Horinouchi S, Kito M, Nishiyama M, Furuya K, Hong S K, Miyake K, Beppu T
Department of Agricultural Chemistry, University of Tokyo, Japan.
Gene. 1990 Oct 30;95(1):49-56. doi: 10.1016/0378-1119(90)90412-k.
The afsR gene of Streptomyces coelicolor A3(2) complements afsB mutations affecting production of pigmented antibiotics. It also directs pigment production in Streptomyces lividans when carried on a plasmid vector. Nucleotide sequencing of the afsR gene revealed that it codes for a 993-amino acid protein (Mr 105,600) with A- and B-type ATP-binding consensus sequences at its N-terminal portion and two DNA-binding consensus sequences with a helix-turn-helix motif at its C-terminal portion. Each of the N- and C-terminal halves was capable of conferring pigment production, to some extent, in S. lividans, when carried separately on a multicopy plasmid. In addition, expression in trans of the two regions on the same plasmid conferred pigment production to almost the same extent as did the intact afsR gene. Mutations at the two ATP-binding consensus sequences, that were generated by in vitro site-directed mutagenesis, revealed their functional importance. Disruption of the S. coelicolor A3(2) chromosomal afsR gene in either the N- or C-terminal half using phage phi C31 KC515 resulted in significant, but not complete, loss of pigment production. These data suggest that the AfsR protein comprises two domains, viz., an ATP-binding and a DNA-binding domain, each of which could function as a positive regulator for pigment production. These afsR mutants sporulate normally. In addition to an internal promoter, which we previously detected in the middle of the AfsR coding region, S1 nuclease mapping revealed two tandem transcriptional start points, separated by 64 bp, upstream from a putative ATG start codon of the AfsR product.
天蓝色链霉菌A3(2)的afsR基因可弥补影响色素性抗生素产生的afsB突变。当携带在质粒载体上时，它还能指导淡青链霉菌产生色素。afsR基因的核苷酸测序表明，它编码一种993个氨基酸的蛋白质（分子量105,600），在其N端部分具有A和B型ATP结合共有序列，在其C端部分具有两个带有螺旋-转角-螺旋基序的DNA结合共有序列。N端和C端的每一半在多拷贝质粒上单独携带时，都能在一定程度上赋予淡青链霉菌产生色素的能力。此外，同一质粒上两个区域的反式表达赋予色素产生的程度与完整的afsR基因几乎相同。通过体外定点诱变产生的两个ATP结合共有序列的突变揭示了它们的功能重要性。使用噬菌体phi C31 KC515破坏天蓝色链霉菌A3(2)染色体afsR基因的N端或C端半部分，导致色素产生显著但不完全丧失。这些数据表明，AfsR蛋白包含两个结构域，即一个ATP结合结构域和一个DNA结合结构域，每个结构域都可作为色素产生的正调控因子发挥作用。这些afsR突变体正常产孢。除了我们之前在AfsR编码区中部检测到的一个内部启动子外，S1核酸酶作图还揭示了两个串联的转录起始点，它们相距64 bp，位于AfsR产物假定的ATG起始密码子上游。
