A Novel Protein Domain Induces High Affinity Selenocysteine Insertion Sequence Binding and Elongation Factor Recruitment

Donovan, Jesse; Caban, Kelvin; Ranaweera, Ruchira S.; González-Flores, Jonathan; Copeland, Paul R.

doi:10.1074/jbc.m806008200

Cited by 54 publications

(81 citation statements)

References 21 publications

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“…33,34 As shown in Figure 1B, maximal luciferase activity was obtained using 80 nM purified bacterially expressed SBP2-CT, which is comparable to reports in the literature that range from 40-300 nM. [35][36][37][38][39] In contrast, 0.4 nM in vitro translated SBP2-CT generates an equivalent signal to 40 nM of recombinant protein (compare Figs. 1B and C), suggesting that the in vitro translated protein is significantly more active than the bacterially expressed protein.…”

Section: Resultssupporting

confidence: 85%

Characterization of the UGA-recoding and SECIS-binding activities of SECIS-binding protein 2

2014

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Selenium, a micronutrient, is primarily incorporated into human physiology as selenocysteine (Sec). The 25 Seccontaining proteins in humans are known as selenoproteins. Their synthesis depends on the translational recoding of the UGA stop codon to allow Sec insertion. This requires a stem-loop structure in the 3' untranslated region of eukaryotic mRNAs known as the Selenocysteine Insertion Sequence (SECIS). The SECIS is recognized by SECIS-binding protein 2 (SBP2) and this RNA:protein interaction is essential for UGA recoding to occur. Genetic mutations cause SBP2 deficiency in humans, resulting in a broad set of symptoms due to differential effects on individual selenoproteins. Progress on understanding the different phenotypes requires developing robust tools to investigate SBP2 structure and function. In this study we demonstrate that SBP2 protein produced by in vitro translation discriminates among SECIS elements in a competitive UGA recoding assay and has a much higher specific activity than bacterially expressed protein. We also show that a purified recombinant protein encompassing amino acids 517-777 of SBP2 binds to SECIS elements with high affinity and selectivity. The affinity of the SBP2:SECIS interaction correlated with the ability of a SECIS to compete for UGA recoding activity in vitro. The identification of a 250 amino acid sequence that mediates specific, selective SECIS-binding will facilitate future structural studies of the SBP2:SECIS complex. Finally, we identify an evolutionarily conserved core cysteine signature in SBP2 sequences from the vertebrate lineage. Mutation of multiple, but not single, cysteines impaired SECIS-binding but did not affect protein localization in cells.

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Section: Resultssupporting

confidence: 85%

Characterization of the UGA-recoding and SECIS-binding activities of SECIS-binding protein 2

2014

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“…VERVM 466 -470 ). We successfully employed a similar method to identify critical residues in SBP2 (10,11). A structural model of archaeal eEFSec is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…We have described previously an EMSA that revealed a direct tRNA-independent but SECIS-dependent interaction between SBP2 and eEFSec (10). Using this assay, we sought to determine whether the Domain IV mutant proteins are able to form a stable complex with SBP2/SECIS.…”

Section: Resultsmentioning

confidence: 99%

“…Paradoxically, however, the two factors could not be co-immunoprecipitated from rabbit reticulocyte lysate, despite this being an active Sec incorporation system. In contrast, a native electrophoretic mobility shift assay (EMSA) demonstrated that eEFSec can form a complex with SBP2 in the presence of a SECIS element without the requirement of SectRNA Sec (10). This study also showed that the RNA-binding domain (RBD) of SBP2 and the SECIS element are sufficient for eEFSec recruitment.…”

mentioning

confidence: 85%

“…All constructs were confirmed by DNA sequence analysis. SBP2 constructs were made as described previously (10,11).…”

Section: Methodsmentioning

confidence: 99%

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The Selenocysteine-specific Elongation Factor Contains a Novel and Multi-functional Domain

González-Flores¹,

Gupta²,

DeMong³

et al. 2012

Journal of Biological Chemistry

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Background: Selenocysteine (Sec) incorporation requires the function of a unique translation elongation factor, eEFSec. Results: The novel Domain IV of eEFSec is required for at least three functions. Conclusion: Domain IV of eEFSec is the key site for dictating specificity in the conversion of the UGA stop codon into a Sec codon. Significance: Understanding elongation factor function is critical to deciphering the mechanism of Sec incorporation.

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Molecular Mechanism of Eukaryotic Selenocysteine Incorporation

2011

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A Novel Protein Domain Induces High Affinity Selenocysteine Insertion Sequence Binding and Elongation Factor Recruitment

Cited by 54 publications

References 21 publications

Characterization of the UGA-recoding and SECIS-binding activities of SECIS-binding protein 2

Characterization of the UGA-recoding and SECIS-binding activities of SECIS-binding protein 2

The Selenocysteine-specific Elongation Factor Contains a Novel and Multi-functional Domain

Molecular Mechanism of Eukaryotic Selenocysteine Incorporation

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