Formation of repressor-inducer-operator ternary complex: negative cooperativity of d-camphor binding to CamR

Abstract

A repressor composed of homodimeric subunits, as is often found in bacteria, possesses two effector-binding sites per molecule, enabling sophisticated regulation by the cooperative binding of two effector molecules. Positive cooperativity generates a narrower region of effector concentration for switching, but little is known about the role of negative cooperativity. D-camphor, an inducer for Pseudomonas putida cytochrome P450cam hydroxylase operon (camDCAB), binds to the homodimeric cam repressor (CamR). Here, we report solid evidence that the complex of CamR and an operator DNA is not dissociated by the first binding of D-camphor but, at a higher concentration, is dissociated by the second binding. D-camphor thus binds to the CamR in two steps with negative cooperativity, yielding two distinct dissociation constants of K d1 = 0.064 ± 0.030 and K d2 = 14 ± 0.3 lM, as well as the Hill coefficient of 0.56 ± 0.05 (<1). The first binding guarantees the high specificity of the inducer by the high affinity, although the second binding turns on the gene expression at a 200-fold higher concentration, a more suitable switching point for the catabolism of D-camphor.

Modulation of Global Low-Frequency Motions Underlies Allosteric Regulation: Demonstration in CRP/FNR Family Transcription Factors

“…In metabolism, recent modelling suggests that there is a significant overall advantage for metabolic pathway flux with components showing negative cooperativity [51],[52]. In transcriptional regulation, negative cooperativity in the binding of D-camphor to the CamR repressor of Pseudomonas putida is proposed to enable coupling of high specificity for D-camphor with a physiological response to high concentrations of the metabolite [53]. Against this framework, it is reasonable to conjecture that negative cooperativity in CAP offers a selective advantage by increasing the concentration range over which a transcriptional response can be generated [54].…”

Cooperative Binding

“…Conversely, examples of negative cooperativity for the binding of transcription factors were also documented, as for the homodimeric repressor of the Pseudomonas putida cytochrome P450cam hydroxylase operon (n = 0.56) [35].…”

Development of a Tightly Controlled Off Switch for Saccharomyces cerevisiae Regulated by Camphor, a Low-Cost Natural Product

“…CamR, a component of cam-TA, binds to a specific sequence, 5′-CAGGCTCTATATCTGCGATATACTGAGCAT ( Fujita et al 1993 ). We assembled six repeats of the operator sequence (camO) separated by a junction sequence, 5′-CCCCC, used previously ( Aramaki et al 2011 ) between the alcohol dehydrogenase ADH1 terminator and a CYC1 (cytochrome c) promoter from which the endogenous UAS had been previously removed. The architecture, consisting of a terminator, an operator, and a UAS-less promoter, was analogous to a promoter used in the yeast Tet-system ( Gari et al 1997 ).…”