Sarubbi E, Rudd K E, Xiao H, Ikehara K, Kalman M, Cashel M
Section on Molecular Regulation, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
J Biol Chem. 1989 Sep 5;264(25):15074-82.
The Escherichia coli spoT gene encodes a guanosine-3',5'-bispyrophosphate (ppGpp) 3'-pyrophosphohydrolase known to be responsible for cellular (ppGpp) degradation. The DNA sequence of the spoT region is presented. The spoT gene is deduced to be 702 codons long, with a probable UUG initiation codon, and a deduced mass of 79,342 daltons. Two spoT mutations (spoT202 and spoT203) have been localized to an open reading frame by complementation of function as well as by genetic marker rescue. The ability to overexpress the spoT gene is limited, but enough ppGppase activity can be made to reverse ppGpp accumulation during the stringent response to amino acid starvation. The spoT gene is located within a larger spo operon and is flanked by two smaller genes. The first gene in the operon encodes omega, a protein that copurifies with RNA polymerase (Gentry, D. R., and Burgess, R. R. (1986) Gene (Amst.) 48, 33-40). The spoT gene is the second gene in the operon; it is followed by a third open reading frame deduced to encode a protein with a mass of 25,343 daltons. Insertion of a kanamycin resistance gene in the omega gene reduces spoT gene expression as judged by lowered ppGppase activity, relA-dependent reduction of growth rate, and abolition of spoT mutant complementation activity. These effects are reversed by expression of the spoT gene, but not the omega gene, in trans. Transcription of the spo operon occurs in a clockwise direction on the E. coli chromosome and is probably directed by at least two promoters.
大肠杆菌的spoT基因编码一种鸟苷-3',5'-双焦磷酸（ppGpp）3'-焦磷酸水解酶，已知该酶负责细胞内（ppGpp）的降解。本文给出了spoT区域的DNA序列。推测spoT基因长度为702个密码子，起始密码子可能为UUG，推测分子量为79,342道尔顿。通过功能互补以及遗传标记拯救，已将两个spoT突变（spoT202和spoT203）定位到一个开放阅读框。spoT基因的过表达能力有限，但在对氨基酸饥饿的严格应答过程中，可产生足够的ppGpp酶活性来逆转ppGpp的积累。spoT基因位于一个更大的spo操纵子内，两侧是两个较小的基因。操纵子中的第一个基因编码ω蛋白，该蛋白与RNA聚合酶共纯化（Gentry, D. R., and Burgess, R. R. (1986) Gene (Amst.) 48, 33 - 40）。spoT基因是操纵子中的第二个基因；其后是第三个开放阅读框，推测编码一个分子量为25,343道尔顿的蛋白质。根据ppGpp酶活性降低、relA依赖的生长速率降低以及spoT突变体互补活性的丧失判断，在ω基因中插入卡那霉素抗性基因会降低spoT基因的表达。通过反式表达spoT基因而非ω基因，可逆转这些效应。spo操纵子在大肠杆菌染色体上按顺时针方向转录，可能由至少两个启动子指导。
