Loss of housekeeping selenoprotein expression in mouse liver modulates lipoprotein metabolism

Selenium is incorporated into proteins as selenocysteine (Sec), which is dependent on its specific tRNA, designated tRNA^[Ser]Sec. Targeted removal of the tRNA^[Ser]Sec gene (Trsp) in mouse hepatocytes previously demonstrated the importance of selenoproteins in liver function. Herein, analysis of plasma proteins in this Trsp knockout mouse revealed increases in apolipoprotein E (ApoE) that was accompanied by elevated plasma cholesterol levels. The expression of genes involved in cholesterol biosynthesis, metabolism and transport were also altered in knockout mice. Additionally, in two transgenic Trsp mutant mouse lines (wherein only housekeeping selenoprotein synthesis was restored), the expression of ApoE, as well as genes involved in cholesterol biosynthesis, metabolism and transport were similar to those observed in wild type mice. These data correlate with reports that selenium deficiency results in increased levels of ApoE, indicating for the first time that housekeeping selenoproteins have a role in regulating lipoprotein biosynthesis and metabolism.     

Osteo-Chondroprogenitor–Specific Deletion of the Selenocysteine tRNA Gene,Trsp, Leads to Chondronecrosis and Abnormal Skeletal Development: A Putative Model for Kashin-Beck Disease

Kashin-Beck disease, a syndrome characterized by short stature, skeletal deformities, and arthropathy of multiple joints, is highly prevalent in specific regions of Asia. The disease has been postulated to result from a combination of different environmental factors, including contamination of barley by mold mycotoxins, iodine deficiency, presence of humic substances in drinking water, and, importantly, deficiency of selenium. This multifunctional trace element, in the form of selenocysteine, is essential for normal selenoprotein function, including attenuation of excessive oxidative stress, and for the control of redox-sensitive molecules involved in cell growth and differentiation. To investigate the effects of skeletal selenoprotein deficiency, a Cre recombinase transgenic mouse line was used to trigger Trsp gene deletions in osteo-chondroprogenitors. Trsp encodes selenocysteine tRNA^[Ser]Sec, required for the incorporation of selenocysteine residues into selenoproteins. The mutant mice exhibited growth retardation, epiphyseal growth plate abnormalities, and delayed skeletal ossification, as well as marked chondronecrosis of articular, auricular, and tracheal cartilages. Phenotypically, the mice thus replicated a number of the pathological features of Kashin-Beck disease, supporting the notion that selenium deficiency is important to the development of this syndrome.     

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.     

Expression of Selenoproteins Is Maintained in Mice Carrying Mutations in SECp43, the tRNA Selenocysteine 1 Associated Protein (Trnau1ap)

Selenocysteine tRNA 1 associated protein (Trnau1ap) has been characterized as a tRNA^[Ser]Sec-binding protein of 43 kDa, hence initially named SECp43. Previous studies reported its presence in complexes containing tRNA^[Ser]Sec implying a role of SECp43 as a co-factor in selenoprotein expression. We generated two conditionally mutant mouse models targeting exons 3+4 and exons 7+8 eliminating parts of the first RNA recognition motif or of the tyrosine-rich domain, respectively. Constitutive inactivation of exons 3+4 of SECp43 apparently did not affect the mice or selenoprotein expression in several organs. Constitutive deletion of exons 7+8 was embryonic lethal. We therefore generated hepatocyte-specific Secp43 knockout mice and characterized selenoprotein expression in livers of mutant mice. We found no significant changes in the levels of ⁷⁵Se-labelled hepatic proteins, selenoprotein levels as determined by Western blot analysis, enzymatic activity or selenoprotein mRNA abundance. The methylation pattern of tRNA^[Ser]Sec remained unchanged. Truncated Secp43 ^Δ7,8mRNA increased in Secp43-mutant livers suggesting auto-regulation of Secp43 mRNA abundance. We found no signs of liver damage in Secp433-mutant mice, but neuron-specific deletion of exons 7+8 impaired motor performance, while not affecting cerebral selenoprotein expression or cerebellar development. These findings suggest that the targeted domains in the SECp43 protein are not essential for selenoprotein biosynthesis in hepatocytes and neurons. Whether the remaining second RNA recognition motif plays a role in selenoprotein biosynthesis and which other cellular process depends on SECp43 remains to be determined.     

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.     

