Fidelity of secondary and tertiary interactions in tRNA.

Contingency table analysis has previously been used to detect sequence correlations in RNAs caused by either secondary or tertiary interactions. An approach known as matrix reduction is developed here as an alternative to the usual Chi square test. This approach is especially sensitive to covariation between equivalent positions and is effective at detecting tertiary interactions that exhibit such covariation. Matrix reduction was also effective at detecting Watson-Crick base-pairs that exhibit a high degree of pairing fidelity. The method was applied to five closely related structural classes of tRNA and a number of tertiary interactions were detected in each class.     

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.     

Unique secondary and tertiary structural features of the eucaryotic selenocysteine tRNA(Sec).

Cotranslational insertion of selenocysteine into selenoenzymes is mediated by a specialized transfer RNA, the tRNA(Sec). We have carried out the determination of the solution structure of the eucaryotic tRNA(Sec). Based on the enzymatic and chemical probing approach, we show that the secondary structure bears a few unprecedented features like a 9 bp aminoacid-, a 4 bp thymine- and a 6 bp dihydrouridine-stems. Surprisingly, the eighth nucleotide, although being a uridine, is base-paired and cannot therefore correspond to the single-stranded invariant U8 found in all tRNAs. Rather, experimental evidence led us to propose that the role of the invariant U8 is actually played by the tenth nucleotide which is an A, numbered A8 to indicate this fact. The experimental data therefore demonstrate that the cloverleaf structure we derived experimentally resembles the hand-folded model proposed by Böck et al (ref. 3). Using the solution data and computer modelling, we derived a three-dimensional structure model which shows some unique aspects. Basically, A8, A14, U21 form a novel type of tertiary interaction in which A8 interacts with the Hoogsteen sites of A14 which itself forms a Watson-Crick pair with U21. No coherent model containing the canonical 15-48 interaction could be derived. Thus, the number of tertiary interactions appear to be limited, leading to an uncoupling of the variable stem from the rest of the molecule.     

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.     

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.     

Selective inhibition of selenocysteine tRNA maturation and selenoprotein synthesis in transgenic mice expressing isopentenyladenosine-deficient selenocysteine tRNA

Selenocysteine (Sec) tRNA (tRNA([Ser]Sec)) serves as both the site of Sec biosynthesis and the adapter molecule for donation of this amino acid to protein. The consequences on selenoprotein biosynthesis of overexpressing either the wild type or a mutant tRNA([Ser]Sec) lacking the modified base, isopentenyladenosine, in its anticodon loop were examined by introducing multiple copies of the corresponding tRNA([Ser]Sec) genes into the mouse genome. Overexpression of wild-type tRNA([Ser]Sec) did not affect selenoprotein synthesis. In contrast, the levels of numerous selenoproteins decreased in mice expressing isopentenyladenosine-deficient (i(6)A(-)) tRNA([Ser]Sec) in a protein- and tissue-specific manner. Cytosolic glutathione peroxidase and mitochondrial thioredoxin reductase 3 were the most and least affected selenoproteins, while selenoprotein expression was most and least affected in the liver and testes, respectively. The defect in selenoprotein expression occurred at translation, since selenoprotein mRNA levels were largely unaffected. Analysis of the tRNA([Ser]Sec) population showed that expression of i(6)A(-) tRNA([Ser]Sec) altered the distribution of the two major isoforms, whereby the maturation of tRNA([Ser]Sec) by methylation of the nucleoside in the wobble position was repressed. The data suggest that the levels of i(6)A(-) tRNA([Ser]Sec) and wild-type tRNA([Ser]Sec) are regulated independently and that the amount of wild-type tRNA([Ser]Sec) is determined, at least in part, by a feedback mechanism governed by the level of the tRNA([Ser]Sec) population. This study marks the first example of transgenic mice engineered to contain functional tRNA transgenes and suggests that i(6)A(-) tRNA([Ser]Sec) transgenic mice will be useful in assessing the biological roles of selenoproteins.     

Bar to normal UGA translation by the selenocysteine tRNA.

The selC gene product, tRNA(Sec), inserts selenocysteine at UGA (opal) codons in a specialized mRNA context. We have investigated the action of the tRNA at ordinary UGA codons, normally not translated, by changing the unusual structural features of tRNA(Sec). Sequences in the D arm, CCA arm and variable arm of the tRNA all contribute to the prohibition against translation of ordinary UGA codons. One multiple mutant is a moderately efficient serine-inserting UGA suppressor tRNA.     

