科研之友
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摘要
We report a cadmium- and lead-detecting transcriptional repressor from Mycobacterium tuberculosis designated CmtR. Two genes were co-transcribed with cmtR, one encoding a deduced P-1 type ATPase. Purified CmtR bound to the cmt operator-promoter, and repression of transcription was lost after introduction of a stop codon into cmtR. Assays of metal-dependent expression from cmt and nmt operator-promoters established that the metal specificity of CmtR in vivo was perfectly inverted relative to the nickel-cobalt sensor NmtR from the same organism, with CmtR totally insensitive to Co(II) or Ni(II) and NmtR totally insensitive to Cd(II) or Pb(II). Absorption spectroscopy of Cd(II)-, Co( II)-, and Ni( II)substituted CmtR revealed S- to metal-charge-transfer which was absent in NmtR, providing diagnostic metal-difference spectra that discriminated between metal-binding to these two proteins. Ni( II)- binding isothermal titrations of CmtR are complex, with K-app = 1.8 x 10(4) M-1 for site(1), three orders of magnitude weaker than K-Ni for NmtR. Mixing equimolar apo-NmtR and apo-CmtR with 0.9 equivalents of Cd( II) gave Cd( II)- dependent difference spectra almost identical to Cd( II)(0.9)-CmtR. Thus, Cd( II) bound to CmtR in preference to NmtR, whereas the converse was true for Ni( II); this correlates faithfully with and provides a simplistic basis for metal-sensing preferences. In contrast, CmtR and NmtR had similar affinities for Co( II), and alternative explanations for Co(II) sensitivities are invoked. ArsR-SmtB repressors detect metals through derivatives of one or both of two possible allosteric sites at either carboxyl-terminal alpha5 helices or helix alpha3 proximal to the DNA-binding site. Unexpectedly, neither site was required for inducer recognition by CmtR. The mutants in potential metal ligands in, or near, these regions, Cys(4), Cys(35), Asp(79), His(81), Asp(97), Asp(99), Glu(105), Glu(111), and Glu(114), retained both repression and inducer recognition. Crucially, substitution of Cys(57), Cys(61), and Cys(102) with Ser revealed that each of these three residues is obligatory for Cd( II) detection, and this defines completely new sensory sites.
- 出版日期2003-11-7
