Characterization and evolutionary history of an archaeal kinase involved in selenocysteinyl-tRNA formation

Selenocysteine (Sec)-decoding archaea and eukaryotes employ a unique route of Sec-tRNA^Sec synthesis in which O-phosphoseryl-tRNA^Sec kinase (PSTK) phosphorylates Ser-tRNA^Sec to produce the O-phosphoseryl-tRNA^Sec (Sep-tRNA^Sec) substrate that Sep-tRNA:Sec-tRNA synthase (SepSecS) converts to Sec-tRNA^Sec. This study presents a biochemical characterization of Methanocaldococcus jannaschii PSTK, including kinetics of Sep-tRNA^Sec formation (K_m for Ser-tRNA^Sec of 40 nM and ATP of 2.6 mM). PSTK binds both Ser-tRNA^Sec and tRNA^Sec with high affinity (K_d values of 53 nM and 39 nM, respectively). The ATPase activity of PSTK may be activated via an induced fit mechanism in which binding of tRNA^Sec specifically stimulates hydrolysis. Albeit with lower activity than ATP, PSTK utilizes GTP, CTP, UTP and dATP as phosphate-donors. Homology with related kinases allowed prediction of the ATPase active site, comprised of phosphate-binding loop (P-loop), Walker B and RxxxR motifs. Gly14, Lys17, Ser18, Asp41, Arg116 and Arg120 mutations resulted in enzymes with decreased activity highlighting the importance of these conserved motifs in PSTK catalysis both in vivo and in vitro. Phylogenetic analysis of PSTK in the context of its ‘DxTN’ kinase family shows that PSTK co-evolved precisely with SepSecS and indicates the presence of a previously unidentified PSTK in Plasmodium species.     

C-terminal domain of archaeal O-phosphoseryl-tRNA kinase displays large-scale motion to bind the 7-bp D-stem of archaeal tRNASec

O-Phosphoseryl-tRNA kinase (PSTK) is the key enzyme in recruiting selenocysteine (Sec) to the genetic code of archaea and eukaryotes. The enzyme phosphorylates Ser-tRNA^Sec to produce O-phosphoseryl-tRNA^Sec (Sep-tRNA^Sec) that is then converted to Sec-tRNA^Sec by Sep-tRNA:Sec-tRNA synthase. Earlier we reported the structure of the Methanocaldococcus jannaschii PSTK (MjPSTK) complexed with AMPPNP. This study presents the crystal structure (at 2.4-Å resolution) of MjPSTK complexed with an anticodon-stem/loop truncated tRNA^Sec (Mj*tRNA^Sec), a good enzyme substrate. Mj*tRNA^Sec is bound between the enzyme’s C-terminal domain (CTD) and N-terminal kinase domain (NTD) that are connected by a flexible 11 amino acid linker. Upon Mj*tRNA^Sec recognition the CTD undergoes a 62-Å movement to allow proper binding of the 7-bp D-stem. This large reorganization of the PSTK quaternary structure likely provides a means by which the unique tRNA^Sec species can be accurately recognized with high affinity by the translation machinery. However, while the NTD recognizes the tRNA acceptor helix, shortened versions of MjPSTK (representing only 60% of the original size, in which the entire CTD, linker loop and an adjacent NTD helix are missing) are still active in vivo and in vitro, albeit with reduced activity compared to the full-length enzyme.     

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.     

New Developments in Selenium Biochemistry: Selenocysteine Biosynthesis in Eukaryotes and Archaea