Crystal structure of human selenocysteine tRNA

Selenocysteine (Sec) is the 21st amino acid in translation. Sec tRNA (tRNA^Sec) has an anticodon complementary to the UGA codon. We solved the crystal structure of human tRNA^Sec. tRNA^Sec has a 9-bp acceptor stem and a 4-bp T stem, in contrast with the 7-bp acceptor stem and the 5-bp T stem in the canonical tRNAs. The acceptor stem is kinked between the U6:U67 and G7:C66 base pairs, leading to a bent acceptor-T stem helix. tRNA^Sec has a 6-bp D stem and a 4-nt D loop. The long D stem includes unique A14:U21 and G15:C20a pairs. The D-loop:T-loop interactions include the base pairs G18:U55 and U16:U59, and a unique base triple, U20:G19:C56. The extra arm comprises of a 6-bp stem and a 4-nt loop. Remarkably, the D stem and the extra arm do not form tertiary interactions in tRNA^Sec. Instead, tRNA^Sec has an open cavity, in place of the tertiary core of a canonical tRNA. The linker residues, A8 and U9, connecting the acceptor and D stems, are not involved in tertiary base pairing. Instead, U9 is stacked on the first base pair of the extra arm. These features might allow tRNA^Sec to be the target of the Sec synthesis/incorporation machineries.     

Tertiary structure of bacterial selenocysteine tRNA

Selenocysteine (Sec) is translationally incorporated into proteins in response to the UGA codon. The tRNA specific to Sec (tRNA^Sec) is first ligated with serine by seryl-tRNA synthetase (SerRS). In the present study, we determined the 3.1 Å crystal structure of the tRNA^Sec from the bacterium Aquifex aeolicus, in complex with the heterologous SerRS from the archaeon Methanopyrus kandleri. The bacterial tRNA^Sec assumes the L-shaped structure, from which the long extra arm protrudes. Although the D-arm conformation and the extra-arm orientation are similar to those of eukaryal/archaeal tRNA^Secs, A. aeolicus tRNA^Sec has unique base triples, G14:C21:U8 and C15:G20a:G48, which occupy the positions corresponding to the U8:A14 and R15:Y48 tertiary base pairs of canonical tRNAs. Methanopyrus kandleri SerRS exhibited serine ligation activity toward A. aeolicus tRNA^Sec in vitro. The SerRS N-terminal domain interacts with the extra-arm stem and the outer corner of tRNA^Sec. Similar interactions exist in the reported tRNA^Ser and SerRS complex structure from the bacterium Thermus thermophilus. Although the catalytic C-terminal domain of M. kandleri SerRS lacks interactions with A. aeolicus tRNA^Sec in the present complex structure, the conformational flexibility of SerRS is likely to allow the CCA terminal region of tRNA^Sec to enter the SerRS catalytic site.     

Decoding apparatus for eukaryotic selenocysteine insertion

Decoding UGA as selenocysteine requires a unique tRNA, a specialized elongation factor, and specific secondary structures in the mRNA, termed SECIS elements. Eukaryotic SECIS elements are found in the 3′ untranslated region of selenoprotein mRNAs while those in prokaryotes occur immediately downstream of UGA. Consequently, a single eukaryotic SECIS element can serve multiple UGA codons, whereas prokaryotic SECIS elements only function for the adjacent UGA, suggesting distinct mechanisms for recoding in the two kingdoms. We have identified and characterized the first eukaryotic selenocysteyl-tRNA-specific elongation factor. This factor forms a complex with mammalian SECIS binding protein 2, and these two components function together in selenocysteine incorporation in mammalian cells. Expression of the two functional domains of the bacterial elongation factor–SECIS binding protein as two separate proteins in eukaryotes suggests a mechanism for rapid exchange of charged for uncharged selenocysteyl-tRNA–elongation factor complex, allowing a single SECIS element to serve multiple UGA codons.     

