UGA suppression by normal tRNA Trp in Escherichia coli: codon context effects.

The nucleotide sequences at the 3' side of in-phase UGA termination codons in mRNAs of various prokaryotic genes were re-examined. An adenine (A) residue is found to be adjacent to the 3' side of UGA in mRNAs which code for readthrough proteins by the suppression of UGA by normal Escherichia coli tRNA Trp. It is suggested that the nature of the nucleotide following a UGA codon determines whether the UGA signals inefficiently or efficiently the termination of polypeptide chain synthesis: an A residue at this position permits the UGA readthrough process.     

Modeling the tertiary interactions in the eukaryotic selenocysteine tRNA.

A novel three-dimensional model of tertiary interactions in the core region of the eukaryotic selenocysteine tRNA is proposed based on the analysis of available nucleotide sequences. The model features the 7/5 tRNA(Sec) secondary structure characterized by seven and five base pairs in the acceptor and T-stems, respectively, and four nucleotides in the connector region between the acceptor and D-stems. The model suggests a unique system of tertiary interactions in the area between the major groove of the D-stem and the first base pair of the extra arm that provides a rigid orientation of the extra arm and contributes to the overall stability of the molecule. The model is consistent with available experimental data on serylation, selenylation, and phosphorylation of different tRNA(Sec) mutants. The important similarity between the proposed model and the structure of the tRNA(Ser) is shown. Based on this similarity, the ability of some tRNA(Ser) mutants to be serylated, selenylated, and phosphorylated was evaluated and found to be in a good agreement with experimental data.     

Identification of a protein component of a mammalian tRNA(Sec) complex implicated in the decoding of UGA as selenocysteine

This report describes a novel RNA-binding protein, SECp43, that associates specifically with mammalian selenocysteine tRNA (tRNA(Sec)). SECp43, identified from a degenerate PCR screen, is a highly conserved protein with two ribonucleoprotein-binding domains and a polar/acidic carboxy terminus. The protein and corresponding mRNA are generally expressed in rat tissues and mammalian cell lines. To gain insight into the biological role of SECp43, affinity-purified antibody was employed to identify its molecular partners. Surprisingly, the application of native HeLa cell extracts to a SECp43 antibody column results in the purification of a 90-nt RNA species identified by direct sequencing and Northern blot analysis as tRNA(Sec). The purification of tRNA(Sec) by the antibody column is striking, based on the low abundance of this tRNA species. Using recombinant SECp43 as a probe for interacting protein partners, we also identify a 48-kDa interacting protein, which is a possible component of the mammalian selenocysteine insertion (SECIS) pathway. To our knowledge, SECp43 is the first cloned protein demonstrated to associate specifically with eukaryotic tRNA(Sec).     

Search for a selenocysteine tRNA in Bacillus subtilis.

Activity to convert serine to selenocysteine in B. subtilis was studied but no activity was detected. In addition, although we tried to find its selenocysteine tRNA (tRNA(SeCys)) gene from a total genome sequence (1) by the computer search with FASTA against E. coli selC (2), no convincing candidate was found. These results suggest that in B. subtilis, selenium-related system is considerably different from known one like E. coli.     

Primary structure of an unusual glycine tRNA UGA suppressor.

We have determined the nucleotide sequences of two UGA-suppressing glycine transfer RNAs. The suppressor tRNAs were previously shown to translate both UGA and UGG and to have arisen as a consequence of mutation in glyT, the gene for the GGA/G-reading glycine tRNA of Escherichia coli. In each mutant tRNA, the primary sequence change was the substitution of adenine for cytosine in the 3' position of the anticodon. In addition, a portion of mutant glyT tRNA molecules contained N6-(delta 2-isopentenyl)-2-thiomethyl adenine adjacent to the 3' end of the anticodon (nucleotide 37). The presence or absence of this hypermodification may be a determinant in some of the biological properties of the mutant tRNA.     

Selenium metabolism in Drosophila. Characterization of the selenocysteine tRNA population.

The selenocysteine (Sec) tRNA population in Drosophila melanogaster is aminoacylated with serine, forms selenocysteyl-tRNA, and decodes UGA. The Km of Sec tRNA and serine tRNA for seryl-tRNA synthetase is 6.67 and 9.45 nM, respectively. Two major bands of Sec tRNA were resolved by gel electrophoresis. Both tRNAs were sequenced, and their primary structures were indistinguishable and colinear with that of the corresponding single copy gene. They are 90 nucleotides in length and contain three modified nucleosides, 5-methylcarboxymethyluridine, N6-isopentenyladenosine, and pseudouridine, at positions 34, 37, and 55, respectively. Neither form contains 1-methyladenosine at position 58 or 5-methylcarboxymethyl-2'-O-methyluridine, which are characteristically found in Sec tRNA of higher animals. We conclude that the primary structures of the two bands of Sec tRNA resolved by electrophoresis are indistinguishable by the techniques employed and that Sec tRNAs in Drosophila may exist in different conformational forms. The Sec tRNA gene maps to a single locus on chromosome 2 at position 47E or F. To our knowledge, Drosophila is the lowest eukaryote in which the Sec tRNA population has been characterized to date.     

