Seril-tRNA-sintetaze iz metanogenih arheja: supresija bakterijskih amber mutacija i heterologna toksičnost

Methanogenic archaea possess unusual seryl-tRNA synthetases (SerRS), evolutionarily distinct from the SerRSs found in other archaea, eucaryotes and bacteria. Our recent X-ray structural analysis of Methanosarcina barkeri SerRS revealed an idiosyncratic N-terminal domain and catalytic zinc ion in the active site. To shed further light on substrate discrimination by methanogenic-type SerRS, we set up to explore in vivo the interaction of methanogenic-type SerRSs with their cognate tRNAs in Escherichia coli or Saccharomyces cerevisiae. The expression of various methanogenic-type SerRSs was toxic for E. coli, resulting in the synthesis of erroneous proteins, as revealed by β-galactosidase stability assay. Although SerRSs from methanogenic archaea recognize tRNAsSer from all three domains of life in vitro, the toxicity presumably precluded the complementation of endogenous SerRS function in both, E. coli and S. cerevisiae. However, despite the observed toxicity, coexpression of methanogenic-type SerRS with its cognate tRNA suppressed bacterial amber mutation.Metanogene arheje imaju neobične seril-tRNA-sintetaze (SerRS), evolucijski udaljene od SerRS koje se mogu naći u drugih arheja, eukariota i bakterija. Naša nedavna analiza kristalne strukture SerRS iz metanogene arheje Methanosarina barkeri otkrila je karakterističnu N-terminalnu domenu i katalitički ion cinka na aktivnom mjestu. Da bi se rasvijetlio način na koji metanogeni tip SerRS diskriminira supstrate, autori su istraživali in vivo interakciju metanogenog tipa SerRS s pripadnim molekulama tRNA u bakteriji Escherichia coli ili kvascu Saccharomyces cerevisiae. Ekspresija raznih SerRS metanogenoga tipa bila je toksična za bakteriju E. coli, te je rezultirala sintezom proteina s greškama u aminokiselinskom sastavu, što se vidi iz testa stabilnosti β-galaktozidaze. Iako je SerRS iz metanogenih arheja mogla prepoznati tRNASer iz sve tri domene života in vitro, vjerojatno je toksičnost zasjenila komplementaciju funkcije endogene SerRS u bakteriji E. coli i kvascu S. cerevisiae. Međutim, unatoč toksičnosti, koekspresija metanogenoga tipa SerRS s pripadnom tRNA suprimirala je bakterijsku amber mutaciju

Peroxin Pex21p Interacts with C-terminal Noncatalytic Domain of Yeast Seryl-tRNA Synthetase and Forms a Specific Ternary Complex with tRNA\u3csup\u3eSer\u3c/sup\u3e

The seryl‐tRNA synthetase from Saccharomyces cerevisiae interacts with the peroxisome biogenesis‐related factor Pex21p. Several deletion mutants of seryl‐tRNA synthetase were constructed and inspected for their ability to interact with Pex21p in a yeast two‐hybrid assay, allowing mapping of the synthetase domain required for complex assembly. Deletion of the 13 C‐terminal amino acids abolished Pex21p binding to seryl‐tRNA synthetase. The catalytic parameters of purified truncated seryl‐tRNA synthetase, determined in the serylation reaction, were found to be almost identical to those of the native enzyme. In vivo loss of interaction with Pex21p was confirmed in vitro by coaffinity purification. These data indicate that the C‐terminally appended domain of yeast seryl‐tRNA synthetase does not participate in substrate binding, but instead is required for association with Pex21p. We further determined that Pex21p does not directly bind tRNA, and nor does it possess a tRNA‐binding motif, but it instead participates in the formation of a specific ternary complex with seryl‐tRNA synthetase and tRNASer, strengthening the interaction of seryl‐tRNA synthetase with its cognate tRNASer

