Dinçbas-Renqvist V, Engström A, Mora L, Heurgué-Hamard V, Buckingham R, Ehrenberg M
Department of Cell and Molecular Biology and Department of Medical Biochemistry and Microbiology, Uppsala University, BMC, S-751 24, Uppsala, Sweden.
EMBO J. 2000 Dec 15;19(24):6900-7. doi: 10.1093/emboj/19.24.6900.
A post-translational modification affecting the translation termination rate was identified in the universally conserved GGQ sequence of release factor 2 (RF2) from Escherichia coli, which is thought to mimic the CCA end of the tRNA molecule. It was shown by mass spectrometry and Edman degradation that glutamine in position 252 is N:(5)-methylated. Overexpression of RF2 yields protein lacking the methylation. RF2 from E.coli K12 is unique in having Thr246 near the GGQ motif, where all other sequenced bacterial class 1 RFs have alanine or serine. Sequencing the prfB gene from E.coli B and MRE600 strains showed that residue 246 is coded as alanine, in contrast to K12 RF2. Thr246 decreases RF2-dependent termination efficiency compared with Ala246, especially for short peptidyl-tRNAs. Methylation of Gln252 increases the termination efficiency of RF2, irrespective of the identity of the amino acid in position 246. We propose that the previously observed lethal effect of overproducing E.coli K12 RF2 arises through accumulating the defects due to lack of Gln252 methylation and Thr246 in place of alanine.
在大肠杆菌释放因子2（RF2）普遍保守的GGQ序列中发现了一种影响翻译终止速率的翻译后修饰，该序列被认为模拟了tRNA分子的CCA末端。质谱分析和埃德曼降解表明，第252位的谷氨酰胺发生了N:(5)-甲基化。RF2的过表达产生缺乏甲基化的蛋白质。来自大肠杆菌K12的RF2在GGQ基序附近有苏氨酸246，这一点很独特，而所有其他已测序的细菌1类RFs在此处具有丙氨酸或丝氨酸。对大肠杆菌B和MRE600菌株的prfB基因进行测序表明，与K12 RF2相反，第246位残基编码为丙氨酸。与丙氨酸246相比，苏氨酸246降低了RF2依赖性终止效率，尤其是对于短肽基-tRNA。无论第246位氨基酸的身份如何，谷氨酰胺252的甲基化都会提高RF2的终止效率。我们认为，先前观察到的过量生产大肠杆菌K12 RF2的致死效应是由于缺乏谷氨酰胺252甲基化和苏氨酸246取代丙氨酸而积累的缺陷所致。