We used comparative genomics and experimental analyses to show that (1) eukaryotes and archaea, which possess the selenocysteine (Sec) protein insertion machinery contain an enzyme, O-phosphoseryl-transfer RNA (tRNA)^[Ser]Sec kinase (designated PSTK), which phosphorylates seryl-tRNA^[Ser]Sec to form O-phosphoseryl-tRNA^[Ser]Sec and (2) the Sec synthase (SecS) in mammals is a pyridoxal phosphate-containing protein previously described as the soluble liver antigen (SLA). SecS uses the product of PSTK, O-phosphoseryl-tRNA^[Ser]Sec, and selenophosphate as substrates to generate selenocysteyl-tRNA^[Ser]Sec. Sec could be synthesized on tRNA^[Ser]Sec from selenide, adenosine triphosphate (ATP), and serine using tRNA^[Ser]Sec, seryl-tRNA synthetase, PSTK, selenophosphate synthetase, and SecS. The enzyme that synthesizes monoselenophosphate is a previously identified selenoprotein, selenophosphate synthetase 2 (SPS2), whereas the previously identified mammalian selenophosphate synthetase 1 did not serve this function. Monoselenophosphate also served directly in the reaction replacing ATP, selenide, and SPS2, demonstrating that this compound was the active selenium donor. Conservation of the overall pathway of Sec biosynthesis suggests that this pathway is also active in other eukaryotes and archaea that contain selenoproteins. X.-M. Xu and B. A. Carlson contributed equally to the studies described herein.     

A tRNA-dependent cysteine biosynthesis enzyme recognizes the selenocysteine-specific tRNA in Escherichia coli

The essential methanogen enzyme Sep-tRNA:Cys-tRNA synthase (SepCysS) converts O-phosphoseryl-tRNA^Cys (Sep-tRNA^Cys) into Cys-tRNA^Cys in the presence of a sulfur donor. Likewise, Sep-tRNA:Sec-tRNA synthase converts O-phosphoseryl-tRNA^Sec (Sep-tRNA^Sec) to selenocysteinyl-tRNA^Sec (Sec-tRNA^Sec) using a selenium donor. While the Sep moiety of the aminoacyl-tRNA substrates is the same in both reactions, tRNA^Cys and tRNA^Sec differ greatly in sequence and structure. In an Escherichia coli genetic approach that tests for formate dehydrogenase activity in the absence of selenium donor we show that Sep-tRNA^Sec is a substrate for SepCysS. Since Sec and Cys are the only active site amino acids known to sustain FDH activity, we conclude that SepCysS converts Sep-tRNA^Sec to Cys-tRNA^Sec, and that Sep is crucial for SepCysS recognition.     

SerRS-tRNASec complex structures reveal mechanism of the first step in selenocysteine biosynthesis

Selenocysteine (Sec) is found in the catalytic centers of many selenoproteins and plays important roles in living organisms. Malfunctions of selenoproteins lead to various human disorders including cancer. Known as the 21st amino acid, the biosynthesis of Sec involves unusual pathways consisting of several stages. While the later stages of the pathways are well elucidated, the molecular basis of the first stage—the serylation of Sec-specific tRNA (tRNA^Sec) catalyzed by seryl-tRNA synthetase (SerRS)—is unclear. Here we present two cocrystal structures of human SerRS bound with tRNA^Sec in different stoichiometry and confirm the formation of both complexes in solution by various characterization techniques. We discovered that the enzyme mainly recognizes the backbone of the long variable arm of tRNA^Sec with few base-specific contacts. The N-terminal coiled-coil region works like a long-range lever to precisely direct tRNA 3′ end to the other protein subunit for aminoacylation in a conformation-dependent manner. Restraints of the flexibility of the coiled-coil greatly reduce serylation efficiencies. Lastly, modeling studies suggest that the local differences present in the D- and T-regions as well as the characteristic U20:G19:C56 base triple in tRNA^Sec may allow SerRS to distinguish tRNA^Sec from closely related tRNA^Ser substrate.     

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.     

The 1.2 Å crystal structure of an E. coli tRNA acceptor stem microhelix reveals two magnesium binding sites

tRNA identity elements assure the correct aminoacylation of tRNAs by the cognate aminoacyl-tRNA synthetases. tRNA^Ser belongs to the so-called class II system, in which the identity elements are rather simple and are mostly located in the acceptor stem region, in contrast to ‘class I’, where tRNA determinants are more complex and are located within different regions of the tRNA.The structure of an Escherichia coli tRNA^Ser acceptor stem microhelix was solved by high resolution X-ray structure analysis. The RNA crystallizes in the space group C2, with one molecule per asymmetric unit and with the cell constants a = 35.79, b = 39.13, c = 31.37 Å, and β = 111.1°. A defined hydration pattern of 97 water molecules surrounds the tRNA^Ser acceptor stem microhelix. Additionally, two magnesium binding sites were detected in the tRNA^Ser aminoacyl stem.     

