RESUMO

tRNA synthetases are responsible for decoding the molecular information, from codons to amino acids. Seryl-tRNA synthetase (SerRS), besides the five isoacceptors of tRNASer, recognizes tRNA[Ser]Sec for the incorporation of selenocysteine (Sec, U) into selenoproteins. The selenocysteine synthesis pathway is known and is dependent on several protein-protein and protein-RNA interactions. Those interactions are not fully described, in particular, involving tRNA[Ser]Sec and SerRS. Here we describe the molecular interactions between the Escherichia coli Seryl-tRNA synthetase (EcSerRS) and tRNA[Ser]Sec in order to determine their specificity, selectivity and binding order, leading to tRNA aminoacylation. The dissociation constant of EcSerRS and tRNA[Ser]Sec was determined as (126 ± 20) nM. We also demonstrate that EcSerRS binds initially to tRNA[Ser]Sec in the presence of ATP for further recognition by E. coli selenocysteine synthetase (EcSelA) for Ser to Sec conversion. The proposed studies clarify the mechanism of tRNA[Ser]Sec incorporation in Bacteria as well as of other domains of life.

Assuntos

Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , RNA de Transferência Aminoácido-Específico/metabolismo , RNA de Transferência de Cisteína/metabolismo , Serina-tRNA Ligase/metabolismo , Transferases/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Escherichia coli/genética , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA de Transferência Aminoácido-Específico/genética , RNA de Transferência de Cisteína/genética , Serina-tRNA Ligase/genética , Termodinâmica , Aminoacilação de RNA de Transferência/genética , Transferases/genética