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Category: Microbial Genetics and Molecular Biology
The Selenocysteine-Inserting tRNA Species: Structure and Function, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555818333/9781555810733_Chap26-1.gif /docserver/preview/fulltext/10.1128/9781555818333/9781555810733_Chap26-2.gifAbstract:
The occurrence of the amino acid selenocysteine in proteins was first demonstrated for protein A of glycine reductase from Clostridium sticklandii in 1976, and questions were immediately raised on its mechanism of incorporation. At that time, the universality of the 20 proteinogenic amino acids was taken for granted, as was the fact that the 64 codons of the "universal" genetic code are assigned either to code for one of these 20 amino acids or to serve as termination signals. Thus, it seemed unlikely that selenocysteine would be considered as a classical amino acid. In principle, the definition of such a 21st amino acid would require (i) that its incorporation proceeds via a cotranslational mechanism, (ii) that it is directed by a specific codon, and (iii) that a specific tRNA mediates its transport to the ribosome. This chapter illustrates that selenocysteine fulfills these criteria. It first describes the unusual structural properties of tRNASec, and then discusses the unique pathway of selenocysteine insertion that has been worked out for Escherichia coli, which has finally led to the proposal of a model for the co-translational incorporation process at the ribosome. The chapter further compares the pathway established in E. coli with the current knowledge on the mammalian system. Finally, it addresses the interesting question of the evolution of the pathway for the incorporation of selenocysteine that differs from that of the 20 standard amino acids in many respects.
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Cloverleaf structures of tRNASec from Escherichia coli (A) and Xenopus laevis (B). The numbering of E. coli tRNASSec is according to Sprinzl et al. ( 68 ); the nucleotides 5a and 67a designate extra nucleotides renamed in order to keep the standard numbering system ( 7 ). The numbering of X. laevis tRNASec is according to Sturchler et al. ( 70 ); the extra nucleotides in the acceptor stem were renamed to keep the standard numbering system.
Cloverleaf structures of tRNASec from Escherichia coli (A) and Xenopus laevis (B). The numbering of E. coli tRNASSec is according to Sprinzl et al. ( 68 ); the nucleotides 5a and 67a designate extra nucleotides renamed in order to keep the standard numbering system ( 7 ). The numbering of X. laevis tRNASec is according to Sturchler et al. ( 70 ); the extra nucleotides in the acceptor stem were renamed to keep the standard numbering system.
Stereo view of three-dimensional model of solution structure of tRNASec from Escherichia coli ( 7 ).
Stereo view of three-dimensional model of solution structure of tRNASec from Escherichia coli ( 7 ).
Biosynthesis and incorporation of selenocysteine into proteins of Escherichia coli compared with that of the 20 standard amino acids. The selenocysteine pathway involves the action of seryl-tRNA synthetase and the sel gene products. The discrimination of the UGA (selenocysteine) from UGA (stop) codons is mediated by a stem-loop structure immediately 3' to the UGA codons in the fdhF and fdnG mRNAs. Selenium is also incorporated in the anticodon of tRNAclu and tRNALys isoacceptors; the modification at U34 is 5-methylaminomethyl-2-selenouridine (mnm5se2U).
Biosynthesis and incorporation of selenocysteine into proteins of Escherichia coli compared with that of the 20 standard amino acids. The selenocysteine pathway involves the action of seryl-tRNA synthetase and the sel gene products. The discrimination of the UGA (selenocysteine) from UGA (stop) codons is mediated by a stem-loop structure immediately 3' to the UGA codons in the fdhF and fdnG mRNAs. Selenium is also incorporated in the anticodon of tRNAclu and tRNALys isoacceptors; the modification at U34 is 5-methylaminomethyl-2-selenouridine (mnm5se2U).
Postulated reaction mechanism for conversion of seryl-tRNASec to selenocysteyl-tRNASec in active site of selenocysteine synthase ( 28 ).
