ABSTRACT
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.
Subject(s)
Archaeal Proteins/chemistry , Phosphotransferases/chemistry , RNA, Archaeal/chemistry , RNA, Transfer, Amino Acid-Specific/chemistry , Anticodon/chemistry , Archaeal Proteins/genetics , Base Sequence , Binding Sites , Crystallography , Methanococcales/enzymology , Models, Molecular , Molecular Sequence Data , Motion , Mutation , Phosphotransferases/genetics , Protein Binding , Protein Structure, TertiaryABSTRACT
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.
Subject(s)
Cysteine/biosynthesis , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Escherichia coli/metabolism , RNA, Transfer, Amino Acyl/metabolism , Base Sequence , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Formate Dehydrogenases/genetics , Formate Dehydrogenases/metabolism , Genetic Complementation Test , Molecular Sequence Data , Nucleic Acid Conformation , Substrate Specificity/geneticsABSTRACT
Selenocysteine and pyrrolysine, known as the 21st and 22nd amino acids, are directly inserted into growing polypeptides during translation. Selenocysteine is synthesized via a tRNA-dependent pathway and decodes UGA (opal) codons. The incorporation of selenocysteine requires the concerted action of specific RNA and protein elements. In contrast, pyrrolysine is ligated directly to tRNA(Pyl) and inserted into proteins in response to UAG (amber) codons without the need for complex re-coding machinery. Here we review the latest updates on the structure and mechanisms of molecules involved in Sec-tRNA(Sec) and Pyl-tRNA(Pyl) formation as well as the distribution of the Pyl-decoding trait.
Subject(s)
Genetic Code , Lysine/analogs & derivatives , RNA, Transfer, Amino Acyl/metabolism , Selenocysteine/genetics , Transfer RNA Aminoacylation , Codon, Terminator/genetics , Lysine/biosynthesis , Lysine/genetics , Selenocysteine/biosynthesisABSTRACT
Compared to bacteria, archaea and eukaryotes employ an additional enzyme for the biosynthesis of selenocysteine (Sec), the 21(st) natural amino acid (aa). An essential RNA-dependent kinase, O-phosphoseryl-tRNA(Sec) kinase (PSTK), converts seryl-tRNA(Sec) to O-phosphoseryl-tRNA(Sec), the immediate precursor of selenocysteinyl-tRNA(Sec). The sequence of Methanocaldococcus jannaschii PSTK (MjPSTK) suggests an N-terminal kinase domain (177 aa) followed by a presumed tRNA binding region (75 aa). The structures of MjPSTK complexed with ADP and AMPPNP revealed that this enzyme belongs to the P-loop kinase class, and that the kinase domain is closely related to gluconate kinase and adenylate kinase. ATP is bound by the P-loop domain (residues 11-18). Formed by antiparallel dimerization of two PSTK monomers, the enzyme structure shows a deep groove with positive electrostatic potential. Located in this groove is the enzyme's active site, which biochemical and genetic data suggest is composed of Asp-41, Arg-44, Glu-55, Tyr-82, Tyr-83, Met-86, and Met-132. Based on structural comparison with Escherichia coli adenylate kinase a docking model was generated that assigns these amino acids to the recognition of the terminal A76-Ser moieties of Ser-tRNA(Sec). The geometry and electrostatic environment of the groove in MjPSTK are perfectly complementary to the unusually long acceptor helix of tRNA(Sec).
Subject(s)
Archaeal Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , RNA, Transfer, Amino Acyl/metabolism , Selenocysteine/metabolism , Archaeal Proteins/chemistry , Archaeal Proteins/genetics , Binding Sites/genetics , Crystallization , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/growth & development , Escherichia coli/metabolism , Genetic Complementation Test , Methanococcales/enzymology , Methanococcales/genetics , Methanococcales/metabolism , Models, Molecular , Mutation , Protein Binding , Protein Conformation , Protein Multimerization , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Protein Structure, Tertiary , RNA, Transfer, Amino Acyl/chemistryABSTRACT
Selenocysteine is the only genetically encoded amino acid in humans whose biosynthesis occurs on its cognate transfer RNA (tRNA). O-Phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS) catalyzes the final step of selenocysteine formation by a poorly understood tRNA-dependent mechanism. The crystal structure of human tRNA(Sec) in complex with SepSecS, phosphoserine, and thiophosphate, together with in vivo and in vitro enzyme assays, supports a pyridoxal phosphate-dependent mechanism of Sec-tRNA(Sec) formation. Two tRNA(Sec) molecules, with a fold distinct from other canonical tRNAs, bind to each SepSecS tetramer through their 13-base pair acceptor-TPsiC arm (where Psi indicates pseudouridine). The tRNA binding is likely to induce a conformational change in the enzyme's active site that allows a phosphoserine covalently attached to tRNA(Sec), but not free phosphoserine, to be oriented properly for the reaction to occur.
