RESUMO
N6-threonylcarbamoyl adenosine (t6A) is a nucleoside modification found in all kingdoms of life at position 37 of tRNAs decoding ANN codons, which functions in part to restrict translation initiation to AUG and suppress frameshifting at tandem ANN codons. In Bacteria the proteins TsaB, TsaC (or C2), TsaD, and TsaE, comprise the biosynthetic apparatus responsible for t6A formation. TsaC(C2) and TsaD harbor the relevant active sites, with TsaC(C2) catalyzing the formation of the intermediate threonylcarbamoyladenosine monophosphate (TC-AMP) from ATP, threonine, and CO2, and TsaD catalyzing the transfer of the threonylcarbamoyl moiety from TC-AMP to A37 of substrate tRNAs. Several related modified nucleosides, including hydroxynorvalylcarbamoyl adenosine (hn6A), have been identified in select organisms, but nothing is known about their biosynthesis. To better understand the mechanism and structural constraints on t6A formation, and to determine if related modified nucleosides are formed via parallel biosynthetic pathways or the t6A pathway, we carried out biochemical and biophysical investigations of the t6A systems from E. coli and T. maritima to address these questions. Using kinetic assays of TsaC(C2), tRNA modification assays, and NMR, our data demonstrate that TsaC(C2) exhibit relaxed substrate specificity, producing a variety of TC-AMP analogs that can differ in both the identity of the amino acid and nucleotide component, whereas TsaD displays more stringent specificity, but efficiently produces hn6A in E. coli and T. maritima tRNA. Thus, in organisms that contain modifications such as hn6A in their tRNA, we conclude that their origin is due to formation via the t6A pathway.
Assuntos
Adenosina/análogos & derivados , Vias Biossintéticas/genética , Nucleosídeos/genética , RNA de Transferência/genética , Adenosina/genética , Monofosfato de Adenosina/genética , Trifosfato de Adenosina/genética , Aminoácidos/genética , Domínio Catalítico/genética , Escherichia coli/genética , Conformação Proteica , Especificidade por Substrato/genética , Thermotoga maritima/genética , Treonina/genéticaRESUMO
The universally conserved N6-threonylcarbamoyladenosine (t6A) modification of tRNA is essential for translational fidelity. In bacteria, t6A biosynthesis starts with the TsaC/TsaC2-catalyzed synthesis of the intermediate threonylcarbamoyl adenylate (TC-AMP), followed by transfer of the threonylcarbamoyl (TC) moiety to adenine-37 of tRNA by the TC-transfer complex comprised of TsaB, TsaD and TsaE subunits and possessing an ATPase activity required for multi-turnover of the t6A cycle. We report a 2.5-Å crystal structure of the T. maritima TC-transfer complex (TmTsaB2D2E2) bound to Mg2+-ATP in the ATPase site, and substrate analog carboxy-AMP in the TC-transfer site. Site directed mutagenesis results show that residues in the conserved Switch I and Switch II motifs of TsaE mediate the ATP hydrolysis-driven reactivation/reset step of the t6A cycle. Further, SAXS analysis of the TmTsaB2D2-tRNA complex in solution reveals bound tRNA lodged in the TsaE binding cavity, confirming our previous biochemical data. Based on the crystal structure and molecular docking of TC-AMP and adenine-37 in the TC-transfer site, we propose a model for the mechanism of TC transfer by this universal biosynthetic system.
Assuntos
Adenosina/análogos & derivados , Proteínas de Bactérias/química , RNA de Transferência/metabolismo , Adenosina/biossíntese , Adenosina Trifosfatases/genética , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Modelos Moleculares , Mutagênese , Conformação Proteica , RNA de Transferência/química , Thermotoga maritimaRESUMO
The universal N(6)-threonylcarbamoyladenosine (t6A) modification at position 37 of ANN-decoding tRNAs is central to translational fidelity. In bacteria, t6A biosynthesis is catalyzed by the proteins TsaB, TsaC/TsaC2, TsaD and TsaE. Despite intense research, the molecular mechanisms underlying t6A biosynthesis are poorly understood. Here, we report biochemical and biophysical studies of the t6A biosynthesis system from Thermotoga maritima. Small angle X-ray scattering analysis reveals a symmetric 2:2 stoichiometric complex of TsaB and TsaD (TsaB2D2), as well as 2:2:2 complex (TsaB2D2E2), in which TsaB acts as a dimerization module, similar to the role of Pcc1 in the archaeal system. The TsaB2D2 complex is the minimal platform for the binding of one tRNA molecule, which can then accommodate a single TsaE subunit. Kinetic data demonstrate that TsaB2D2 alone, and a TsaB2D2E1 complex with TsaE mutants deficient in adenosine triphosphatase (ATPase) activity, can catalyze only a single cycle of t6A synthesis, while gel shift experiments provide evidence that the role of TsaE-catalyzed ATP hydrolysis occurs after the release of product tRNA. Based on these results, we propose a model for t6A biosynthesis in bacteria.
Assuntos
Adenosina/análogos & derivados , Proteínas de Bactérias/genética , Ligases/genética , Biossíntese de Proteínas , RNA de Transferência/genética , Thermotoga maritima/enzimologia , Adenosina/biossíntese , Adenosina/química , Adenosina/genética , Adenosina Trifosfatases/deficiência , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Códon , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Ligases/química , Ligases/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA de Transferência/química , RNA de Transferência/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Thermotoga maritima/genéticaRESUMO
tRNA molecules undergo extensive post-transcriptional processing to generate the mature functional tRNA species that are essential for translation in all organisms. These processing steps include the introduction of numerous specific chemical modifications to nucleotide bases and sugars; among these modifications, methylation reactions are by far the most abundant. The tRNA methyltransferases comprise a diverse enzyme superfamily, including members of multiple structural classes that appear to have arisen independently during evolution. Even among closely related family members, examples of unusual substrate specificity and chemistry have been observed. Here we review recent advances in tRNA methyltransferase mechanism and function with a particular emphasis on discoveries of alternative substrate specificities and chemistry associated with some methyltransferases. Although the molecular function for a specific tRNA methylation may not always be clear, mutations in tRNA methyltransferases have been increasingly associated with human disease. The impact of tRNA methylation on human biology is also discussed.