Točnost sinteze seril-tRNA

The high level of translational fidelity is ensured by various types of quality control mechanisms, which are adapted to prevent or correct naturally occurring mistakes. Accurate aminoacyl-tRNA synthesis is mostly dependent on the specificity of the aminoacyl-tRNA synthetases (aaRS), i.e. their ability to choose among competing structurally similar substrates. Our studies have revealed that accurate seryl-tRNA synthesis in yeast and plants is accomplished via tRNA-assisted optimization of amino acid binding to the active site of seryl-tRNA synthetase (SerRS). Based on our recent kinetic data, a mechanism is proposed by which transient protein : RNA complex activates the cognate amino acid more efficiently and more specifically than the apoenzyme alone. This may proceed via a tRNA induced conformational change in the enzyme’s active site. The influence of tRNASer, on the activation of serine by SerRS variants mutated in the active site, is much less pronounced. Although SerRS misactivates structurally similar threonine in vitro, the formation of such erroneous threonyl-adenylate is reduced in the presence of nonchargeable tRNASer analog. Thus, the sequence-specific tRNA : SerRS interactions enhance the accuracy of amino acid recognition. Another type of quality control mechanism in tRNA serylation is assumed to be based on the complex formation between SerRS and a nonsynthetase protein. Using in vivo interaction screen, yeast peroxin Pex21p was identified as SerRS interacting protein. This was confirmed by an in vitro binding assay. Kinetic experiments performed in the presence of Pex21p revealed that this peroxin acts as an activator of seryl-tRNA synthetase in the aminoacylation reaction.Točnost biosinteze proteina nadziru različiti kontrolni mehanizmi koji sprečavaju ili ispravljaju gre{ke u translaciji. Specifičnost aminoacil tRNA-sintetaza (aaRS) pri izboru i kovalentnom povezivanju pripadnih aminokiselina i tRNA ključna je u ovom procesu. Istraživanja u našem laboratoriju pokazala su da se specifičnost i učinkovitost sinteze seril-tRNA u kvascu i biljkama povećavaju tRNA-ovisnim prilagođavanjem veznog mjesta za serin u aktivnom mjestu seril tRNA-sintetaze (SerRS). Dakle, makromolekularni kompleksi tRNA i enzima imaju bolja katalitička svojstva od apoenzima. Naši rezultati kinetike pokazuju da se vezanjem tRNA bitno mijenja konformacija veznog mjesta za serin u enzimu divljeg tipa, dok je konformacijska promjena slabija kod enzima s mutacijama u aktivnom mjestu. Iako SerRS može aktivirati i serinu sličan treonin, stvaranje treonil adenilata smanjeno je u prisutnosti aminoacilacijski inaktivnog analoga tRNA. Time je pokazano da su interakcije između pripadne tRNA i SerRS bitne za točan izbor aminokiseline. Djelotvornost serilacije povećava se i interakcijom SerRS s nesintetaznim proteinom, peroksinom Pex21p. Ta neočekivana interakcija uočena je prvo in vivo, pretragom kvaščeve biblioteke u sustavu dvaju hibrida sa SerRS kao interakcijskim proteinom. Interakcija je potvrđena in vitro. Kinetički eksperimenti pokazali su da Pex21p djeluje kao aktivator SerRS, što ovu neobičnu interakciju čini biološki značajnom jer povećava učinkovitost aminoaciliranja

Archaeal aminoacyl-tRNA synthetases interact with the ribosome to recycle tRNAs

Aminoacyl-tRNA synthetases (aaRS) are essential enzymes catalyzing the formation of aminoacyl-tRNAs, the immediate precursors for encoded peptides in ribosomal protein synthesis. Previous studies have suggested a link between tRNA aminoacylation and high-molecular-weight cellular complexes such as the cytoskeleton or ribosomes. However, the structural basis of these interactions and potential mechanistic implications are not well understood. To biochemically characterize these interactions we have used a system of two interacting archaeal aaRSs: an atypical methanogenic-type seryl-tRNA synthetase and an archaeal ArgRS. More specifically, we have shown by thermophoresis and surface plasmon resonance that these two aaRSs bind to the large ribosomal subunit with micromolar affinities. We have identified the L7/L12 stalk and the proteins located near the stalk base as the main sites for aaRS binding. Finally, we have performed a bioinformatics analysis of synonymous codons in the Methanothermobacter thermautotrophicus genome that supports a mechanism in which the deacylated tRNAs may be recharged by aaRSs bound to the ribosome and reused at the next occurrence of a codon encoding the same amino acid. These results suggest a mechanism of tRNA recycling in which aaRSs associate with the L7/L12 stalk region to recapture the tRNAs released from the preceding ribosome in polysome

Seryl-tRNA Synthetases from Methanogenic Archaea: Suppression of Bacterial Amber Mutation and Heterologous Toxicity

Methanogenic archaea possess unusual seryl-tRNA synthetases (SerRS), evolutionarily distinct from the SerRSs found in other archaea, eucaryotes and bacteria. Our recent X-ray structural analysis of Methanosarcina barkeri SerRS revealed an idiosyncratic N-terminal domain and catalytic zinc ion in the active site. To shed further light on substrate discrimination by methanogenic-type SerRS, we set up to explore in vivo the interaction of methanogenic-type SerRSs with their cognate tRNAs in Escherichia coli or Saccharomyces cerevisiae. The expression of various methanogenic-type SerRSs was toxic for E. coli, resulting in the synthesis of erroneous proteins, as revealed by β-galactosidase stability assay. Although SerRSs from methanogenic archaea recognize tRNAsSer from all three domains of life in vitro, the toxicity presumably precluded the complementation of endogenous SerRS function in both, E. coli and S. cerevisiae. However, despite the observed toxicity, coexpression of methanogenic-type SerRS with its cognate tRNA suppressed bacterial amber mutation

