Kudlicki W, Odom O W, Kramer G, Hardesty B, Merrill G A, Horowitz P M
Department of Chemistry and Biochemistry, University of Texas, Austin 78712, USA.
J Biol Chem. 1995 May 5;270(18):10650-7. doi: 10.1074/jbc.270.18.10650.
Two lines of evidence indicate the importance of the N-terminal portion of rhodanese for correct folding of the nascent ribosome-bound polypeptide. A mutant gene lacking the codons for amino acids 1-23 of the wild-type protein is expressed very efficiently by coupled transcription/translation on Escherichia coli ribosomes; however, the mutant protein that is released from the ribosomes is enzymatically inactive. The mutant protein does not undergo the reaction that is promoted by the bacterial chaperone, DnaJ, which appears to be essential for folding of ribosome-bound rhodanese into the native conformation. The effect of DnaJ is monitored by fluorescence from coumarin cotranslationally incorporated at the N terminus of nascent rhodanese. Secondly, a synthetic peptide corresponding to the N-terminal 17 amino acids of the wild-type protein interferes with the synthesis of wild-type rhodanese but has much less effect on the synthesis of the N-terminal deletion mutant. The N-terminal peptide inhibits the effect of DnaJ on the nascent wild-type rhodanese and blocks the chaperone-mediated release and activation of ribosome-bound full-length rhodanese polypeptides that accumulate during in vitro synthesis. The results lead to the hypothesis that the N-terminal segment of rhodanese is required for its chaperone-dependent folding on the ribosome.
有两条证据表明硫氧还蛋白的N端部分对于新生的核糖体结合多肽的正确折叠很重要。一个缺少野生型蛋白第1至23位氨基酸密码子的突变基因，通过在大肠杆菌核糖体上进行的偶联转录/翻译能非常高效地表达；然而，从核糖体释放出来的突变蛋白没有酶活性。该突变蛋白不会发生由细菌伴侣蛋白DnaJ促进的反应，而DnaJ对于核糖体结合的硫氧还蛋白折叠成天然构象似乎至关重要。DnaJ的作用通过共翻译掺入新生硫氧还蛋白N端的香豆素发出的荧光来监测。其次，一个对应于野生型蛋白N端17个氨基酸的合成肽会干扰野生型硫氧还蛋白的合成，但对N端缺失突变体的合成影响小得多。该N端肽抑制DnaJ对新生野生型硫氧还蛋白的作用，并阻断伴侣蛋白介导的在体外合成过程中积累的核糖体结合全长硫氧还蛋白多肽的释放和激活。这些结果引出了一个假说，即硫氧还蛋白的N端片段是其在核糖体上依赖伴侣蛋白折叠所必需的。