Biosynthesis of selenocysteine on its tRNA in eukaryotes

Selenocysteine (Sec) is cotranslationally inserted into protein in response to UGA codons and is the 21st amino acid in the genetic code. However, the means by which Sec is synthesized in eukaryotes is not known. Herein, comparative genomics and experimental analyses revealed that the mammalian Sec synthase (SecS) is the previously identified pyridoxal phosphate-containing protein known as the soluble liver antigen. SecS required selenophosphate and O-phosphoseryl-tRNA^[Ser]Sec as substrates to generate selenocysteyl-tRNA^[Ser]Sec. Moreover, it was found that Sec was synthesized on the tRNA scaffold from selenide, ATP, and serine using tRNA^[Ser]Sec, seryl-tRNA synthetase, O-phosphoseryl-tRNA^[Ser]Sec kinase, selenophosphate synthetase, and SecS. By identifying the pathway of Sec biosynthesis in mammals, this study not only functionally characterized SecS but also assigned the function of the O-phosphoseryl-tRNA^[Ser]Sec kinase. In addition, we found that selenophosphate synthetase 2 could synthesize monoselenophosphate in vitro but selenophosphate synthetase 1 could not. Conservation of the overall pathway of Sec biosynthesis suggests that this pathway is also active in other eukaryotes and archaea that synthesize selenoproteins.     

A role for the bulged nucleotide 47 in the facilitation of tertiary interactions in the tRNA structure.

Based on computer modeling and with the use of energy minimisation procedure, we show that the bulged nucleotide 47 in the yeast tRNA(Phe) structure plays an important steric role, allowing the formation of canonical tertiary interactions 15-48 and 22-46 within the D-domain. The absence of nucleotide 47 can be compensated by the presence of a wobble pair U13-G22, whose unusual stereochemistry permits as well the formation of the canonical tertiary interactions. The tRNA database show that the vast majority of the cytosolic tRNAs have either a nucleotide at position 47 or a wobble pair U13-G22. On the contrary, many mitochondrial tRNAs, having a Watson-Crick pair 13-22, do not have nucleotide in position 47, which suggests that their tertiary interactions within the D-domain must differ from those in cytosolic tRNAs.     

A selenocysteine tRNA and SECIS element in Plasmodium falciparum

The molecular machinery for incorporating selenocysteine into proteins is present in both prokaryotes and eukaryotes. Although selenocysteine insertion has been reported in animals, plants, and protozoans, known eukaryotic selenocysteine tRNA sequences and selenocysteine insertion sequences are limited to animals and plants. Here we present clear indications of the presence of selenocysteine-tRNA and a selenocysteine insertion sequence in Plasmodium falciparum. To our knowledge, this is the first report of an identification of protozoan selenocysteine insertion machinery at the sequence level.     

Chlamydomonas reinhardtii selenocysteine tRNA[Ser]Sec

Eukaryotic selenocysteine (Sec) protein insertion machinery was thought to be restricted to animals, but the occurrence of both Sec-containing proteins and the Sec insertion system was recently found in Chlamydomonas reinhardtii, a member of the plant kingdom. Herein, we used RT-PCR to determine the sequence of C. reinhardtii Sec tRNA[Ser]Sec, the first non-animal eukaryotic Sec tRNA[Ser]Sec sequence. Like its animal counterpart, it is 90 nucleotides in length, is aminoacylated with serine by seryl-tRNA synthetase, and decodes specifically UGA. Evolutionary analyses of known Sec tRNAs identify the C. reinhardtii form as the most diverged eukaryotic Sec tRNA[Ser]Sec and reveal a common origin for this tRNA in bacteria, archaea, and eukaryotes.     

Analysis of eukaryotic mRNA structures directing cotranslational incorporation of selenocysteine.

Translation of an mRNA encoding a selenoprotein requires that at least one UGA codon in the reading frame is recoded as a site for the insertion of selenocysteine. In eukaryotes, the termination codon recoding event is directed by a cis-acting signal element located in the 3' untranslated region of the gene. This 'selenocysteine insertion sequence' (SECIS) comprises conserved sequences in a region of extensive base-pairing. In order to study the structure-function relationships of the SECIS structure, we have applied a newly developed reporter gene system which allows analysis of stop codon suppression in animal cell lines. This system obviates the need for enzymatic or immunological estimation of selenoprotein synthesis, relying instead on the simple quantification of translational readthrough from the lacZ gene into the luciferase gene. The 3'-UTR of the phospholipid hydroperoxide glutathione peroxidase (PHGPx) gene was shown to contain a highly active SECIS element. Mutations in the base-paired sequences of other SECIS elements were used to analyse the significance of primary structure, secondary structure and pairing stability in the stem regions. The results demonstrate that the exact sequences of the paired nucleotides are comparatively unimportant, provided that a consensus combination of length and thermodynamic stability of the base-paired structures is maintained.     

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.