Postulated reaction mechanism for conversion of seryl-tRNASec to selenocysteyl-tRNASec in active site of selenocysteine synthase ( 28 ).
Cloverleaf structure of mutated tRNASec variant (tRNASecdelAc), where extra base pair 5a-67a in acceptor stem has been deleted, and functional consequences of this deletion.
Cloverleaf structure of mutated tRNASec variant (tRNASecdelAc), where extra base pair 5a-67a in acceptor stem has been deleted, and functional consequences of this deletion.
Electron micrographs of selenocysteine synthase from different angles free and complexed with aminoacryloyl-tRNASec ( 26 ). Selenocysteine synthase is shown symmetrized fivefold in top view orientation (A) and in side view projection (B). Selenocysteine synthase is also shown bound with five molecules of aminoacryloyl-tRNASec in top view projection (C) and in side view projection (D). Note the additional densities under and above the plane, which correspond to the additional densities of the tRNA.
Electron micrographs of selenocysteine synthase from different angles free and complexed with aminoacryloyl-tRNASec ( 26 ). Selenocysteine synthase is shown symmetrized fivefold in top view orientation (A) and in side view projection (B). Selenocysteine synthase is also shown bound with five molecules of aminoacryloyl-tRNASec in top view projection (C) and in side view projection (D). Note the additional densities under and above the plane, which correspond to the additional densities of the tRNA.
Dissection of mRNA determinants for selenocysteine insertion into proteins of Escherichia coli. A DNA segment corresponding to the UGA codon and the 3' adjacent stem-loop structure of the fdhF mRNA was fused in the correct reading frame into the lacZ gene. Selenocysteine insertion was assessed by the measurement of β-galactosidase activity and selenium labeling experiments (not shown). The values displayed correspond to the read-through activities of constructs in which the mutations indicated had been introduced, in percent relative to that of a wild-type construct ( 43 ).
Dissection of mRNA determinants for selenocysteine insertion into proteins of Escherichia coli. A DNA segment corresponding to the UGA codon and the 3' adjacent stem-loop structure of the fdhF mRNA was fused in the correct reading frame into the lacZ gene. Selenocysteine insertion was assessed by the measurement of β-galactosidase activity and selenium labeling experiments (not shown). The values displayed correspond to the read-through activities of constructs in which the mutations indicated had been introduced, in percent relative to that of a wild-type construct ( 43 ).
Quaternary complex formation of the SELB protein with GTP, selenocysteyl-tRNASec, and mRNA recognition element of fdhF mRNA demonstrated by gel retardation assays in nondenaturing polyacrylamide gels ( 6 ). A: 5'-[32P]-labeled mRNA transcript was incubated with increasing amounts of SELB • GTP or SELB-GTP• selenocysteyl-tRNASec complex as indicated and subjected to electrophoretic separation. B: Parallel experiment performed on unlabeled mRNA and [14C]-amino acid-labeled tRNASec.
Quaternary complex formation of the SELB protein with GTP, selenocysteyl-tRNASec, and mRNA recognition element of fdhF mRNA demonstrated by gel retardation assays in nondenaturing polyacrylamide gels ( 6 ). A: 5'-[32P]-labeled mRNA transcript was incubated with increasing amounts of SELB • GTP or SELB-GTP• selenocysteyl-tRNASec complex as indicated and subjected to electrophoretic separation. B: Parallel experiment performed on unlabeled mRNA and [14C]-amino acid-labeled tRNASec.
Model for co-translational incorporation of selenocysteine into Escherichia coli proteins.
Model for co-translational incorporation of selenocysteine into Escherichia coli proteins.
Selenocysteine incorporation into proteins of all three lines of descent
a This table lists only those selenoproteins where the presence of the uga codon was confirmed by sequencing of the corresponding genes.
Selenocysteine incorporation into proteins of all three lines of descent
a This table lists only those selenoproteins where the presence of the uga codon was confirmed by sequencing of the corresponding genes.