The Accuracy of Seryl-tRNA Synthesis

The high level of translational fidelity is ensured by various types of quality control mechanisms, which are adapted to prevent or correct naturally occurring mistakes. Accurate aminoacyl-tRNA synthesis is mostly dependent on the specificity of the aminoacyl-tRNA synthetases (aaRS), i.e. their ability to choose among competing structurally similar substrates. Our studies have revealed that accurate seryl-tRNA synthesis in yeast and plants is accomplished via tRNA-assisted optimization of amino acid binding to the active site of seryl-tRNA synthetase (SerRS). Based on our recent kinetic data, a mechanism is proposed by which transient protein : RNA complex activates the cognate amino acid more efficiently and more specifically than the apoenzyme alone. This may proceed via a tRNA induced conformational change in the enzyme’s active site. The influence of tRNASer, on the activation of serine by SerRS variants mutated in the active site, is much less pronounced. Although SerRS misactivates structurally similar threonine in vitro, the formation of such erroneous threonyl-adenylate is reduced in the presence of nonchargeable tRNASer analog. Thus, the sequence-specific tRNA : SerRS interactions enhance the accuracy of amino acid recognition. Another type of quality control mechanism in tRNA serylation is assumed to be based on the complex formation between SerRS and a nonsynthetase protein. Using in vivo interaction screen, yeast peroxin Pex21p was identified as SerRS interacting protein. This was confirmed by an in vitro binding assay. Kinetic experiments performed in the presence of Pex21p revealed that this peroxin acts as an activator of seryl-tRNA synthetase in the aminoacylation reaction

Cryo-EM structure of the archaeal 50S ribosomal subunit in complex with initiation factor 6 and implications for ribosome evolution

Translation of mRNA into proteins by the ribosome is universally conserved in all cellular life. The composition and complexity of the translation machinery differ markedly between the three domains of life. Organisms from the domain Archaea show an intermediate level of complexity, sharing several additional components of the translation machinery with eukaryotes that are absent in bacteria. One of these translation factors is initiation factor 6 (IF6), which associates with the large ribosomal subunit. We have reconstructed the 50S ribosomal subunit from the archaeon Methanothermobacter thermautotrophicus in complex with archaeal IF6 at 6.6 Å resolution using cryo-electron microscopy (EM). The structure provides detailed architectural insights into the 50S ribosomal subunit from a methanogenic archaeon through identification of the rRNA expansion segments and ribosomal proteins that are shared between this archaeal ribosome and eukaryotic ribosomes but are mostly absent in bacteria and in some archaeal lineages. Furthermore, the structure reveals that, in spite of highly divergent evolutionary trajectories of the ribosomal particle and the acquisition of novel functions of IF6 in eukaryotes, the molecular binding of IF6 on the ribosome is conserved between eukaryotes and archaea. The structure also provides a snapshot of the reductive evolution of the archaeal ribosome and offers new insights into the evolution of the translation system in archaea

Archaeal aminoacyl-tRNA synthetases interact with the ribosome to recycle tRNAs

Aminoacyl-tRNA synthetases (aaRS) are essential enzymes catalyzing the formation of aminoacyl-tRNAs, the immediate precursors for encoded peptides in ribosomal protein synthesis. Previous studies have suggested a link between tRNA aminoacylation and high-molecular-weight cellular complexes such as the cytoskeleton or ribosomes. However, the structural basis of these interactions and potential mechanistic implications are not well understood. To biochemically characterize these interactions we have used a system of two interacting archaeal aaRSs: an atypical methanogenic-type seryl-tRNA synthetase and an archaeal ArgRS. More specifically, we have shown by thermophoresis and surface plasmon resonance that these two aaRSs bind to the large ribosomal subunit with micromolar affinities. We have identified the L7/L12 stalk and the proteins located near the stalk base as the main sites for aaRS binding. Finally, we have performed a bioinformatics analysis of synonymous codons in the Methanothermobacter thermautotrophicus genome that supports a mechanism in which the deacylated tRNAs may be recharged by aaRSs bound to the ribosome and reused at the next occurrence of a codon encoding the same amino acid. These results suggest a mechanism of tRNA recycling in which aaRSs associate with the L7/L12 stalk region to recapture the tRNAs released from the preceding ribosome in polysomes.ISSN:1362-4962ISSN:0301-561