Rutherford J C, Cavet J S, Robinson N J
Biochemistry and Genetics, The Medical School, University of Newcastle, NE2 4HH, United Kingdom.
J Biol Chem. 1999 Sep 3;274(36):25827-32. doi: 10.1074/jbc.274.36.25827.
CoaR associates with and confers cobalt-dependent activation of the coaT operator-promoter. A CoaR mutant (Ser-Asn-Ser) in a carboxyl-terminal Cys-His-Cys motif bound the coaT operator-promoter but did not activate expression in response to cobalt, implicating thiolate and/or imidazole ligands at these residues in an allosteric cobalt binding site. Deletion of 1 or 2 nucleotides from between near consensus, but with aberrant (20 base pairs) spacing, -10 and -35 elements enhanced expression from the coaT operator-promoter but abolished activation by cobalt-CoaR. It is inferred that cobalt effects a transition in CoaR that underwinds the coaT operator-promoter to realign promoter elements. In the absence of cobalt, CoaR represses expression (approximately 50%). CoaR is a fusion of ancestral MerR (mercury-responsive transcriptional activator)- and precorrin isomerase (enzyme of vitamin B(12) biosynthesis)-related sequences. Expression from the coaT operator-promoter was enhanced in a partial mutant of cbiE (encoding an enzyme preceding precorrin isomerase in B(12) biosynthesis), revealing that this pathway "inhibits" coaT expression. Disruption of coaT reduced cobalt tolerance and increased cytoplasmic (57)Co accumulation. coaT-mediated restoration of cobalt tolerance has been used as a selectable marker.
CoaR与coaT操纵子启动子结合并赋予其钴依赖性激活。羧基末端Cys-His-Cys基序中的CoaR突变体（Ser-Asn-Ser）结合coaT操纵子启动子，但不响应钴激活表达，这表明这些残基处的硫醇盐和/或咪唑配体存在于变构钴结合位点中。从接近共有序列但间距异常（20个碱基对）的-10和-35元件之间缺失1或2个核苷酸，增强了coaT操纵子启动子的表达，但消除了钴-CoaR的激活作用。据推测，钴会导致CoaR发生转变，使coaT操纵子启动子解旋以重新排列启动子元件。在没有钴的情况下，CoaR抑制表达（约50%）。CoaR是祖先MerR（汞响应转录激活因子）和前咕啉异构酶（维生素B12生物合成酶）相关序列的融合体。cbiE（编码B12生物合成中前咕啉异构酶之前的一种酶）的部分突变体中，coaT操纵子启动子的表达增强，这表明该途径“抑制”coaT表达。破坏coaT会降低钴耐受性并增加细胞质中（57）Co的积累。coaT介导的钴耐受性恢复已被用作选择标记。
