Crombie T, Swaffield J C, Brown A J
University of Aberdeen Marischal College, U.K.
J Mol Biol. 1992 Nov 5;228(1):7-12. doi: 10.1016/0022-2836(92)90486-4.
Previous studies have proposed that specific translational pauses have evolved to promote protein folding inside the cell by temporally separating the folding of specific regions of some polypeptide chains during their synthesis. Here we show that this is the case for a bifunctional protein in Saccharomyces cerevisiae. The yeast TRP3 gene contains a translational pause comprising ten contiguous non-preferred codons within its second functional domain (indoleglycerol phosphate synthase). Site-directed mutagenesis was used to remove this translational pause by increasing the codon bias of the region without changing the amino acid sequence of the protein (to create the gene TRP3pr: pause replaced). The TRP3pr gene was able to complement a trp3:: URA3 null mutation in yeast. No significant differences in the doubling times of TRP3 or TRP3pr yeast transformants were observed during growth at 25 degrees C, 30 degrees C or 37 degrees C, or in the presence of sublethal concentrations of the analogue, 5-methyltryptophan. However, further analysis of TRP3 and TRP3pr transformants revealed that the removal of the translational pause causes a 1.5-fold decrease in indoleglycerol phosphate synthase activity per TRP3 mRNA. This observation which is statistically significant (P < 0.05) and reproducible, suggests that translational pausing promotes the correct intracellular folding of the TRP3 protein.
以往的研究提出，特定的翻译暂停已经进化出来，通过在某些多肽链合成过程中暂时分离其特定区域的折叠，来促进细胞内的蛋白质折叠。在这里我们表明，酿酒酵母中的一种双功能蛋白就是这种情况。酵母TRP3基因在其第二个功能域（吲哚甘油磷酸合酶）内包含一个由十个连续的非优选密码子组成的翻译暂停。通过增加该区域的密码子偏好性而不改变蛋白质的氨基酸序列，使用定点诱变来消除这种翻译暂停（以创建TRP3pr基因：暂停被替换）。TRP3pr基因能够弥补酵母中的trp3::URA3无效突变。在25℃、30℃或37℃生长期间，或在存在亚致死浓度的类似物5-甲基色氨酸的情况下，未观察到TRP3或TRP3pr酵母转化体的倍增时间有显著差异。然而，对TRP3和TRP3pr转化体的进一步分析表明，翻译暂停的消除导致每个TRP3 mRNA的吲哚甘油磷酸合酶活性降低1.5倍。这一具有统计学显著性（P<0.05）且可重复的观察结果表明，翻译暂停促进了TRP3蛋白在细胞内的正确折叠。