Structural Asymmetry of the Terminal Catalytic Complex in Selenocysteine Synthesis

Selenocysteine (Sec), the 21^st amino acid, is synthesized from a serine precursor in a series of reactions that require selenocysteine tRNA (tRNA^Sec). In archaea and eukaryotes, O-phosphoseryl-tRNA^Sec:selenocysteinyl-tRNA^Sec synthase (SepSecS) catalyzes the terminal synthetic reaction during which the phosphoseryl intermediate is converted into the selenocysteinyl moiety while being attached to tRNA^Sec. We have previously shown that only the SepSecS tetramer is capable of binding to and recognizing the distinct fold of tRNA^Sec. Because only two of the four tRNA-binding sites were occupied in the crystal form, a question was raised regarding whether the observed arrangement and architecture faithfully recapitulated the physiologically relevant ribonucleoprotein complex important for selenoprotein formation. Herein, we determined the stoichiometry of the human terminal synthetic complex of selenocysteine by using small angle x-ray scattering, multi-angle light scattering, and analytical ultracentrifugation. In addition, we provided the first estimate of the ratio between SepSecS and tRNA^Sec in vivo. We show that SepSecS preferentially binds one or two tRNA^Sec molecules at a time and that the enzyme is present in large molar excess over the substrate tRNA in vivo. Moreover, we show that in a complex between SepSecS and two tRNAs, one enzyme homodimer plays a role of the noncatalytic unit that positions CCA ends of two tRNA^Sec molecules into the active site grooves of the other, catalytic, homodimer. Finally, our results demonstrate that the previously determined crystal structure represents the physiologically and catalytically relevant complex and suggest that allosteric regulation of SepSecS might play an important role in regulation of selenocysteine and selenoprotein synthesis.     

Human Cells Have a Limited Set of tRNA Anticodon Loop Substrates of the tRNA Isopentenyltransferase TRIT1 Tumor Suppressor

Human TRIT1 is a tRNA isopentenyltransferase (IPTase) homologue of Escherichia coli MiaA, Saccharomyces cerevisiae Mod5, Schizosaccharomyces pombe Tit1, and Caenorhabditis elegans GRO-1 that adds isopentenyl groups to adenosine 37 (i6A37) of substrate tRNAs. Prior studies indicate that i6A37 increases translation fidelity and efficiency in codon-specific ways. TRIT1 is a tumor suppressor whose mutant alleles are associated with cancer progression. We report the systematic identification of i6A37-containing tRNAs in a higher eukaryote, performed using small interfering RNA knockdown and other methods to examine TRIT1 activity in HeLa cells. Although several potential substrates contained the IPTase recognition sequence A36A37A38 in the anticodon loop, only tRNA^SerAGA, tRNA^SerCGA, tRNA^SerUGA, and selenocysteine tRNA with UCA (tRNA^[Ser]SecUCA) contained i6A37. This subset is a significantly more restricted than that for two distant yeasts (S. cerevisiae and S. pombe), the only other organisms comprehensively examined. Unlike the fully i6A37-modified tRNAs for Ser, tRNA^[Ser]SecUCA is partially (∼40%) modified. Exogenous selenium and other treatments that decreased the i6A37 content of tRNA^[Ser]SecUCA led to increased levels of the tRNA^[Ser]SecUCA. Of the human mitochondrion (mt)-encoded tRNAs with A36A37A38, only mt tRNAs tRNA^SerUGA and tRNA^TrpUCA contained detectable i6A37. Moreover, while tRNA^Ser levels were unaffected by TRIT1 knockdown, the tRNA^[Ser]SecUCA level was increased and the mt tRNA^SerUGA level was decreased, suggesting that TRIT1 may control the levels of some tRNAs as well as their specific activity.     