Osteo-Chondroprogenitor–Specific Deletion of the Selenocysteine tRNA Gene, Trsp, Leads to Chondronecrosis and Abnormal Skeletal Development: A Putative Model for Kashin-Beck Disease

Kashin-Beck disease, a syndrome characterized by short stature, skeletal deformities, and arthropathy of multiple joints, is highly prevalent in specific regions of Asia. The disease has been postulated to result from a combination of different environmental factors, including contamination of barley by mold mycotoxins, iodine deficiency, presence of humic substances in drinking water, and, importantly, deficiency of selenium. This multifunctional trace element, in the form of selenocysteine, is essential for normal selenoprotein function, including attenuation of excessive oxidative stress, and for the control of redox-sensitive molecules involved in cell growth and differentiation. To investigate the effects of skeletal selenoprotein deficiency, a Cre recombinase transgenic mouse line was used to trigger Trsp gene deletions in osteo-chondroprogenitors. Trsp encodes selenocysteine tRNA^[Ser]Sec, required for the incorporation of selenocysteine residues into selenoproteins. The mutant mice exhibited growth retardation, epiphyseal growth plate abnormalities, and delayed skeletal ossification, as well as marked chondronecrosis of articular, auricular, and tracheal cartilages. Phenotypically, the mice thus replicated a number of the pathological features of Kashin-Beck disease, supporting the notion that selenium deficiency is important to the development of this syndrome.     

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.     

大肠杆菌tRNASec关键核苷酸位点

在原核生物中,硒蛋白合成需要tRNA~(Sec) (SelC)与硒代半胱氨酸合成(Sec synthase, SelA)、硒代半胱氨酸特异性延伸因子(Sec-specificelongationfactor,SelB)之间相互作用。【目的】基于大肠杆菌掺硒机器,寻找tRNA~(Sec)骨架上关键核苷酸位点,为解决硒蛋白目前面临的掺硒效率较低、产量低的问题提供新思路。【方法】以大鼠细胞质型硫氧还蛋白还原酶(thioredoxinreductase1,TrxR1)为掺硒模式蛋白为定点突变tRNA~(Sec),转化至BL21 (DE3) gor-获得阳性重组菌株(携带pET-TRSter/pSUABC’),用于表达大鼠硒蛋白TrxR1,然后使用2￠,5￠ADP-Sepharose亲和层析和凝胶过滤两步法分离纯化TrxR1,最后利用经典硒依赖型DTNB还原反应测定TrxR1的酶活,分析关键核苷酸位点,评价掺硒效率。【结果】在存在SECIS元件的前提下,当SelA、SelB、tRNA~(Sec)共表达时,与野生型相比,携带突变型tRNA~(Sec)所共表达的TrxR1酶活力呈现不同程度的降低,其中E.colitRNA~(Sec)的G18、G19这两个位点的所有的TrxR1酶活远低于野生型(10%);然而,a26和b7的酶活相对较高。【结论】E. coli tRNA~(Sec)骨架上G18和G19位点对于维持tRNA稳定性和灵活性发挥了关键作用,位点突变引起tRNA结构变化会影响tRNA~(Sec)与掺硒元件的互作,因此有望通过改造tRNA核苷酸位点来提高硒蛋白的掺硒效率。     

Inhibition of Cellular Methyltransferases Promotes Endothelial Cell Activation by Suppressing Glutathione Peroxidase 1 Protein Expression

S-Adenosylhomocysteine (SAH) is a negative regulator of most methyltransferases and the precursor for the cardiovascular risk factor homocysteine. We have previously identified a link between the homocysteine-induced suppression of the selenoprotein glutathione peroxidase 1 (GPx-1) and endothelial dysfunction. Here we demonstrate a specific mechanism by which hypomethylation, promoted by the accumulation of the homocysteine precursor SAH, suppresses GPx-1 expression and leads to inflammatory activation of endothelial cells. The expression of GPx-1 and a subset of other selenoproteins is dependent on the methylation of the tRNA^Sec to the Um34 form. The formation of methylated tRNA^Sec facilitates translational incorporation of selenocysteine at a UGA codon. Our findings demonstrate that SAH accumulation in endothelial cells suppresses the expression of GPx-1 to promote oxidative stress. Hypomethylation stress, caused by SAH accumulation, inhibits the formation of the methylated isoform of the tRNA^Sec and reduces GPx-1 expression. In contrast, under these conditions, the expression and activity of thioredoxin reductase 1, another selenoprotein, is increased. Furthermore, SAH-induced oxidative stress creates a proinflammatory activation of endothelial cells characterized by up-regulation of adhesion molecules and an augmented capacity to bind leukocytes. Taken together, these data suggest that SAH accumulation in endothelial cells can induce tRNA^Sec hypomethylation, which alters the expression of selenoproteins such as GPx-1 to contribute to a proatherogenic endothelial phenotype.     