Subject(s)
Amino Acyl-tRNA Synthetases/chemistry , Amino Acyl-tRNA Synthetases/metabolism , RNA, Transfer, Amino Acid-Specific/chemistry , RNA, Transfer, Amino Acid-Specific/metabolism , RNA, Transfer, Amino Acyl/metabolism , Selenocysteine/biosynthesis , Base Sequence , Biocatalysis , Catalytic Domain , Crystallography, X-Ray , Humans , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Phosphates/chemistry , Phosphates/metabolism , Phosphoserine/chemistry , Phosphoserine/metabolism , Protein Conformation , Protein Multimerization , Protein Structure, Secondary , Selenocysteine/geneticsABSTRACT
Selenocysteine (Sec) is the 21st genetically encoded amino acid found in organisms from all three domains of life. Sec biosynthesis is unique in that it always proceeds from an aminoacyl-tRNA precursor. Even though Sec biosynthesis in bacteria was established almost two decades ago, only recently the pathway was elucidated in archaea and eukaryotes. While other aspects of Sec biology have been reviewed previously (Allmang and Krol, Biochimie 2006;88:1561-1571, Hatfield et al., Prog Nucleic Acid Res Mol Biol 2006;81:97-142, Squires and Berry, IUBMB Life 2008;60:232-235), here we review the biochemistry and evolution of Sec biosynthesis and coding and show how the knowledge of an archaeal cysteine biosynthesis pathway helped to uncover the route to Sec formation in archaea and eukaryotes.
Subject(s)
Archaea/genetics , Cysteine/biosynthesis , Evolution, Molecular , Genetic Code/genetics , Genetics/history , Selenocysteine/biosynthesis , Selenocysteine/genetics , Archaea/metabolism , History, 20th Century , History, 21st Century , HumansABSTRACT
Although nascent noncoding RNAs can undergo maturation to functional RNAs or degradation by quality control pathways, the events that influence the choice of pathway are not understood. We report that the targeting of pre-tRNAs and certain other noncoding RNAs for decay by the TRAMP pathway is strongly influenced by competition between the La protein and the Rex1 exonuclease for access to their 3' ends. The La protein binds the 3' ends of many nascent noncoding RNAs, protecting them from exonucleases. We demonstrate that unspliced, end-matured, partially aminoacylated pre-tRNAs accumulate in yeast lacking the TRAMP subunit Trf4p, indicating that these pre-tRNAs normally undergo decay. By comparing RNA extracted from wild-type and mutant yeast strains, we show that Rex1p is the major exonuclease involved in pre-tRNA trailer trimming and may also function in nuclear CCA turnover. As the accumulation of end-matured pre-tRNAs in trf4Delta cells requires Rex1p, these pre-tRNAs are formed by exonucleolytic trimming. Accumulation of truncated forms of 5S rRNA and SRP RNA in trf4Delta cells also requires Rex1p. Overexpression of the La protein Lhp1p reduces both exonucleolytic pre-tRNA trimming in wild-type cells and the accumulation of defective RNAs in trf4Delta cells. Our experiments reveal that one consequence of Rex1p-dependent 3' trimming is the generation of aberrant RNAs that are targeted for decay by TRAMP.
Subject(s)
DNA-Directed DNA Polymerase/metabolism , Exoribonucleases/metabolism , RNA 3' End Processing , RNA Precursors/metabolism , RNA, Transfer/metabolism , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , DNA-Directed DNA Polymerase/genetics , Exoribonucleases/genetics , RNA Stability , RNA, Fungal/metabolism , RNA, Untranslated/metabolism , RNA-Binding Proteins/genetics , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Many prokaryotes form the amide aminoacyl-tRNAs glutaminyl-tRNA and asparaginyl-tRNA by tRNA-dependent amidation of the mischarged tRNA species, glutamyl-tRNA(Gln) or aspartyl-tRNA(Asn). Archaea employ two such amidotransferases, GatCAB and GatDE, while bacteria possess only one, GatCAB. The Methanothermobacter thermautotrophicus GatDE is slightly more efficient using Asn as an amide donor than Gln (k(cat)/K(M) of 5.4 s(-1)/mM and 1.2 s(-1)/mM, respectively). Unlike the bacterial GatCAB enzymes studied to date, the M. thermautotrophicus GatCAB uses Asn almost as well as Gln as an amide donor (k(cat)/K(M) of 5.7 s(-1)/mM and 16.7 s(-1)/mM, respectively). In contrast to the initial characterization of the M. thermautotrophicus GatCAB as being able to form Asn-tRNA(Asn) and Gln-tRNA(Gln), our data demonstrate that while the enzyme is able to transamidate Asp-tRNA(Asn) (k(cat)/K(M) of 125 s(-1)/mM) it is unable to transamidate M. thermautotrophicus Glu-tRNA(Gln). However, M. thermautotrophicus GatCAB is capable of transamidating Glu-tRNA(Gln) from H. pylori or B. subtilis, and M. thermautotrophicus Glu-tRNA(Asn). Thus, M. thermautotrophicus encodes two amidotransferases, each with its own activity, GatDE for Gln-tRNA and GatCAB for Asn-tRNA synthesis.
