High-level Expression And Single-step Purification Of Leucyl-tRNA Synthetase From Aquifex Aeolicus The Structure And Function Of Aminoacyl-tRNA Synthetase Complex

A. aeolicus leucyl-tRNA synthetase is the only known heterodimeric LeuRS,consisting of two subunits with molecular masses of 74.0KDa and 33.5KDa, andnamed αβ-LeuRS. The gene encoding α subunit was cloned into pSBET-b vector.Synthetic oligonucleotide encoding six histidine residues was also inserted in frontof α subunit. PSBET-b vector containing argU gene, which encodes a rare E. colitRNA^Arg(AGA/AGG). The argU gene helps A. aeolicus LeuRS, which containsAGA/AGG codons in exceptionally high frequency, express well in E. coli. Thegene encoding β subunit was inserted into pET-15b vector. E. coli BL21-CodonPlus(DE3) cells were transformed with the two recombinant plasmids to produceαβ-LeuRS with a His6 tag at the N-terminus of α subunit. The enzyme was purifiedby affinity chromatography on Ni-NTA Superflow. About 7 mg purified αβ-LeuRSwas obtained from 250 ml culture. The His6-tag at the N-terminus did not affect theaminoacylation activity of the enzyme.Human cytosolic leucyl-tRNA synthetase (HcLeuRS) is one component of amacromolecular aminoacyl-tRNA synthetase complex (aaRS complex). This isunlike prokaryotic and lower eukaryotic LeuRSs that exist as free soluble enzymes.HcLeuRS has been purified in our laboratory before, and several characters of ithave been determined. Since hcLeuRS has a long C-terminal extension that is absentfrom bacterial and yeast LeuRSs, a C-terminal 89-amino acid truncated humancytosolic leucyl-tRNA synthetase (?ChcLeuRS) was heterologously expressed in abaculovirus system and purified to homogeneity. It was found that the kineticconstants of hcLeuRS and ?ChcLeuRS for ATP, leucine and tRNALeu in the ATP-PPiexchange and tRNA leucylation reactions were similar. The two enzymes also havesimilar thermal stabilities and identical subcellular localizations in 293 T cells. Invivo and in vitro experiments, however, show that the C-terminal extension ofhuman cytosolic leucyl-tRNA synthetase is indispensable for its interaction with theN-terminal of human cytosolic Arginyl-tRNA synthetase (hcArgRS) in themacromolecular complex. Our results also indicate that the two molecules interactwith each other only through their appended peptides.The functions of aminoacyl-tRNA synthetase complex (aaRS complex) are littleknown untill now. p43, p38 and p18 are the three important structural proteins inaaRS complex. Herein, the three proteins were purified, and their impacts on theaminoacylation activies of hcLeuRS (human cytosolic LeuRS), ?ChcLeuRS(C-terminal truncated hcLeuRS), hcArgRS (human cytosolic ArgRS), ?NhcArgRS(N-terminal truncated hcArgRS) and LeuRSs from yeast, E. coli and A. aeolicuswere detected. It was proved for the first time that the three auxiliary factorsimprove the activies of aaRS, with or without the terminal extension peptides ofaaRS. Three factors only modulate the activity of LeuRS from human, not LeuRSsfrom yeast, E. coli or A. aeolicus. It was proposed that one of the reasons for aaRScomplex formation is to facilitate the aminoacylation of tRNA. Combined with otherexperiment results, the possible functions of aaRS complex were discussed.