Leyh T S, Taylor J C, Markham G D
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
J Biol Chem. 1988 Feb 15;263(5):2409-16.
The sulfate activation locus of Escherichia coli K12 has been cloned by complementation. The genes and gene products of this locus have been characterized by correlating the enzyme activity, complementation patterns, and polypeptides associated with subclones of the cloned DNA. The enzymes of the sulfate activation pathway, ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) and APS kinase (ATP:adenosine-5'-phosphosulfate 3'-phosphotransferase, EC 2.7.1.25) have been overproduced approximately 100-fold. Overproduction of ATP sulfurylase requires the expression of both the cysD gene, encoding a 27-kDa polypeptide, and a previously unidentified gene, denoted cysN, which encodes a 62-kDa polypeptide. Purification of ATP sulfurylase to homogeneity reveals that the enzyme is composed of two types of subunits which are encoded by cysD and cysN. Insertion of a kanamycin resistance gene into plasmid or chromosomal cysN prevents sulfate activation and decreases expression of the downstream cysC gene. cysC appears to be the APS kinase structural gene and encodes a 21-kDa polypeptide. The genes are adjacent and are transcribed counterclockwise on the E. coli chromosome in the order cysDNC. cysN and cysC are within the same operon and cysDNC are not in an operon containing cysHIJ.
大肠杆菌K12的硫酸盐激活位点已通过互补作用克隆出来。通过将酶活性、互补模式以及与克隆DNA亚克隆相关的多肽进行关联，对该位点的基因和基因产物进行了表征。硫酸盐激活途径的酶，即ATP硫酸化酶（ATP：硫酸腺苷酰转移酶，EC 2.7.7.4）和APS激酶（ATP：腺苷-5'-磷酸硫酸3'-磷酸转移酶，EC 2.7.1.25）的产量增加了约100倍。ATP硫酸化酶产量的增加需要编码27 kDa多肽的cysD基因和一个先前未鉴定的基因（称为cysN）的表达，cysN编码62 kDa的多肽。将ATP硫酸化酶纯化至同质状态表明，该酶由两种类型的亚基组成，分别由cysD和cysN编码。将卡那霉素抗性基因插入质粒或染色体cysN中会阻止硫酸盐激活，并降低下游cysC基因的表达。cysC似乎是APS激酶结构基因，编码21 kDa的多肽。这些基因相邻，在大肠杆菌染色体上按cysDNC的顺序逆时针转录。cysN和cysC在同一个操纵子内，而cysDNC不在包含cysHIJ的操纵子中。
