Translation factor SelB is the key component for the specific decoding of UGA codons with selenocysteine at the ribosome. SelB binds selenocysteyl-tRNASec, guanine nucleotides and a secondary structure of the selenopr...Translation factor SelB is the key component for the specific decoding of UGA codons with selenocysteine at the ribosome. SelB binds selenocysteyl-tRNASec, guanine nucleotides and a secondary structure of the selenoprotein mRNA following the UGA at the 3' side. A comparison of the amino acid sequences of SelB species from E. coli,Desulfomicrobium baculatum, Clostridium thermoaceticum and Haemophilus influenzae showed that the proteins consist of at least four structural domains from which the Nterminal three are well conserved and share homology with elongation factor Tu whereas the C-terminal one is more variable and displays no similarity to any protein known. With the aid of the coordinates of EF-Tu the N-terminal part has been modelled into a 3D structure which exhibits intriguing features concerning its interaction with guanine nucleotides and other components of the translational apparatus. Cloning and expression of fragments of SelB and biochemical analysis of the purified truncated proteins showed that the C-terminal 19 kDa protein fragment is able to specifically bind to the selenoprotein mRNA. SelB, thus, is a translation factor functionally homologous to EF-Tu hooked up to the mRNA with its C-terminal end. The formation by SelB of a quaternary complex in vivo has been proven by overexpression of truncated genes of SelB and by demonstration that fragments comprising the mRNA or the tRNA binding domain inhibit selenocysteine insertion展开更多
In Escherichia coil four gene products（selA, selB, selC and selD） and a selenocysteine（Sec） insertion sequence（SECIS） are required for the correct translation of UGA codons encoding Sec. Previous studies have sh...In Escherichia coil four gene products（selA, selB, selC and selD） and a selenocysteine（Sec） insertion sequence（SECIS） are required for the correct translation of UGA codons encoding Sec. Previous studies have shown that the stoichiometry of selenoproteine mRNA and elongation factor SelB affect the efficiency of Sec incorporation. Herein lies a detailed analysis of the effects of co-expressing selA, selB and selC genes under inducible promoters on the incorporation efficiency of Sec. Over-expression of either selA or selB reduced the efficiency of Sec incorporation by 61.1% or 11.6%, respectively, compared to the over-expression of the reporter vector alone did. Concomitant over-expression ofselC with selA or selB completely reversed the reduce of the efficiency of Sec but still reduced the efficiency of the incorporation relative to that observed for expression of selC alone. Over-expression of selC gene alone under L-arabinose induction reduced the efficiency of the incorporation relative to that observed for co-expressing selC with selA and selB under the control of its endogenous promoter in the absence of L-arabinose. Co-expression of selA, selB and selC with selA or selB under the control of inducible promoters increased the effi- ciency of Sec incorporation by 69.7%. Moreover, inducing selenoprotein-related gene expression during the late exponential phase increased the efficiency of Sec incorporation by a factor of 5.4 relative to that observed for the reporter vector alone. These results suggest that the co-expression of selA, selB and selC in Escherichia coli under the control of inducible promoters is a viable and promising strategy for increasing the yields of selenoproteins.展开更多
The genetic codon UGA has a dual function： serving as a terminator and encoding selenocysteine. However, most popular gene annotation programs only take it as a stop signal, resulting in misannotation or completely m...The genetic codon UGA has a dual function： serving as a terminator and encoding selenocysteine. However, most popular gene annotation programs only take it as a stop signal, resulting in misannotation or completely missing selenoprotein genes. We developed a computational method named Asec-Prediction that is specific for the prediction of archaeal selenoprotein genes. To evaluate its effectiveness, we first applied it to 14 archaeal genomes with previously known selenoprotein genes, and Asec-Prediction identified all reported selenoprotein genes without redundant results. When we applied it to 12 archaeal genomes that had not been researched for selenoprotein genes, Asec-Prediction detected a novel selenoprotein gene in Methanosarcina acetivorans. Further evidence was also collected to support that the predicted gene should Asec-Prediction is effective be a real selenoprotein gene. for the prediction of archaeal The result shows that selenoprotein genes.展开更多
