RESUMEN
Early work on aminoacylation of alanine-specific tRNA (tRNA(Ala)) by alanyl-tRNA synthetase (AlaRS) gave rise to the concept of an early "second genetic code" imbedded in the acceptor stems of tRNAs. A single conserved and position-specific G:U base pair in the tRNA acceptor stem is the key identity determinant. Further understanding has been limited due to lack of a crystal structure of the enzyme. We determined a 2.14 A crystal structure of the 453 amino acid catalytic fragment of Aquifex aeolicus AlaRS. It contains the catalytic domain characteristic of class II synthetases, a helical domain with a hairpin motif critical for acceptor-stem recognition, and a C-terminal domain of a mixed alpha/beta fold. Docking of tRNA(Ala) on AlaRS shows critical contacts with the three domains, consistent with previous mutagenesis and functional data. It also suggests conformational flexibility within the C domain, which might allow for the positional variation of the key G:U base pair seen in some tRNA(Ala)s.
Asunto(s)
Alanina-ARNt Ligasa/química , Proteínas Bacterianas/química , Alanina-ARNt Ligasa/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Emparejamiento Base , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/enzimología , Escherichia coli/genética , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ARN Bacteriano/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The crystal structure of a catalytic fragment of Aquifex aeolicus AlaRS and additional data suggest how the critical G3:U70 identity element of its cognate tRNA acceptor stem is recognized. Though this identity element is conserved from bacteria to the cytoplasm of eukaryotes, Drosophila melanogaster mitochondrial (Dm mt) tRNA(Ala) contains a G:U base pair that has been translocated to the adjacent 2:71 position. This G2:U71 is the major determinant for identity of Dm mt tRNA(Ala). Sequence alignments showed that Dm mt AlaRS is differentiated from G3:U70-recognizing AlaRSs by an insertion of 27 amino acids in the region of the protein that contacts the acceptor stem. Precise deletion of this insertion from Dm mt AlaRS gave preferential recognition to a G3:U70-containing substrate. Larger or smaller deletions were ineffective. The crystal structure of the orthologous A. aeolicus protein places this insertion on the surface, where it can act as a hinge that provides positional switching of G:U recognition.
