Operator Design and Mechanism for CarA Repressor-mediated Down-regulation of the Photoinducible carB Operon in Myxococcus xanthus

Abstract

The carB operon encodes all except one of the enzymes involved in light-induced carotenogenesis in Myxococcus xanthus. Expression of its promoter (P B ) is repressed in the dark by sequence-specific DNA binding of CarA to a palindrome (pI) located between positions ؊47 and ؊64 relative to the transcription start site. This promotes subsequent binding of CarA to additional sites that remain to be defined. CarS, produced in the light, interacts physically with CarA, abrogates CarA-DNA binding, and thereby derepresses P B . In this study, we delineate the operator design that exists for CarA by precisely mapping out the second operator element. For this, we examined how stepwise deletions and site-directed mutagenesis in the region between the palindrome and the transcription start site affect CarA binding around P B in vitro and expression of P B in vivo. These revealed the second operator element to be an imperfect interrupted palindrome (pII) spanning positions ؊26 to ؊40. In vitro assays using purified M. xanthus RNA polymerase showed that CarA abolishes P B -RNA polymerase binding and runoff transcription and that both were restored by CarS, thus rationalizing the observations in vivo. CarA binding to pII (after association with pI) effectively occludes RNA polymerase from P B and so provides the operative mechanism for the repression of the carB operon by CarA. The bipartite operator design, whereby transcription is blocked by the low affinity CarA-pII binding and is readily restored by CarS, may have evolved to match the needs for a rapid and an effective response to light.

Light-dependent gene regulation by a coenzyme B 12 -based photoreceptor

“…S2D). In DNase I footprinting, CarA protects a stretch extending from the −19 to −70 positions relative to the transcription start site, with hypersensitivities at −55 and −63 (27). AdoB 12 enabled CTt1 and, more markedly, CTt2 to protect against DNase I the same operator stretch mapped for CarA (Fig.…”

“…CarA binds cooperatively to its operator, first to a high-affinity, palindromic site upstream of P B and then to a low-affinity site straddling the −35 region of P B to block access to RNA polymerase (Fig. S2A) (27). This is observed in EMSA by the progressive appearance of a lower band followed by an upper band at higher CarA concentrations (Fig.…”

A Vitamin B12-Based System for Conditional Expression RevealsdksATo Be an Essential Gene inMyxococcus xanthus

“…In the dark, P B is repressed by CarA or CarH-B 12 binding to a bipartite operator that consists of a high-affinity site pI (a perfect interrupted palindrome located upstream of P B ) and a low-affinity site pII (a pI-like pseudopalindrome that straddles the Ϫ35 region of P B ) (30). CarS, produced on illumination, relieves repression of P B by physically interacting with CarA or CarH to dismantle repressoroperator DNA complexes ( Fig.…”

“…In the pull-down assay to confirm interaction between DksA and M. xanthus RNA polymerase (obtained as described previously [30]), 200 l of Talon in two 2-ml SigmaPrep (Sigma-Aldrich) columns was washed five times with 600 l of low-salt buffer (buffer A plus 50 mM NaCl). A total of 400 l of His 6 -DksA (ϳ25 nmol) was applied to one column but not to the other, which served as a control.…”

“…In addition to repression by B 12 and CarH, expression of P B is also repressed in the dark by CarA, another MerR-type transcriptional factor remarkably similar to CarH in domain organization but whose action does not depend on B 12 . CarH (in the presence of B 12 ) and CarA bind to the same bipartite operator to impede promoter access to RNA polymerase (RNAP) and thereby repress P B (29,30,44,45). RNAP holoenzyme containing the extracytoplasmic function factor CarQ, together with a host of transcriptional factors (11), drives expression of the carQRS operon to produce CarS, an antirepressor that physically binds to CarA and CarH to derepress P B in the light.…”