Thermodynamic and Kinetic Framework of Selenocysteyl-tRNASec Recognition by Elongation Factor SelB

SelB is a specialized translation elongation factor that delivers selenocysteyl-tRNA^Sec (Sec-tRNA^Sec) to the ribosome. Here we show that Sec-tRNA^Sec binds to SelB·GTP with an extraordinary high affinity (K_d = 0.2 pm). The tight binding is driven enthalpically and involves the net formation of four ion pairs, three of which may involve the Sec residue. The dissociation of tRNA from the ternary complex SelB·GTP·Sec-tRNA^Sec is very slow (0.3 h⁻¹), and GTP hydrolysis accelerates the release of Sec-tRNA^Sec by more than a million-fold (to 240 s⁻¹). The affinities of Sec-tRNA^Sec to SelB in the GDP or apoforms, or Ser-tRNA^Sec and tRNA^Sec to SelB in any form, are similar (K_d = 0.5 μm). Thermodynamic coupling in binding of Sec-tRNA^Sec and GTP to SelB ensures at the same time the specificity of Sec- versus Ser-tRNA^Sec selection and rapid release of Sec-tRNA^Sec from SelB after GTP cleavage on the ribosome. SelB provides an example for the evolution of a highly specialized protein-RNA complex toward recognition of unique set of identity elements. The mode of tRNA recognition by SelB is reminiscent of another specialized factor, eIF2, rather than of EF-Tu, the common delivery factor for all other aminoacyl-tRNAs, in line with a common evolutionary ancestry of SelB and eIF2.     

Structural basis for recognition of cognate tRNA by tyrosyl-tRNA synthetase from three kingdoms

The specific aminoacylation of tRNA by tyrosyl-tRNA synthetases (TyrRSs) relies on the identity determinants in the cognate tRNA^Tyrs. We have determined the crystal structure of Saccharomyces cerevisiae TyrRS (SceTyrRS) complexed with a Tyr-AMP analog and the native tRNA^Tyr(GΨA). Structural information for TyrRS–tRNA^Tyr complexes is now full-line for three kingdoms. Because the archaeal/eukaryotic TyrRSs–tRNA^Tyrs pairs do not cross-react with their bacterial counterparts, the recognition modes of the identity determinants by the archaeal/eukaryotic TyrRSs were expected to be similar to each other but different from that by the bacterial TyrRSs. Interestingly, however, the tRNA^Tyr recognition modes of SceTyrRS have both similarities and differences compared with those in the archaeal TyrRS: the recognition of the C1-G72 base pair by SceTyrRS is similar to that by the archaeal TyrRS, whereas the recognition of the A73 by SceTyrRS is different from that by the archaeal TyrRS but similar to that by the bacterial TyrRS. Thus, the lack of cross-reactivity between archaeal/eukaryotic and bacterial TyrRS-tRNA^Tyr pairs most probably lies in the different sequence of the last base pair of the acceptor stem (C1-G72 vs G1-C72) of tRNA^Tyr. On the other hand, the recognition mode of Tyr-AMP is conserved among the TyrRSs from the three kingdoms.     

Processing of plant mitochondrial tRNAGly and tRNASer(GCU) is independent of RNA editing

The genes encoding pea and potato mitochondrial tRNA^Gly and pea mitochondrial tRNA^Ser(GCU) were analyzed with particular respect to their expression. Secondary-structure models deduced from the identical potato and pea tRNA^Gly gene sequences revealed A₇:C₆₆ mismatches in the seventh base pair at the base of the acceptor stems of both tRNAs. Sequence analyses of tRNA^Gly cDNA clones showed that these mispairings are not corrected by C₆₆ to U₆₆ conversions, as observed in plant mitochondrial tRNA^Phe. Likewise, a U₆:C₆₇ mismatch identified in the acceptor stem of the pea tRNA^Ser(GCU) is not altered by RNA editing to a mismatched U:U pair, which is created by RNA editing in Oenothera mitochondrial tRNA^Cys. In vitro processing reactions with the respective tRNA^Gly and tRNA^Ser(GCU) precursors show that such conversions are not necessary for 5′ and 3′ end maturation of these tRNAs. These results demonstrate that not all C:A (A:C) or U:C (C:U) mismatches in double-stranded regions of tRNAs are altered by RNA editing. An RNA editing event in plant mitochondrial tRNAs is thus not generally indicated by the presence of a mismatch but may depend on additional parameters.     

