Protein splicing involving the Saccharomyces cerevisiae VMA intein - The steps in the splicing pathway, side reactions leading to protein cleavage, and establishment of an in vitro splicing system
Protein splicing involving the Saccharomyces cerevisiae VMA intein - The steps in the splicing pathway, side reactions leading to protein cleavage, and establishment of an in vitro splicing system
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DOI：
10.1074/jbc.271.36.22159
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发表时间：
1996-09-06
影响因子：
4.8
通讯作者：
Xu, MQ
中科院分区：
文献类型：
--
作者：
Chong, SR;Shao, Y;Xu, MQ
蛋白质剪接涉及从前体蛋白质中切除内部蛋白质片段（内含肽），并伴随连接侧翼N-和C-末端区域。它存在于嗜温细菌、酵母和嗜热古菌中。在外源蛋白背景下，通过温度和pH控制嗜热内含肽的蛋白质剪接的能力促进了嗜热菌中蛋白质剪接机制的研究。另一方面，没有直接的研究已经做了蛋白质剪接在嗜温细胞的机制。我们研究了含有内含肽的空泡ATP酶亚基（VMA）的酿酒酵母，涉及半胱氨酸，而不是丝氨酸在反应中心的嵌合蛋白的剪接。剪接过程中的步骤是通过分析中间体和副产物来推断的，这些中间体和副产物是由于氨基酸取代而积累的，并且被发现与嗜热菌中发生的那些类似。此外，适当的氨基酸替换使我们能够开发第一个嗜温体外蛋白质剪接系统以及用于调节蛋白质剪接速率和用于将剪接反应转化为在任一剪接点处的有效蛋白质切割反应的策略。
Protein splicing involves the excision of an internal protein segment, the intein, from a precursor protein and the concomitant ligation of the flanking N- and C-terminal regions. It occurs in mesophilic bacteria, yeast, and thermophilic archaea. The ability to control protein splicing of a thermophilic intein by temperature and pH in a foreign protein context facilitated the study of the mechanism of protein splicing in thermophiles. On the other hand, no direct studies have been done on the mechanism of protein splicing in mesophiles. We examined the splicing of a chimeric protein containing the intein of the vacuolar ATPase subunit (VMA) of Saccharomyces cerevisiae that involves cysteines rather than serines at the reaction center. The steps in the splicing process were deduced by analyzing intermediates and side products that accumulated as a result of amino acid substitutions and were found to be analogous to those occurring in thermophiles. Moreover, appropriate amino acid replacements allowed us to develop the first mesophilic in vitro protein splicing system as well as strategies for modulating the rate of protein splicing and for converting the splicing reaction to an efficient protein cleavage reaction at either splice junction.