The tRNA-Interacting Factor p43 Associates with Mammalian Arginyl-tRNA Synthetase but Does Not Modify Its tRNA Aminoacylation Properties

摘要

Arginyl-tRNA synthetase (ArgRS)is one of the nine synthetase components of a multienzyme complex containing three auxiliary proteins as well.We previously established that the N-terminal moiety of the auxiliary protein p43 associates with the N-terminal,eukaryotic-specific polypeptide extension of ArgRS.Because p43 is homologous to Arclp,a yeast general RNA-binding protein that associates with MetRS and GluRS and plays the role of tRNA-binding cofactor in the aminoacylation reaction,we analyzed the functional significance of p43-ArgRS association.We had previously showed that full-length ArgRS,corresponding to the ArgRS species associated within the multisynthetase complex,and ArgRS with a deletion of 73 N-terminal amino acid residues,corresponding to a free species of ArgRS,both produced in yeast,have similar catalytic parameters (Lazard,M.,Kerjan,P.,Agou,F.,and Mirande,M.(2000)J.Mol.Biol 302,991-1004).However,a recent study had suggested that association of p43 to ArgRS reduces the apparent K_M of ArgRS to tRNA (Park,S.G.,Jung,K.H.,Lee,J.S.,Jo,Y.J.,Motegi,H.,Kim,S.,and Shiba,K.(1999)J.Biol.Chem.274,16673-16676).In this study,we analyzed in detail,by gel retardation assays and enzyme kinetics,the putative role of p43 as a tRNA-binding cofactor of ArgRS.The association of p43 with ArgRS neither strengthened tRNA-binding nor changed kinetic parameters in the amino acid activation or in the tRNA aminoacylation reaction.Furthermore,selective removal of the C-terminal RNA-binding domain of p43 from the multisynthetase complex did not affect kinetic parameters for ArgRS.Therefore,p43 has a dual function.It promotes association of ArgRS to the complex via its N-terminal domain,but its C-terminal RNA-binding domain may act as a tRNA-interacting factor for an as yet unidentified component of the complex.