MINOR-GROOVE RECOGNITION OF THE CRITICAL ACCEPTOR HELIX BASE-PAIR BY AN APPENDED MODULE OF A CLASS-II TRANSFER-RNA SYNTHETASE

The class-defining active site domain of the 10 class II tRNA syntheta ses is well conserved and, based on the crystal structure of aspartyl- tRNA synthetase, approaches the end of the tRNA acceptor stem from the major groove side of the helix. Paradoxically, for the class II alany l-tRNA synthetase (AlaRS), aminoacylation is dependent on minor groove recognition of an acceptor helix G3 . U70 base pair. Additional contr ibutions to aminoacylation efficiency are made by the A73 ''discrimina tor'' base and G2 . C71 base pair located at the end of the acceptor s tem. Using microhelix substrates containing only the first four base p airs of the alanine tRNA acceptor helix, we demonstrated that the cata lytic center of AlaRS with the three class-defining sequence motifs co ntains determinants for recognition of A73 and G2 . C71. However, this structural unit does not discriminate between different base pairs at the critical 3.70 position. Discrimination at G3 . U70 was mapped to a 76 amino acid polypeptide outside the catalytic center. We propose t hat the G3 . U70 recognition motif is a structural appendage that fold s back to the catalytic center to make contact with the bound acceptor stem. A ''fold-back'' appendage provides a specific mechanism for min or groove recognition of the acceptor helix by a class II tRNA synthet ase.