Functional characterization of the eukaryotic SECIS elements which direct selenocysteine insertion at UGA codons.

We investigated the requirements for selenocysteine insertion at single or multiple UGA codons in eukaryotic selenoproteins. Two functional SECIS elements were identified in the 3' untranslated region of the rat selenoprotein P mRNA, with predicted stem-loops and critical nucleotides similar to those in the SECIS elements in the type I iodothyronine 5' deiodinase (5'DI) and glutathione peroxidase selenoprotein mRNAs. Site-directed mutational analyses of three SECIS elements confirmed that conserved nucleotides in the loop and in unpaired regions of the stem are critical for activity. This indicates that multiple contact sites are required for SECIS function. Stop codon function at any of five out-of-context UGA codons in the 5'DI mRNA was suppressed by SECIS elements from the 5'DI or selenoprotein P genes linked downstream. Thus, the presence of SECIS elements in eukaryotic selenoprotein mRNAs permits complete flexibility in UGA codon position.     

A study of the method to pick up a selenocysteine tRNA in Bacillus subtilis.

In Bacillus subtilis, selenocysteine tRNA has not been identified in a total genome sequence so far (1). To explore the system of selenocysteine incorporation in B. subtilis, we screened serine-acceptable tRNAs to find an unknown tRNA for selenocysteine by the combined method of specific biotinylation of aa-tRNA (2) and RT-PCR (3). cDNAs obtained from the serine-acceptable tRNA pool were amplified and cloned into plasmid to read its sequence. This procedure gave cDNA library corresponding known serine tRNAs, but no candidate for selenocysteine has been found. Thus, this result, together with the previous data (4), might reveal that there is no selenocysteine tRNA in B. subtilis and/or metabolism of selenium is considerably different from known one as seen in other bacteria.     

Divergence of selenocysteine tRNA recognition by archaeal and eukaryotic O-phosphoseryl-tRNASec kinase

Selenocysteine (Sec) biosynthesis in archaea and eukaryotes requires three steps: serylation of tRNA^Sec by seryl-tRNA synthetase (SerRS), phosphorylation of Ser-tRNA^Sec by O-phosphoseryl-tRNA^Sec kinase (PSTK), and conversion of O-phosphoseryl-tRNA^Sec (Sep-tRNA^Sec) by Sep-tRNA:Sec-tRNA synthase (SepSecS) to Sec-tRNA^Sec. Although SerRS recognizes both tRNA^Sec and tRNA^Ser species, PSTK must discriminate Ser-tRNA^Sec from Ser-tRNA^Ser. Based on a comparison of the sequences and secondary structures of archaeal tRNA^Sec and tRNA^Ser, we introduced mutations into Methanococcus maripaludis tRNA^Sec to investigate how Methanocaldococcus jannaschii PSTK distinguishes tRNA^Sec from tRNA^Ser. Unlike eukaryotic PSTK, the archaeal enzyme was found to recognize the acceptor stem rather than the length and secondary structure of the D-stem. While the D-arm and T-loop provide minor identity elements, the acceptor stem base pairs G2-C71 and C3-G70 in tRNA^Sec were crucial for discrimination from tRNA^Ser. Furthermore, the A5-U68 base pair in tRNA^Ser has some antideterminant properties for PSTK. Transplantation of these identity elements into the tRNA^Ser_UGA scaffold resulted in phosphorylation of the chimeric Ser-tRNA. The chimera was able to stimulate the ATPase activity of PSTK albeit at a lower level than tRNA^Sec, whereas tRNA^Ser did not. Additionally, the seryl moiety of Ser-tRNA^Sec is not required for enzyme recognition, as PSTK efficiently phosphorylated Thr-tRNA^Sec.     

