RESUMO
Purine nucleosides on position 9 of eukaryal and archaeal tRNAs are frequently modified in vivo by the post-transcriptional addition of a methyl group on their N1 atom. The methyltransferase Trm10 is responsible for this modification in both these domains of life. While certain Trm10 orthologues specifically methylate either guanosine or adenosine at position 9 of tRNA, others have a dual specificity. Until now structural information about this enzyme family was only available for the catalytic SPOUT domain of Trm10 proteins that show specificity toward guanosine. Here, we present the first crystal structure of a full length Trm10 orthologue specific for adenosine, revealing next to the catalytic SPOUT domain also N- and C-terminal domains. This structure hence provides crucial insights in the tRNA binding mechanism of this unique monomeric family of SPOUT methyltransferases. Moreover, structural comparison of this adenosine-specific Trm10 orthologue with guanosine-specific Trm10 orthologues suggests that the N1 methylation of adenosine relies on additional catalytic residues.
Assuntos
Adenosina/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , RNA de Transferência/metabolismo , Sulfolobus acidocaldarius/enzimologia , tRNA Metiltransferases/metabolismo , Adenosina/química , Proteínas Arqueais/genética , Domínio Catalítico , Cristalografia por Raios X , Metilação , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , RNA de Transferência/química , RNA de Transferência de Metionina/química , RNA de Transferência de Metionina/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios X , tRNA Metiltransferases/química , tRNA Metiltransferases/genéticaRESUMO
The parDE family of toxin-antitoxin (TA) operons is ubiquitous in bacterial genomes and, in Vibrio cholerae, is an essential component to maintain the presence of chromosome II. Here, we show that transcription of the V. cholerae parDE2 (VcparDE) operon is regulated in a toxin:antitoxin ratio-dependent manner using a molecular mechanism distinct from other type II TA systems. The repressor of the operon is identified as an assembly with a 6:2 stoichiometry with three interacting ParD2 dimers bridged by two ParE2 monomers. This assembly docks to a three-site operator containing 5'- GGTA-3' motifs. Saturation of this TA complex with ParE2 toxin results in disruption of the interface between ParD2 dimers and the formation of a TA complex of 2:2 stoichiometry. The latter is operator binding-incompetent as it is incompatible with the required spacing of the ParD2 dimers on the operator.