Metzger S, Dror I B, Aizenman E, Schreiber G, Toone M, Friesen J D, Cashel M, Glaser G
Department of Cellular Biochemistry, Hadassah Medical School, Hebrew University, Jerusalem, Israel.
J Biol Chem. 1988 Oct 25;263(30):15699-704.
The relA gene product of Escherichia coli is known to be responsible for the synthesis of guanosine 3',5'-bispyrophosphate (ppGpp) during the stringent response to amino acid starvation. This report presents the sequence of the relA gene region and assignment of its 743-codon open reading frame by the following criteria: 1) genetic complementation of ppGpp synthesis in a relaxed (relA1) mutant during the stringent response; 2) changes in 3-aminotriazole resistance during growth to mimic a relA+ phenotype; 3) verification of the presence of an amber codon at the normal carboxyl terminus of the relA gene; and 4) immunological assays of expression of the RelA protein. The apparent molecular mass of the cloned relA gene product is calculated to be 83,856 daltons and as visualized by immunoblotting is identical to that of the previously characterized protein. A promoter has been identified that directs relA gene transcription towards the pyrG gene, in a counterclockwise direction on the E. coli chromosome. Genomic Southern blot analyses verify that the relA regions cloned and subjected to nucleotide sequence analysis correspond to homologous regions on the E. coli chromosome.
已知大肠杆菌的relA基因产物在对氨基酸饥饿的严紧反应过程中负责合成鸟苷3',5'-双焦磷酸（ppGpp）。本报告给出了relA基因区域的序列，并根据以下标准确定了其743个密码子的开放阅读框：1）在严紧反应期间，对松弛型（relA1）突变体中ppGpp合成的遗传互补作用；2）生长过程中3-氨基三唑抗性的变化，以模拟relA+表型；3）验证relA基因正常羧基末端存在琥珀密码子；4）对RelA蛋白表达进行免疫测定。计算得出克隆的relA基因产物的表观分子量为83,856道尔顿，通过免疫印迹观察到其与先前鉴定的蛋白质相同。已鉴定出一个启动子，它在大肠杆菌染色体上以逆时针方向指导relA基因向pyrG基因转录。基因组Southern印迹分析证实，克隆并进行核苷酸序列分析的relA区域与大肠杆菌染色体上的同源区域相对应。
