Initiation of protein synthesis from a termination codon.
Initiation of protein synthesis from a termination codon.
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从终止密码子开始蛋白质合成。
DOI：
10.1073/pnas.87.4.1586
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发表时间：
1990
影响因子：
11.1
通讯作者：
RajBhandary,UL
中科院分区：
文献类型：
--
作者：
Varshney,U;RajBhandary,UL
我们发现，琥珀终止密码子UAG可以启动蛋白质合成在大肠杆菌。我们将氯霉素乙酰转移酶（CAT）基因的起始密码子AUG突变为UAG（CATam 1），并在大肠杆菌中翻译来自突变CAT基因的mRNA。coli S-30提取物。在tRNA（fMetCUA）存在下合成全长CAT多肽，突变的E.大肠杆菌起始子tRNA，其反密码子序列从CAU变为CUA。添加纯化的E.大肠杆菌氨酰-tRNA合成酶基本上刺激CAT多肽的合成。因此，用UAG和tRNA（fMetCUA）启动蛋白质合成最可能发生在谷氨酰胺而不是甲硫氨酸。UAG密码子还启动体内蛋白质合成。为了消除第五密码子AUC的弱二级起始位点，我们进一步在密码子2（GAG-GAC）和5（AUC-ACC）处诱变CATam 1基因。转化大肠用所得到的CATam 1.2.5基因转化大肠杆菌，产生了合成CAT多肽的转化子，并且只有当它们也用突变体tRNA（fMetCUA）基因转化时才对氯霉素具有抗性。从转化细胞提取物中的CAT酶活性的免疫印迹分析和测定表明，从UAG起始是有效的，从AUC获得的60-70%。使用突变体起始剂tRNA从UAG起始蛋白质合成允许严格调控特定基因的表达。这可能对E.大肠杆菌和其他真细菌的蛋白质，这些蛋白质对这些细胞有毒。
We show that the amber termination codon UAG can initiate protein synthesis in Escherichia coli. We mutated the initiation codon AUG of the chloramphenicol acetyltransferase (CAT) gene to UAG (CATam1) and translated mRNA derived from the mutant CAT gene in E. coli S-30 extracts. A full-length CAT polypeptide was synthesized in the presence of tRNA(fMetCUA), a mutant E. coli initiator tRNA which has a change in the anticodon sequence from CAU to CUA. Addition of purified E. coli glutaminyl-tRNA synthetase substantially stimulated synthesis of the CAT polypeptide. Thus, initiation of protein synthesis with UAG and tRNA(fMetCUA) most likely occurs with glutamine and not methionine. The UAG codon also initiates protein synthesis in vivo. To eliminate a weak secondary site of initiation from AUC, the fifth codon, we further mutagenized the CATam1 gene at codons 2 (GAG----GAC) and 5 (AUC----ACC). Transformation of E. coli with the resultant CATam1.2.5 gene yielded transformants that synthesized CAT polypeptide and were resistant to chloramphenicol only when they were also transformed with the mutant tRNA(fMetCUA) gene. Immunoblot analyses and assays for CAT enzyme activity in extracts from transformed cells indicate that initiation from UAG is efficient, 60-70% of that obtained from AUG. Initiation of protein synthesis from UAG using a mutant initiator tRNA allows tightly regulated expression of specific genes. This may be generally useful for overproduction in E. coli and other eubacteria of proteins which are toxic to these cells.