Four sites in the acceptor helix and one site in the variable pocket of tRNA(Ala) determine the molecule's acceptor identity.

The structural features that determine tRNA(Ala) acceptor identity have been studied with amber-suppressor tRNAs in Escherichia coli cells. Previous work established that a wobble pair composed of guanosine at position 3 and uridine at position 70 (G3-U70) in the acceptor helix of tRNA(Ala) is a determinant of the molecule's acceptor identity. We show that additional determinants are located at three other sites in the acceptor helix and at one site in the variable pocket of tRNA(Ala). These latter determinants are less important than G3.U70 since their individual alterations in mutants of tRNA(Ala) have smaller degrading effects on the functions of the molecules, and subsets of the determinants, when combined with G3.U70, are sufficient to switch the identities of several other tRNAs to that of tRNA(Ala). Other workers are using fragments of the tRNA(Ala) acceptor helix to study the molecule's acceptor identity. Our demonstration that the variable pocket contributes to tRNA(Ala) acceptor identity means that such fragments do not faithfully replicate the structure-function relationship of the cellular process.