The Specific Elongation Factor to Selenocysteine Incorporation in Escherichia coli: Unique tRNASec Recognition and its Interactions

Several molecular mechanisms are involved in the genetic code interpretation during translation, as codon degeneration for the incorporation of rare amino acids. One mechanism that stands out is selenocysteine (Sec), which requires a specific biosynthesis and incorporation pathway. In Bacteria, the Sec biosynthesis pathway has unique features compared with the eukaryote pathway as Ser to Sec conversion mechanism is accomplished by a homodecameric enzyme (selenocysteine synthase, SelA) followed by the action of an elongation factor (SelB) responsible for delivering the mature Sec-tRNA^Sec into the ribosome by the interaction with the Selenocysteine Insertion Sequence (SECIS). Besides this mechanism being already described, the sequential events for Sec-tRNA^Sec and SECIS specific recognition remain unclear. In this study, we determined the order of events of the interactions between the proteins and RNAs involved in Sec incorporation. Dissociation constants between SelB and the native as well as unacylated-tRNA^Sec variants demonstrated that the acceptor stem and variable arm are essential for SelB recognition. Moreover, our data support the sequence of molecular events where GTP-activated SelB strongly interacts with SelA.tRNA^Sec. Subsequently, SelB.GTP.tRNA^Sec recognizes the mRNA SECIS to deliver the tRNA^Sec to the ribosome. SelB in complex with its specific RNAs were examined using Hydrogen/Deuterium exchange mapping that allowed the determination of the molecular envelopes and its secondary structural variations during the complex assembly. Our results demonstrate the ordering of events in Sec incorporation and contribute to the full comprehension of the tRNA^Sec role in the Sec amino acid biosynthesis, as well as extending the knowledge of synthetic biology and the expansion of the genetic code.     

Selenoprotein Expression in Macrophages Is Critical for Optimal Clearance of Parasitic Helminth Nippostrongylus brasiliensis

The plasticity of macrophages is evident in helminthic parasite infections, providing protection from inflammation. Previously we demonstrated that the micronutrient selenium induces a phenotypic switch in macrophage activation from a classically activated (pro-inflammatory; M1/CAM) toward an alternatively activated (anti-inflammatory; M2/AAM) phenotype, where cyclooxygenase (COX)-dependent cyclopentenone prostaglandin J₂ (15d-PGJ₂) plays a key role. Here, we hypothesize that dietary selenium modulates macrophage polarization toward an AAM phenotype to assist in the increasing clearance of adult Nippostrongylus brasiliensis, a gastrointestinal nematode parasite. Mice on a selenium-adequate (0.08 ppm) diet significantly augmented intestinal AAM presence while decreasing adult worms and fecal egg production when compared with infection of mice on selenium-deficient (<0.01 ppm) diet. Further increase in dietary selenium to supraphysiological levels (0.4 ppm) had very little or no impact on worm expulsion. Normal adult worm clearance and enhanced AAM marker expression were observed in the selenium-supplemented Trsp^fl/flCre^WT mice that express selenoproteins driven by tRNA^Sec (Trsp), whereas N. brasiliensis-infected Trsp^fl/flCre^LysM selenium-supplemented mice showed a decreased clearance, with lowered intestinal expression of several AAM markers. Inhibition of the COX pathway with indomethacin resulted in delayed worm expulsion in selenium-adequate mice. This was rescued with 15d-PGJ₂, which partially recapitulated the effect of selenium supplementation on fecal egg output in addition to increasing markers of AAMs in the small intestine. Antagonism of PPARγ blocked the effect of selenium. These results suggest that optimal expression of selenoproteins and selenium-dependent production of COX-derived endogenous prostanoids, such as Δ¹²-PGJ₂ and 15d-PGJ₂, may regulate AAM activation to enhance anti-helminthic parasite responses.     

