Kudlicki W, Chirgwin J, Kramer G, Hardesty B
Department of Chemistry and Biochemistry, University of Texas at Austin 78712, USA.
Biochemistry. 1995 Nov 7;34(44):14284-7. doi: 10.1021/bi00044a003.
Rhodanese bound to bacterial ribosomes as peptidyl-tRNA can be folded into an enzymatically active conformation by generating C-terminal extensions of the wild-type enzyme. Rhodanese was synthesized by coupled transcription/translation in a cell-free Escherichia coli system from plasmids containing the coding sequences for the wild-type enzyme or its C-terminally extended mutants. Two proteins with extensions of 23 amino acids or longer were enzymatically active while bound to the ribosomes whereas wild-type protein and a 13-amino acid extension were not. All forms of the enzyme were active after termination and release of the full-length protein from the ribosomes. All five of the bacterial chaperones were required to substantially increase the specific enzymatic activity of the extended rhodanese while the nascent protein was bound to ribosomes. The results provide direct support for the hypothesis that proteins acquire tertiary structure as they are formed in ribosomes.
作为肽基 - tRNA与细菌核糖体结合的硫氰酸酶，可以通过生成野生型酶的C末端延伸片段折叠成具有酶活性的构象。硫氰酸酶是在无细胞大肠杆菌系统中通过偶联转录/翻译从含有野生型酶或其C末端延伸突变体编码序列的质粒合成的。两种具有23个或更长氨基酸延伸的蛋白质在与核糖体结合时具有酶活性，而野生型蛋白质和一个13个氨基酸的延伸片段则没有。所有形式的酶在全长蛋白质从核糖体终止和释放后都具有活性。当新生蛋白质与核糖体结合时，所有五种细菌伴侣蛋白都是大幅提高延伸硫氰酸酶比酶活性所必需的。这些结果为蛋白质在核糖体中形成时获得三级结构这一假说提供了直接支持。
