A BIOLOGICALLY-ACTIVE 53-KDA FRAGMENT OF OVERPRODUCED ALANYL-TRANSFER-RNA SYNTHETASE FROM THERMUS-THERMOPHILUS-HB8 SPECIFICALLY INTERACTS WITH TRNA(ALA) ACCEPTOR HELIX

The alaS gene encoding the alanyl-tRNA synthetase (AlaRS) from Thermus thermophilus HB8 was cloned and sequenced, The gene comprises 2646 bp , corresponding to 882 amino acids, 45% of which are identical to the enzyme from Escherichia coli, The T.thermophilus AlaRS was overproduce d in E.coli, purified and characterized, It has high thermal stability up to similar to 65 degrees C, with a temperature optimum of aminoacy lation activity at similar to 60 degrees C, and will be valuable for c rystallization. The purified enzyme appears as a dimer with a specific activity of 220 U/mg and k(cat)/K-M values of 118 000/s/M for alanine and 114 000/s/M for ATP, By genetic engineering a 53 kDa fragment of AlaRS comprising the N-terminal 470 amino acids (AlaN470) was also ove rproduced and purified, It is as stable as entire AlaRS and sufficient for specific aminoacylation of intact tRNA(Ala), as well as acceptor stem microhelices with a G3-U70, but not U3-A70, I3-U70 or C3-U70, bas e pair, The reduced binding strength of such microhelices to AlaN470 e nabled, due to the resulting fast exchange of the microhelices between free and complexed states, preliminary NMR analyses of the binding mo de and intermolecular recognition.