RECONSTITUTION IN-VITRO OF THE VALYL-TRANSFER RNA SYNTHETASE-ELONGATION FACTOR (EF) 1-BETA-GAMMA-DELTA COMPLEX - ESSENTIAL ROLES OF THE NH2-TERMINAL EXTENSION OF VALYL-TRANSFER RNA-SYNTHETASE AND OF THE EF-1-DELTA SUBUNIT IN COMPLEX-FORMATION

Valyl-tRNA synthetase from mammalian cells is isolated exclusively as a complex with elongation factor (EF) 1H (the ''heavy'' form of eukary otic EF-1, composed of subunits alpha, beta, gamma, and delta). In a p revious study, the 140-kDa valyl-tRNA synthetase subunit dissociated f rom the purified rabbit liver complex was shown to display hydrophobic properties, unlike the corresponding yeast cytoplasmic enzyme of 125 kDa (Bec, G., and Waller, J.-P. (1989) J. Biol. Chem. 264, 21138-21143 ). Compared to the sequence of yeast cytoplasmic valyl-tRNA synthetase , that of the human enzyme displays an NH2-terminal extension of appro ximately 200 amino acid residues that bears strong sequence similarity to the NH2-terminal moiety of EF-1gamma (Hsieh, S. L., and Campbell, R. D. (1991) Biochem. J. 278, 809-816). We now show that this NH2-term inal extension can be selectively excised by elastase treatment of the isolated rabbit valyl-tRNA synthetase, without impairing catalytic ac tivity. To examine the role of the NH2-terminal extension of mammalian valyl-tRNA synthetase in complex formation and to identify the subuni t(s) of EF-1H responsible for binding the enzyme, reconstitution exper iments were undertaken. Native or truncated valyl-tRNA synthetases wer e incubated with the isolated EF-1 subunits betagamma and delta, eithe r separately or in combination, and the ensuing products were analyzed by chromatography on DEAE-Sepharose FF and Superose 6. The results de monstrate that the NH2-terminal extension of valyl-tRNA synthetase is required for complex formation and that the enzyme-binding site(s) res ides on the EF-1delta subunit. Moreover, although the EF-1betagamma bi nary complex does not bind valyl-tRNA synthetase, it is nevertheless r equired for assembly of a complex of defined quaternary structure by p reventing the formation of high molecular weight aggregates generated in the presence of EF-1delta alone.