ from those predicted from sequence.     

HxlR, a member of the DUF24 protein family, is a DNA-binding protein that acts as a positive regulator of the formaldehyde-inducible hxlAB operon in Bacillus subtilis

The HxlR protein from Bacillus subtilis belongs to the DUF24 protein family (InterPro No. IPR002577) of unknown function. The hxlR gene that encodes this protein is located upstream of the hxlAB operon. This operon encodes two key enzymes in the ribulose monophosphate pathway that are involved in formaldehyde fixation, 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. Expression of the hxlAB operon is induced by the presence of formaldehyde. Recombinant HxlR prepared from Escherichia coli showed specific binding to a region of DNA upstream of the hxlAB operon. Using gel-retardation and DNase I footprinting assays, we identified two 25 bp binding regions for HxlR within the upstream DNA. Surface plasmon resonance analyses suggested that two HxlR dimers sequentially bound to the DNA. Finally, we demonstrated that each of the two binding regions for HxlR was necessary for formaldehyde-induced expression of the hxlAB operon in B. subtilis. Thus, we have shown that HxlR is a DNA-binding protein that is necessary for formaldehyde-induced expression of hxlAB in B. subtilis.     

Regulation of open complex formation at the Escherichia coli galactose operon promoters. Simultaneous interaction of RNA polymerase, gal repressor and CAP/cAMP.

The regulation of open complex formation at the Escherichia coli galactose operon promoters by galactose repressor and catabolite activator protein/cyclic AMP (CAP/cAMP) was investigated in DNA-binding and kinetic experiments performed in vitro. We found that gal repressor and CAP/cAMP bind to the gal regulatory region independently, resulting in simultaneous occupancy of the two gal operators and the CAP/cAMP binding site. Both CAP/cAMP and gal repressor altered the partitioning of RNA polymerase between the two overlapping gal promoters. Open complexes formed in the absence of added regulatory proteins were partitioned between gal P1 and P2 with occupancies of 25% and 75%, respectively. CAP/cAMP caused open complexes to be formed nearly exclusively at P1 (98% occupancy). gal repressor caused a co-ordinated, but incomplete, switch in promoter partitioning from P1 to P2 in both the absence and presence of CAP/cAMP. We measured the kinetic constants governing open complex formation and decay at the gal promoters in the absence and presence of gal repressor and CAP/cAMP. CAP/cAMP had the largest effect on the kinetics of open complex formation, resulting in a 30-fold increase in the apparent binding constant. We conclude that the regulation of open complex formation at the gal promoters does not result from competition between gal repressor, CAP/cAMP and RNA polymerase for binding at the gal operon regulatory region, but instead results from the interactions of the three proteins during the formation of a nucleoprotein complex on the gal DNA fragment. Finally, we present a kinetic model for the regulation of open complex formation at the gal operon.