Translational nonsense codon suppression as indicator for functional pre-tRNA splicing in transformed Arabidopsis hypocotyl-derived calli

The transient expression of three novel plant amber suppressors derived from a cloned Nicotiana tRNA^Ser(CGA), an Arabidopsis intron-containing tRNA^Tyr(GTA) and an Arabidopsis intron-containing tRNA^Met(CAT) gene, respectively, was studied in a homologous plant system that utilized the Agro bacterium-mediated gene transfer to Arabidopsis hypocotyl explants. This versatile system allows the detection of β-glucuronidase (GUS) activity by histochemical and enzymatic analyses. The activity of the suppressors was demonstrated by the ability to suppress a premature amber codon in a modified GUS gene. Co-transformation of Arabidopsis hypocotyls with the amber suppressor tRNA^Ser gene and the GUS reporter gene resulted in ~10% of the GUS activity found in the same tissue transformed solely with the functional control GUS gene. Amber suppressor tRNAs derived from intron-containing tRNA^Tyr or tRNA^Met genes were functional in vivo only after some additional gene manipulations. The G3:C70 base pair in the acceptor stem of tRNA^Met(CUA) had to be converted to a G3:U70 base pair, which is the major determinant for alanine tRNA identity. The inability of amber suppressor tRNA^Tyr to show any activity in vivo predominantly results from a distorted intron secondary structure of the corresponding pre-tRNA that could be cured by a single nucleotide exchange in the intervening sequence. The improved amber suppressors tRNA^Tyr and tRNA^Met were subsequently employed for studying various aspects of the plant-specific mechanism of pre-tRNA splicing as well as for demonstrating the influence of intron-dependent base modifications on suppressor activity.     

Dimer–Dimer Interaction of the Bacterial Selenocysteine Synthase SelA Promotes Functional Active-Site Formation and Catalytic Specificity

The 21st amino acid, selenocysteine (Sec), is incorporated translationally into proteins and is synthesized on its specific tRNA (tRNA^Sec). In Bacteria, the selenocysteine synthase SelA converts Ser-tRNA^Sec, formed by seryl-tRNA synthetase, to Sec-tRNA^Sec. SelA, a member of the fold-type-I pyridoxal 5′-phosphate-dependent enzyme superfamily, has an exceptional homodecameric quaternary structure with a molecular mass of about 500 kDa. Our previously determined crystal structures of Aquifex aeolicus SelA complexed with tRNA^Sec revealed that the ring-shaped decamer is composed of pentamerized SelA dimers, with two SelA dimers arranged to collaboratively interact with one Ser-tRNA^Sec. The SelA catalytic site is close to the dimer–dimer interface, but the significance of the dimer pentamerization in the catalytic site formation remained elusive. In the present study, we examined the quaternary interactions and demonstrated their importance for SelA activity by systematic mutagenesis. Furthermore, we determined the crystal structures of “depentamerized” SelA variants with mutations at the dimer–dimer interface that prevent pentamerization. These dimeric SelA variants formed a distorted and inactivated catalytic site and confirmed that the pentamer interactions are essential for productive catalytic site formation. Intriguingly, the conformation of the non-functional active site of dimeric SelA shares structural features with other fold-type-I pyridoxal 5′-phosphate-dependent enzymes with native dimer or tetramer (dimer-of-dimers) quaternary structures.     

