Reconstitution of Selenocysteine Incorporation Reveals Intrinsic Regulation by SECIS Elements

JOURNAL OF MOLECULAR BIOLOGY

卷:425

Article

Gupta, Nirupama¹  DeMong, Louise W.¹  Banda, Sowmya¹  Copeland, Paul R.¹ 

[1] Univ Med & Dent New Jersey, Robert Wood Johnson Med Sch, Piscataway, NJ 08854 USA

关键词: ribosome;    translation elongation;    selenocysteine;    SECIS;   

DOI  :  10.1016/j.jmb.2013.04.016

来源: Elsevier

【 摘 要 】

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. (C) 2013 Elsevier Ltd. All rights reserved.

【 授权许可】

Free