Subject(s)
Methanobacterium/enzymology , Nitrogenous Group Transferases/metabolism , RNA, Transfer, Asn/metabolism , RNA, Transfer, Gln/metabolism , Asparagine/metabolism , Bacillus subtilis/enzymology , Glutamine/metabolism , Helicobacter pylori/enzymologyABSTRACT
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).
Subject(s)
Archaeal Proteins/metabolism , Methanococcales/enzymology , Methanococcus/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , RNA, Transfer, Amino Acid-Specific/chemistry , RNA, Transfer, Ser/chemistry , Adenosine Triphosphatases/metabolism , Animals , Anticodon/chemistry , Bacteria/genetics , Base Pairing , Base Sequence , Humans , Molecular Sequence Data , Phosphorylation , RNA, Transfer, Amino Acyl/metabolism , Substrate SpecificityABSTRACT
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.
Subject(s)
Archaeal Proteins/metabolism , Methanococcales/enzymology , Phosphotransferases/metabolism , RNA, Transfer, Amino Acyl/metabolism , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Archaeal Proteins/chemistry , Archaeal Proteins/genetics , Binding Sites , Evolution, Molecular , Kinetics , Models, Molecular , Mutation , Phosphotransferases/chemistry , Phosphotransferases/genetics , Phylogeny , Protein Structure, Tertiary , Sequence Alignment , Single-Strand Specific DNA and RNA Endonucleases/metabolism , Substrate SpecificityABSTRACT
The micronutrient selenium is present in proteins as selenocysteine (Sec). In eukaryotes and archaea, Sec is formed in a tRNA-dependent conversion of O-phosphoserine (Sep) by O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS). Here, we present the crystal structure of Methanococcus maripaludis SepSecS complexed with PLP at 2.5 A resolution. SepSecS, a member of the Fold Type I PLP enzyme family, forms an (alpha2)2 homotetramer through its N-terminal extension. The active site lies on the dimer interface with each monomer contributing essential residues. In contrast to other Fold Type I PLP enzymes, Asn247 in SepSecS replaces the conserved Asp in binding the pyridinium nitrogen of PLP. A structural comparison with Escherichia coli selenocysteine lyase allowed construction of a model of Sep binding to the SepSecS catalytic site. Mutations of three conserved active site arginines (Arg72, Arg94, Arg307), protruding from the neighboring subunit, led to loss of in vivo and in vitro activity. The lack of active site cysteines demonstrates that a perselenide is not involved in SepSecS-catalyzed Sec formation; instead, the conserved arginines may facilitate the selenation reaction. Structural phylogeny shows that SepSecS evolved early in the history of PLP enzymes, and indicates that tRNA-dependent Sec formation is a primordial process.
Subject(s)
Archaeal Proteins/chemistry , Methanococcus/enzymology , Selenocysteine/metabolism , Transferases/chemistry , Amino Acid Sequence , Archaeal Proteins/classification , Archaeal Proteins/genetics , Archaeoglobus fulgidus/enzymology , Binding Sites , Escherichia coli/enzymology , Humans , Models, Molecular , Molecular Sequence Data , Mutation , Phosphoserine/chemistry , Phylogeny , Sequence Alignment , Transferases/classification , Transferases/geneticsABSTRACT
The important identity elements in tRNA(Gln) and tRNA(Asn) for bacterial GatCAB and in tRNA(Gln) for archaeal GatDE are the D-loop and the first base pair of the acceptor stem. Here we show that Methanothermobacter thermautotrophicus GatCAB, the archaeal enzyme, is different as it discriminates Asp-tRNA(Asp) and Asp-tRNA(Asn) by use of U49, the D-loop and to a lesser extent the variable loop. Since archaea possess the tRNA(Gln)-specific amidotransferase GatDE, the archaeal GatCAB enzyme evolved to recognize different elements in tRNA(Asn) than those recognized by GatDE or by the bacterial GatCAB enzyme in their tRNA substrates.