Mouse Model Reveals the Role of RERE in Cerebellar Foliation and the Migration and Maturation of Purkinje Cells

Nuclear receptors and their coregulators play a critical role in brain development by regulating the spatiotemporal expression of their target genes. The arginine-glutamic acid dipeptide repeats gene (Rere) encodes a nuclear receptor coregulator previously known as Atrophin 2. In the developing cerebellum, RERE is expressed in the molecular layer, the Purkinje cell layer and the granule cell layer but not in granule cell precursors. To study RERE''s role in cerebellar development, we used RERE-deficient embryos bearing a null allele (om) and a hypomorphic allele (eyes3) of Rere (Rere ^om/eyes3). In contrast to wild-type embryos, formation of the principal fissures in these RERE-deficient embryos was delayed and the proliferative activity of granule cell precursors (GCPs) was reduced at E18.5. This reduction in proliferation was accompanied by a decrease in the expression of sonic hedgehog (SHH), which is secreted from Purkinje cells and is required for normal GCP proliferation. The maturation and migration of Purkinje cells in Rere ^om/eyes3 embryos was also delayed with decreased numbers of post-migratory Purkinje cells in the cerebellum. During the postnatal period, RERE depletion caused incomplete division of lobules I/II and III due to truncated development of the precentral fissure in the cerebellar vermis, abnormal development of lobule crus I and lobule crus II in the cerebellar hemispheres due to attenuation of the intercrural fissure, and decreased levels of Purkinje cell dendritic branching. We conclude that RERE-deficiency leads to delayed development of the principal fissures and delayed maturation and migration of Purkinje cells during prenatal cerebellar development and abnormal cerebellar foliation and Purkinje cell maturation during postnatal cerebellar development.     

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.     

GM1 ganglioside as a marker for neuronal differentiation in mouse cerebellum

The distribution of GM1 ganglioside in developing mouse cerebellum was monitored by indirect immunofluorescent detection of choleragenoid receptors. In frozen sections of cerebellum from mice 5 to 10 days old, fluorescence is observed on granule cells in the inner rows of the external granular layer, in the growing molecular layer, the Purkinje cell layer, and the internal granular layer. In sections of adult mice, fluorescence is restricted to the bodies of Purkinje and internal granule neurons. The percentage of fluorescent dissociated or cultured cerebellar cells increases with the postnatal age of the mouse or the duration of time in vitro. No fluorescence is observed in the absence of choleragenoid or if the test material is extracted with chloroform:methanol. To determine whether the expression of surface GM1 ganglioside in culture is a reflection of a developmental program, mice are injected at particular times with [³H]thymidine and cerebellar cultures processed for simultaneous autoradiography and immunofluorescence. Granule cells from 8-day-old mice having cholera toxin receptors at 20 hr in vitro are a distinct population born 1 day or earlier prior to sacrifice. Cells synthesizing DNA on the day of sacrifice are not fluorescent at 20 hr in vitro. This observation correlates well with immunohistological results showing a lack of fluorescence in the outer proliferative rows of the external granular layer. Therefore GM1 ganglioside is not present on granule cell precursors but is expressed at some time after the cells become postmitotic. GM1 ganglioside is detected on growing parallel fibers in situ and neurites in vitro but not on adult axons, suggesting differential localization at a later stage of development.     

Vav3-deficient Mice Exhibit a Transient Delay in Cerebellar Development

Vav3 is a guanosine diphosphate/guanosine triphosphate exchange factor for Rho/Rac GTPases that has been involved in functions related to the hematopoietic system, bone formation, cardiovascular regulation, angiogenesis, and axon guidance. We report here that Vav3 is expressed at high levels in Purkinje and granule cells, suggesting additional roles for this protein in the cerebellum. Consistent with this hypothesis, we demonstrate using Vav3-deficient mice that this protein contributes to Purkinje cell dendritogenesis, the survival of granule cells of the internal granular layer, the timely migration of granule cells of the external granular layer, and to the formation of the cerebellar intercrural fissure. With the exception of the latter defect, the dysfunctions found in Vav3^−/− mice only occur at well-defined postnatal developmental stages and disappear, or become ameliorated, in older animals. Vav2-deficient mice do not show any of those defects. Using primary neuronal cultures, we show that Vav3 is important for dendrite branching, but not for primary dendritogenesis, in Purkinje and granule cells. Vav3 function in the cerebellum is functionally relevant, because Vav3^−/− mice show marked motor coordination and gaiting deficiencies in the postnatal period. These results indicate that Vav3 function contributes to the timely developmental progression of the cerebellum.     

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.