文摘Translation factor SelB is the key component for the specific decoding of UGA codons with selenocysteine at the ribosome. SelB binds selenocysteyl-tRNASec, guanine nucleotides and a secondary structure of the selenoprotein mRNA following the UGA at the 3' side. A comparison of the amino acid sequences of SelB species from E. coli,Desulfomicrobium baculatum, Clostridium thermoaceticum and Haemophilus influenzae showed that the proteins consist of at least four structural domains from which the Nterminal three are well conserved and share homology with elongation factor Tu whereas the C-terminal one is more variable and displays no similarity to any protein known. With the aid of the coordinates of EF-Tu the N-terminal part has been modelled into a 3D structure which exhibits intriguing features concerning its interaction with guanine nucleotides and other components of the translational apparatus. Cloning and expression of fragments of SelB and biochemical analysis of the purified truncated proteins showed that the C-terminal 19 kDa protein fragment is able to specifically bind to the selenoprotein mRNA. SelB, thus, is a translation factor functionally homologous to EF-Tu hooked up to the mRNA with its C-terminal end. The formation by SelB of a quaternary complex in vivo has been proven by overexpression of truncated genes of SelB and by demonstration that fragments comprising the mRNA or the tRNA binding domain inhibit selenocysteine insertion
基金Supported by the National Nature Science Foundation of China（Nos.31070772, 31270907, 21002040, 30970608） and the Doctoral Program of Higher Education of China（No.200901011110136）.
文摘In Escherichia coil four gene products（selA, selB, selC and selD） and a selenocysteine（Sec） insertion sequence（SECIS） are required for the correct translation of UGA codons encoding Sec. Previous studies have shown that the stoichiometry of selenoproteine mRNA and elongation factor SelB affect the efficiency of Sec incorporation. Herein lies a detailed analysis of the effects of co-expressing selA, selB and selC genes under inducible promoters on the incorporation efficiency of Sec. Over-expression of either selA or selB reduced the efficiency of Sec incorporation by 61.1% or 11.6%, respectively, compared to the over-expression of the reporter vector alone did. Concomitant over-expression ofselC with selA or selB completely reversed the reduce of the efficiency of Sec but still reduced the efficiency of the incorporation relative to that observed for expression of selC alone. Over-expression of selC gene alone under L-arabinose induction reduced the efficiency of the incorporation relative to that observed for co-expressing selC with selA and selB under the control of its endogenous promoter in the absence of L-arabinose. Co-expression of selA, selB and selC with selA or selB under the control of inducible promoters increased the effi- ciency of Sec incorporation by 69.7%. Moreover, inducing selenoprotein-related gene expression during the late exponential phase increased the efficiency of Sec incorporation by a factor of 5.4 relative to that observed for the reporter vector alone. These results suggest that the co-expression of selA, selB and selC in Escherichia coli under the control of inducible promoters is a viable and promising strategy for increasing the yields of selenoproteins.
基金supported by the National Natu-ral Science Foundation of China under Grant No.30525037the National 863 Program under Grant No. 2004AA231060
文摘The genetic codon UGA has a dual function： serving as a terminator and encoding selenocysteine. However, most popular gene annotation programs only take it as a stop signal, resulting in misannotation or completely missing selenoprotein genes. We developed a computational method named Asec-Prediction that is specific for the prediction of archaeal selenoprotein genes. To evaluate its effectiveness, we first applied it to 14 archaeal genomes with previously known selenoprotein genes, and Asec-Prediction identified all reported selenoprotein genes without redundant results. When we applied it to 12 archaeal genomes that had not been researched for selenoprotein genes, Asec-Prediction detected a novel selenoprotein gene in Methanosarcina acetivorans. Further evidence was also collected to support that the predicted gene should Asec-Prediction is effective be a real selenoprotein gene. for the prediction of archaeal The result shows that selenoprotein genes.