Subject(s)
Archaeal Proteins/chemistry , Evolution, Molecular , Methanobacteriaceae/enzymology , RNA, Transfer, Asn/chemistry , Transaminases/chemistry , Nucleic Acid Conformation , RNA, Archaeal/chemistry , RNA, Transfer, Amino Acyl/chemistry , RNA, Transfer, Gln/chemistryABSTRACT
Glutaminyl-transfer RNA (Gln-tRNA(Gln)) in archaea is synthesized in a pretranslational amidation of misacylated Glu-tRNA(Gln) by the heterodimeric Glu-tRNA(Gln) amidotransferase GatDE. Here we report the crystal structure of the Methanothermobacter thermautotrophicus GatDE complexed to tRNA(Gln) at 3.15 angstroms resolution. Biochemical analysis of GatDE and of tRNA(Gln) mutants characterized the catalytic centers for the enzyme's three reactions (glutaminase, kinase, and amidotransferase activity). A 40 angstrom-long channel for ammonia transport connects the active sites in GatD and GatE. tRNA(Gln) recognition by indirect readout based on shape complementarity of the D loop suggests an early anticodon-independent RNA-based mechanism for adding glutamine to the genetic code.
Subject(s)
Genetic Code , Glutamine/metabolism , Methanobacteriaceae/enzymology , Nitrogenous Group Transferases/chemistry , Nitrogenous Group Transferases/metabolism , RNA, Archaeal/chemistry , RNA, Transfer, Gln/chemistry , Acylation , Adenosine Triphosphate/metabolism , Ammonia/metabolism , Anticodon , Binding Sites , Catalytic Domain , Computer Simulation , Crystallography, X-Ray , Dimerization , Hydrogen Bonding , Magnesium/metabolism , Methanobacteriaceae/genetics , Models, Molecular , Mutation , Nucleic Acid Conformation , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Structure, Tertiary , RNA, Archaeal/metabolism , RNA, Transfer, Gln/metabolismABSTRACT
Although the La protein stabilizes nascent pre-tRNAs from nucleases, influences the pathway of pre-tRNA maturation, and assists correct folding of certain pre-tRNAs, it is dispensable for growth in both budding and fission yeast. Here we show that the Saccharomyces cerevisiae La shares functional redundancy with both tRNA modification enzymes and other proteins that contact tRNAs during their biogenesis. La is important for growth in the presence of mutations in either the arginyl tRNA synthetase or the tRNA modification enzyme Trm1p. In addition, two pseudouridine synthases, PUS3 and PUS4, are important for growth in strains carrying a mutation in tRNA(Arg)(CCG) and are essential when La is deleted in these strains. Depletion of Pus3p results in accumulation of the aminoacylated mutant tRNA(Arg)(CCG) in nuclei, while depletion of Pus4p results in decreased stability of the mutant tRNA. Interestingly, the degradation of mutant unstable forms of tRNA(Arg)(CCG) does not require the Trf4p poly(A) polymerase, suggesting that yeast cells possess multiple pathways for tRNA decay. These data demonstrate that La functions redundantly with both tRNA modifications and proteins that associate with tRNAs to achieve tRNA structural stability and efficient biogenesis.
Subject(s)
Amino Acyl-tRNA Synthetases/metabolism , Nucleic Acid Conformation , RNA, Transfer, Arg/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Base Sequence , Blotting, Northern , DNA Primers , In Situ Hybridization , Mutagenesis , Plasmids , Saccharomyces cerevisiae/genetics , tRNA Methyltransferases/metabolismABSTRACT
Trypanosoma brucei lacks mitochondrial genes encoding tRNAs and must import nuclearly encoded tRNAs from the cytosol. The mechanism and specificity of this process remain unclear. We have identified a unique sequence motif, YGG(C/A)RRC, upstream of the genes encoding mitochondrially localized tRNAs in T. brucei. Both in vitro import studies and in vivo transfection studies indicate that deletion of the YGG(C/A)RRC sequence alters mitochondrial localization of tRNA(Leu), and in vivo studies also show a decrease in the cellular abundance of tRNA(Leu). These studies provide direct evidence for cis-acting RNA motifs within precursor tRNAs that facilitate the selection of tRNAs for mitochondrial import in trypanosomes. Furthermore, we found that mutations to the YGG(C/A)RRC sequence also altered the intracellular distribution of other endogenous tRNAs, suggesting a general role for this sequence in tRNA trafficking in trypanosomes.
