Abstract：
: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, Sec-tRNA(Sec), the Sec-specific elongation factor eEFSec, and SECIS binding protein 2. 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-tRNA(Sec) 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.
journal_name
J Mol Bioljournal_title
Journal of molecular biologyauthors
Gupta N,DeMong LW,Banda S,Copeland PRdoi
10.1016/j.jmb.2013.04.016subject
Has Abstractpub_date
2013-07-24 00:00:00pages
2415-22issue
14eissn
0022-2836issn
1089-8638pii
S0022-2836(13)00264-7journal_volume
425pub_type
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pub_type： 杂志文章
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pub_type： 杂志文章
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pub_type： 杂志文章,评审
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更新日期：2015-06-05 00:00:00