C-terminal Domain of Leucyl-tRNA Synthetase from Pathogenic Candida albicans Recognizes both tRNASer and tRNALeu

Leucyl-tRNA synthetase (LeuRS) is a multidomain enzyme that catalyzes Leu-tRNA^Leu formation and is classified into bacterial and archaeal/eukaryotic types with significant diversity in the C-terminal domain (CTD). CTDs of both bacterial and archaeal LeuRSs have been reported to recognize tRNA^Leu through different modes of interaction. In the human pathogen Candida albicans, the cytoplasmic LeuRS (CaLeuRS) is distinguished by its capacity to recognize a uniquely evolved chimeric tRNA^Ser (CatRNA^Ser(CAG)) in addition to its cognate CatRNA^Leu, leading to CUG codon reassignment. Our previous study showed that eukaryotic but not archaeal LeuRSs recognize this peculiar tRNA^Ser, suggesting the significance of their highly divergent CTDs in tRNA^Ser recognition. The results of this study provided the first evidence of the indispensable function of the CTD of eukaryotic LeuRS in recognizing non-cognate CatRNA^Ser and cognate CatRNA^Leu. Three lysine residues were identified as involved in mediating enzyme-tRNA interaction in the leucylation process: mutation of all three sites totally ablated the leucylation activity. The importance of the three lysine residues was further verified by gel mobility shift assays and complementation of a yeast leuS gene knock-out strain.     

pGp as the main product of bovine tRNA kinase

One of the Ser-tRNAs, Ser-tRNA^Sec, is converted to Sec-tRNA^Sec by Sec synthase. This Ser-tRNA^Sec is also converted to phosphoser-tRNA^Sec by tRNA kinase. In this study, we analyzed of the products of phosphorylation with tRNA kinase. [³H]Ser-tRNA^Sec purified on Sephacryl S-200 was phosphorylated with [-³²P]ATP by tRNA kinase. The product [³²P][³H]phosphoser-tRNA was purified on Sephacryl S-200 and hydrolyzed with ribonuclease T2. The chromatogram of this hydrolyzate on DEAE-cellulose in 7M urea buffer showed four peaks. The first peak of the pass-through fraction was seryl-adenosine liberated from the 3-terminal of the tRNA. The second peak, eluted before the third peak containing inorganic phosphate, was phosphoseryl-adenosine. The major compound in the fourth peak was pGp. As a control experiment, non-acylated tRNA^Sec was used as a substrate of phosphorylation and the product was analyzed. The chromatogram of the digest with ribonuclease T2 showed no peak of phosphoseryl-adenosine, but a peak of pGp was seen with the peak of inorganic phosphate. Thus, the major product in the presence of tRNA kinase was pGp, and a small but significant proportion of the radioactivity was found as phosphoserine in the presence of seryl residue on the 3-CCA terminal of tRNA^Sec. These results indicated that tRNA kinase phosphorylates not only Ser-tRNA to phosphoser-tRNA but also Gp of the 5-termini of tRNA to pGp. This study gives a new role to mammalian tRNA kinase.     

Crystal structure analysis reveals functional flexibility in the selenocysteine-specific tRNA from mouse

Background

Selenocysteine tRNAs (tRNA^Sec) exhibit a number of unique identity elements that are recognized specifically by proteins of the selenocysteine biosynthetic pathways and decoding machineries. Presently, these identity elements and the mechanisms by which they are interpreted by tRNA^Sec-interacting factors are incompletely understood.

Methodology/Principal Findings

We applied rational mutagenesis to obtain well diffracting crystals of murine tRNA^Sec. tRNA^Sec lacking the single-stranded 3′-acceptor end (^ΔGCCARNA^Sec) yielded a crystal structure at 2.0 Å resolution. The global structure of ^ΔGCCARNA^Sec resembles the structure of human tRNA^Sec determined at 3.1 Å resolution. Structural comparisons revealed flexible regions in tRNA^Sec used for induced fit binding to selenophosphate synthetase. Water molecules located in the present structure were involved in the stabilization of two alternative conformations of the anticodon stem-loop. Modeling of a 2′-O-methylated ribose at position U34 of the anticodon loop as found in a sub-population of tRNA^Sec in vivo showed how this modification favors an anticodon loop conformation that is functional during decoding on the ribosome. Soaking of crystals in Mn²⁺-containing buffer revealed eight potential divalent metal ion binding sites but the located metal ions did not significantly stabilize specific structural features of tRNA^Sec.

Conclusions/Significance

We provide the most highly resolved structure of a tRNA^Sec molecule to date and assessed the influence of water molecules and metal ions on the molecule''s conformation and dynamics. Our results suggest how conformational changes of tRNA^Sec support its interaction with proteins.