The structural features of the G.U wobble pair in Escherichia coli alanine transfer RNA (tRNA(Ala)) that are associated with aminoacylation by alanyl-tRNA synthetase (AlaRS) were investigated in vivo for wild-type tRNA(Ala) and mutant tRNAs with G.U substitutions. tRNA(Ala) with G.U, C.A, or G.A gave similar amounts of charged tRNA(Ala) and supported viability of E. coli lacking chromosomal tRNA(Ala) genes. tRNA(Ala) with G.C was inactive. Recognition of G.U by AlaRS thus requires more than the functional groups on G.U in a regular helix and may involve detection of a helical distortion.

The 2.6 A resolution crystal structure of an inactive complex between yeast tRNA(Asp) and Escherichia coli aspartyl-tRNA synthetase reveals the molecular details of a tRNA-induced mechanism that controls the specificity of the reaction. The dimer is asymmetric, with only one of the two bound tRNAs entering the active site cleft of its subunit. However, the flipping loop, which controls the proper positioning of the amino acid substrate, acts as a lid and prevents the correct positioning of the terminal adenosine. The structure suggests that the acceptor stem regulates the loop movement through sugar phosphate backbone- protein interactions. Solution and cellular studies on mutant tRNAs confirm the crucial role of the tRNA three-dimensional structure versus a specific recognition of bases in the control mechanism.

The previously uncharacterized determinants of the specificity of tRNAPro for aminoacylation (tRNAPro identity) were defined by a computer comparison of all Escherichia coli tRNA sequences and tested by a functional analysis of amber suppressor tRNAs in vivo. We determined the amino acid specificity of tRNA by sequencing a suppressed protein and the aminoacylation efficiency of tRNA by examining the steady-state level of aminoacyl-tRNA. On substituting nucleotides derived from the acceptor end and variable pocket of tRNAPro for the corresponding nucleotides in a tRNAPhe gene, the identity of the resulting tRNA changed substantially but incompletely to that of tRNAPro. The redesigned tRNAPhe was weakly active and aminoacyl-tRNA was not detected. Ethyl methanesulfonate mutagenesis of the redesigned tRNAPhe gene produced a mutant with a wobble pair in place of a base pair in the end of the acceptor-stem helix of the transcribed tRNA. This mutant exhibited both a tRNAPro identity and substantial aminoacyl-tRNA. The results speak for the importance of a distinctive conformation in the acceptor-stem helix of tRNAPro for aminoacylation by the prolyl-tRNA synthetase. The anticodon also contributes to tRNAPro identity but is not necessary in vivo.

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We have identified six new aminoacylation determinants of Escherichia coli tRNA Gln in a genetic and biochemical analysis of suppressor tRNA. The new determinants occupy the interior of the acceptor stem, the inside corner of the L shape, and the anticodon loop of the molecule. They supplement the primary determinants located in the anticodon and acceptor end of tRNA Gln described previously. Remarkably, the three-dimensional structure of the complex between tRNA Gln and glutaminyl-tRNA synthetase shows that the enzyme interacts with the phosphate-sugar backbone but not the base of every new determinant. Moreover, a small protein motif interacts with five of these determinants, and it binds proximal to the sixth. The motif also interacts with the middle base of the anticodon and with the backbones of six other nucleotides. Our results emphasize that synthetase recognition of tRNA is more elaborate than amino acid side chains of the enzyme interacting with nucleotide bases of the tRNA. Recognition also includes synthetase interaction with tRNA backbone functionalities whose distinctive locations in three-dimensional space are exquisitely determined by the tRNA sequence.