RESUMO
The cytidine at the first anticodon position of archaeal tRNA(Ile2), which decodes the isoleucine AUA codon, is modified to 2-agmatinylcytidine (agm(2)C) to guarantee the fidelity of protein biosynthesis. This post-transcriptional modification is catalyzed by tRNA(Ile)-agm(2)C synthetase (TiaS) using ATP and agmatine as substrates. Archaeoglobus fulgidus TiaS was overexpressed in Escherichia coli cells and purified. tRNA(Ile2) was prepared by in vitro transcription with T7 RNA polymerase. TiaS was cocrystallized with both tRNA(Ile2) and ATP by the vapour-diffusion method. The crystals of the TiaS-tRNA(Ile2)-ATP complex diffracted to 2.9 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the primitive hexagonal space group P3(2)21, with unit-cell parameters a = b = 131.1, c = 86.6 Å. The asymmetric unit is expected to contain one TiaS-tRNA(Ile2)-ATP complex, with a Matthews coefficient of 2.8 Å(3) Da(-1) and a solvent content of 61%.
Assuntos
Trifosfato de Adenosina/química , Archaeoglobales/enzimologia , Isoleucina-tRNA Ligase/química , RNA de Transferência de Isoleucina/química , Trifosfato de Adenosina/metabolismo , Cristalização , Cristalografia por Raios X , Isoleucina-tRNA Ligase/metabolismo , Ligação Proteica , RNA de Transferência de Isoleucina/metabolismoRESUMO
The cytidine at the first position of the anticodon (C34) in the AUA codon-specific archaeal tRNA(Ile2) is modified to 2-agmatinylcytidine (agm(2)C or agmatidine), an agmatine-conjugated cytidine derivative, which is crucial for the precise decoding of the genetic code. Agm(2)C is synthesized by tRNA(Ile)-agm(2)C synthetase (TiaS) in an ATP-dependent manner. Here we present the crystal structures of the Archaeoglobus fulgidus TiaS-tRNA(Ile2) complexed with ATP, or with AMPCPP and agmatine, revealing a previously unknown kinase module required for activating C34 by phosphorylation, and showing the molecular mechanism by which TiaS discriminates between tRNA(Ile2) and tRNA(Met). In the TiaS-tRNA(Ile2)-ATP complex, C34 is trapped within a pocket far away from the ATP-binding site. In the agmatine-containing crystals, C34 is located near the AMPCPP γ-phosphate in the kinase module, demonstrating that agmatine is essential for placing C34 in the active site. These observations also provide the structural dynamics for agm(2)C formation.
Assuntos
Anticódon/química , Citidina/análogos & derivados , Conformação de Ácido Nucleico , Conformação Proteica , RNA de Transferência de Isoleucina/química , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Anticódon/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Archaeoglobus fulgidus/enzimologia , Cristalografia por Raios X , Citidina/química , Citidina/metabolismo , Isoleucina-tRNA Ligase/química , Isoleucina-tRNA Ligase/metabolismo , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , RNA Arqueal/genética , RNA Arqueal/metabolismo , RNA de Transferência de Isoleucina/genética , RNA de Transferência de Isoleucina/metabolismoRESUMO
The 5-carboxymethylaminomethyl modification of uridine (cmnm(5)U) at the anticodon first position occurs in tRNAs that read split codon boxes ending with purine. This modification is crucial for correct translation, by restricting codon-anticodon wobbling. Two conserved enzymes, GidA and MnmE, participate in the cmnm(5)U modification process. Here we determined the crystal structure of Aquifex aeolicus GidA at 2.3 A resolution. The structure revealed the tight interaction of GidA with FAD. Structure-based mutation analyses allowed us to identify two conserved Cys residues in the vicinity of the FAD-binding site that are essential for the cmnm(5)U modification in vivo. Together with mutational analysis of MnmE, we propose a mechanism for the cmnm(5)U modification process where GidA, but not MnmE, attacks the C6 atom of uridine by a mechanism analogous to that of thymidylate synthase. We also present a tRNA-docking model that provides structural insights into the tRNA recognition mechanism for efficient modification.
Assuntos
Anticódon/química , Proteínas de Bactérias/química , Cisteína/genética , RNA de Transferência/química , Uridina/análogos & derivados , Sequência de Aminoácidos , Anticódon/metabolismo , Bactérias/enzimologia , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Domínio Catalítico , Sequência Conservada , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Dados de Sequência Molecular , RNA de Transferência/metabolismo , Uridina/química , Uridina/metabolismoRESUMO
Developmental exposure to 17 beta-estradiol (E(2)) induced the death of embryos and fry, malformations, sex reversal, and incomplete ossification of vertebrae and cranial bones in the cyprinodont fish, the mummichog (Fundulus heteroclitus). To clarify the mechanism by which exogenous estrogens caused these developmental effects, we determined the sequence of an estrogen receptor (ER) coding region, encoded by 620 amino acid residues. This region shared 80% identity to that of ER alpha of medaka (Oryzias latipes). Northern blot analysis showed that two ER alpha mRNAs with 5.5 and 4 kb were expressed in the liver. These mRNAs were strongly induced by E(2) stimulation. The 4 kb mRNA was expressed 8 h after treatment, whereas the 5.5 kb mRNA was not induced until 12 h after E(2) stimulation. Vitellogenin (VTG) was expressed 8 h after E(2) stimulation in the male liver. Receptor binding assays using the protein of F. heteroclitus ER alpha (fh ER alpha) ligand binding domain showed that alkylphenols bind to fh ER alpha with a higher affinity (50 times or more) as compared with the human ER alpha. The present results demonstrate that the fh ER alpha has a sequence very similar to that of medaka, and the mRNA for this receptor was induced by E(2)-stimulation, followed subsequently by VTG expression. Furthermore, alkylphenols bind to fh ER alpha more efficiently than to human ER alpha.