Abstract
Sec-tRNA(Sec) is site-specifically delivered at defined UGA codons in selenoprotein mRNAs. This recoding event is specified by the selenocysteine insertion sequence (SECIS) element and requires the selenocysteine (Sec)-specific elongation factor, eEFSec, and the SECIS binding protein, SBP2. Sec-tRNA(Sec) is delivered to the ribosome by eEFSec-GTP, but this ternary complex is not sufficient for Sec incorporation, indicating that its access to the ribosomal A-site is regulated. SBP2 stably associates with ribosomes, and mutagenic analysis indicates that this interaction is essential for Sec incorporation. However, the ribosomal function of SBP2 has not been elucidated. To shed light on the functional relevance of the SBP2-ribosome interaction, we screened the functional centers of the 28 S rRNA in translationally competent 80 S ribosomes using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). We demonstrate that SBP2 specifically alters the reactivity of specific residues in Helix 89 (H89) and expansion segment 31 (ES31). These results are indicative of a conformational change in response to SBP2 binding. Based on the known functions of H89 during translation, we propose that SBP2 allows Sec incorporation by either promoting Sec-tRNA(Sec) accommodation into the peptidyltransferase center and/or by stimulating the ribosome-dependent GTPase activity of eEFSec.
Highlights
Selenocysteine incorporation requires unique translation factors that interact with the ribosome
3 The abbreviations used are: aa-tRNA, aminoacyl-tRNA; Benzoyl cyanide (BzCN), benzoyl cyanide; CTSBP2, C-terminal SBP2; eEFSec, eukaryotic elongation factor for selenocysteine; DMSO, dimethyl sulfoxide; EF, elongation factor; ES31, expansion segment 31; GAC, GTPase-associated center; H, Helix; PTC, peptidyltransferase center; RBD, RNA binding domain; SBP2, SECIS-binding protein 2; Sec, selenocysteine; SECIS, selenocysteine insertion sequence; Ser-tRNASec, serylated Sec tRNA; selective 2-hydroxyl acylation analyzed by primer extension (SHAPE), selective 2Ј-hydroxyl acylation analyzed by primer extension; SID, Sec incorporation domain; sarcin-ricin loop (SRL), sarcinricin loop; TC, ternary complex
Using selective 2Ј-hydroxyl acylation analyzed by primer extension (SHAPE), we demonstrate that CTSBP2 enhances the reactivity of residues U4071, and C4073 in the apical loop of Helix 89 (H89), and between G3802 and C3812, and G3758 and U3768 on expansion segment 31 (ES31; mouse numbering)
Summary
Selenocysteine incorporation requires unique translation factors that interact with the ribosome. Results: SECIS-binding protein 2 alters ribosome conformation at two discrete sites. Significance: Identifying the ribosomal dynamics required for Sec incorporation will enhance our understanding of the translation elongation reaction. We demonstrate that SBP2 alters the reactivity of specific residues in Helix 89 (H89) and expansion segment 31 (ES31). These results are indicative of a conformational change in response to SBP2 binding. Based on the known functions of H89 during translation, we propose that SBP2 allows Sec incorporation by either promoting Sec-tRNASec accommodation into the peptidyltransferase center and/or by stimulating the ribosome-dependent GTPase activity of eEFSec
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