Eukaryotic selenocysteine tRNA has the 9/4 secondary structure

There are two secondary structure models for the eukaryotic selenocysteine (Sec) tRNA(Sec). One model, the 9/4 structure, was experimentally tested and possesses acceptor and T-stems with 9 and 4 bp, respectively [Sturchler et al., 1993; Hubert et al., 1998]. The other one, the 7/5 secondary structure with a bulge in the T-stem, was derived from theoretical calculation [Ioudovitch and Steinberg, 19991. In this report, we show more experimental results supporting the 9/4 secondary structure. Several tRNA(Sec) mutants, whose secondary structure can adopt only the 9/4 structure, were active for serylation and selenylation. Some mutants that cannot base-pair between positions 26 and 44 to provide the 6 bp anticodon stem were still active, inconsistent with the model by Steinberg. We also show that the orientation of the V-arm directly or indirectly influences the selenylation activity, and that the rigid 6 bp D-stem is important. Finally, we conclude that all tRNA(Sec) possess the 13 bp domain II made by the stacking of the colinear AA and T-stems, whether they present the 9/4 structure in Eukarya and Archaea or the 8/5 structure in bacteria.     

The nucleotide sequence of a UGA suppressor serine tRNA from Schizosaccharomyces pombe.

The UGA suppressor tRNA produced by Schizosaccharomyces pombe strain sup3-e was purified to homogeneity. It can be aminoacylated with a serine by a crude aminoacyl-tRNA synthetase preparation from S. pombe cells. By combining post-labeling fingerprinting and gel sequencing methods the nucleotide sequence of this tRNA was determined to be: pG-U-C-A-C-U-A-U-G-U-C-ac4C-G-A-G-D-G-G-D-D-A-A-G-G-A-m2G2-psi-U-A-G-A-N-U-U-C-A-i6A-A-psi-C-U-A-A-U-G-G-G-C-U-U-U-G-C-C-C-G-m5C-G-G-C-A-G-G-T-psi-C-A-m1A-A-U-C-C-U-G-C-U-G-G-U-G-A-C-G-C-C-A OH. The anticodon sequence u ca is complementary to the UGA codon.     

Novel structural determinants in human SECIS elements modulate the translational recoding of UGA as selenocysteine

The selenocysteine insertion sequence (SECIS) element directs the translational recoding of UGA as selenocysteine. In eukaryotes, the SECIS is located downstream of the UGA codon in the 3′-UTR of the selenoprotein mRNA. Despite poor sequence conservation, all SECIS elements form a similar stem-loop structure containing a putative kink-turn motif. We functionally characterized the 26 SECIS elements encoded in the human genome. Surprisingly, the SECIS elements displayed a wide range of UGA recoding activities, spanning several 1000-fold in vivo and several 100-fold in vitro. The difference in activity between a representative strong and weak SECIS element was not explained by differential binding affinity of SECIS binding Protein 2, a limiting factor for selenocysteine incorporation. Using chimeric SECIS molecules, we identified the internal loop and helix 2, which flank the kink-turn motif, as critical determinants of UGA recoding activity. The simultaneous presence of a GC base pair in helix 2 and a U in the 5′-side of the internal loop was a statistically significant predictor of weak recoding activity. Thus, the SECIS contains intrinsic information that modulates selenocysteine incorporation efficiency.     

Regional localization of the selenocysteine tRNA gene (TRSP) on human chromosome 19.

The human selenocysteine tRNA gene (TRSP) has been localized on chromosome 19q13.2-->q13.3 by in situ hybridization and ordered with respect to other genes and anonymous DNA markers in this region by linkage analysis in the forty CEPH pedigrees. These loci span only 10 cM in males and about 30 cM in females. The order of the loci is cen ... D19S7-D19S9-D19S47-CYP2A-CYP2F1-APOC2++ +-(TRSP, CKM). CYP2B flanks the CYP2A and CYP2F1 loci, but it cannot be determined whether it is proximal or distal to the other two cytochrome P450 loci with respect to the centromere.     

Overproduction of selenocysteine tRNA in Chinese hamster ovary cells following transfection of the mouse tRNA[Ser]Sec gene.

Selenocysteine insertion during selenoprotein biosynthesis begins with the aminoacylation of selenocysteine tRNA[ser]sec with serine, the conversion of the serine moiety to selenocysteine, and the recognition of specific UGA codons within the mRNA. Selenocysteine tRNA[ser]sec exists as two major forms, differing by methylation of the ribose portion of the nucleotide at the wobble position of the anticodon. The levels and relative distribution of these two forms of the tRNA are influenced by selenium in mammalian cells and tissues. We have generated Chinese hamster ovary cells that exhibit increased levels of tRNA[ser]sec following transfection of the mouse tRNA[ser]sec gene. The levels of selenocysteine tRNA[ser]sec in transfectants increased proportionally to the number of stably integrated copies of the tRNA[ser]sec gene. Although we were able to generate transfectants overproducing tRNA[ser]sec by as much as tenfold, the additional tRNA was principally retained in the unmethylated form. Selenium supplementation could not significantly affect the relative distributions of the two major selenocysteine tRNA[ser]sec isoacceptors. In addition, increased levels of tRNA[ser]sec did not result in measurable alterations in the levels of selenoproteins, including glutathione peroxidase.