Reconstitution of selenocysteine incorporation reveals intrinsic regulation by SECIS elements.
Reconstitution of selenocysteine incorporation reveals intrinsic regulation by SECIS elements.
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DOI：
10.1016/j.jmb.2013.04.016
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发表时间：
2013-07-24
影响因子：
5.6
通讯作者：
Copeland, Paul R.
中科院分区：
文献类型：
--
作者：
Gupta, Nirupama;DeMong, Louise W.;Banda, Sowmya;Copeland, Paul R.
硒蛋白存在于生命的所有三个领域，并负责细胞对活性氧的抗氧化防御的主要部分。硒蛋白的合成需要将UGA密码子解码为硒代半胱氨酸（Sec）而不是翻译终止。Sec在翻译延伸过程中掺入生长的多肽链中，并且已知需要一组高度特异性的因子：3′非翻译区（3′ UTR）中的Sec插入序列（SECIS）元件、Sec-tRNASec、Sec特异性延伸因子eEFSec和SECIS结合蛋白2（SBP 2）。由于尚未报告复溶，因此尚不清楚这些因素是否足够。在这里，我们报告了一种新的体外翻译系统，其中Sec掺入已重建从纯化的组分引入到Sec幼稚系统。此外，我们还建立了一种新的纯化Sec-tRNASec和活性eEFSec/GTP/tRNA三元复合物的方法。我们发现，已知的基础因素是足够的Sec在体外掺入。使用这种高度可操作的系统，我们还发现，核糖体从非Sec利用生物体不能支持Sec的掺入，一些SECIS元素本质上是效率较低的比别人。在确定了一组重要的因素，这项工作消除了一个重大的障碍，我们的理解的机制，证券交易委员会成立。
Selenoproteins are present in all three domains of life and are responsible for a major part of a cell’s antioxidant defense against reactive oxygen species. Synthesis of selenoproteins requires the decoding of a UGA codon as selenocysteine (Sec) instead of translation termination. Sec is incorporated into the growing polypeptide chain during translation elongation and is known to require a set of highly specific factors: The Sec insertion sequence (SECIS) element in the 3′ untranslated region (3′ UTR), Sec-tRNASec, the Sec-specific elongation factor eEFSec, and SECIS binding protein 2 (SBP2). Since reconstitution has not been reported, whether these factors are sufficient is unknown. Here we report a novel in vitro translation system in which Sec incorporation has been reconstituted from purified components introduced into a Sec naive system. In addition, we developed a novel method to purify Sec-tRNASec and active eEFSec/GTP/tRNA ternary complex. We found that the known basal factors are sufficient for Sec incorporation in vitro. Using this highly manipulable system, we have also found that ribosomes from non-Sec utilizing organisms cannot support Sec incorporation and that some SECIS elements are intrinsically less efficient than others. Having identified the essential set of factors, this work removes a significant barrier to our understanding of the mechanism of Sec incorporation.
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影响因子：
11.4
作者：
Fagegaltier, D;Hubert, N;Krol, A
通讯作者：
Krol, A
影响因子：
14.9
作者：
Fagegaltier, D;Lescure, A;Krol, A
通讯作者：
Krol, A
DOI：
10.1093/bioinformatics/btp020
发表时间：
2009-03-01
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Chapple CE;Guigó R;Krol A
通讯作者：
Krol A
影响因子：
56.9
作者：
Kryukov, GV;Castellano, S;Gladyshev, VN
通讯作者：
Gladyshev, VN
影响因子：
4.8
作者：
Copeland, PR;Driscoll, DM
通讯作者：
Driscoll, DM