The importance of tRNA backbone-mediated interactions with synthetase for aminoacylation

Abstract

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.

Determination of 2′-hydroxyl and phosphate groups important for aminoacylation of Escherichia coli tRNAAsp: A nucleotide analogue interference study

“…A: tRNA of the complex with the synthetase shown in the same orientation as in C+ Small spheres: 29-hydroxyl groups; large spheres (red): critical positions with sequence number+ B: As A, but critical pro Rp-oxygens are in green+ C: Three-dimensional representation of the interaction between tRNA (yellow) and the synthetase (blue) based on the crystal structure of the yeast tRNA Asp complex (Cavarelli et al+, 1993)+ Spheres: phosphate-backbone of tRNA and carbon main-chain atoms of the protein+ experiments for the aminoacylation of tRNA Gln (McClain et al+, 1998)+ In conclusion we have analyzed for the first time full length tRNA transcripts for individual positions where the presence of 29-hydroxyl groups is essential for charging and where phosphates can not be replaced by phosphorothioates+ The degree of reduction in charging efficiency caused by single 29-deoxy and phosphorothioate substitutions is small+ However, multiple occurrences of 29-deoxy substitutions generally leads to complete loss of charging+ The general agreement between the X-ray and interference analyses is encouraging with respect to applying the latter method to the study of other RNA-protein interactions+…”

Revisiting the operational RNA code for amino acids: Ensemble attributes and their implications

“…Determinants facilitate aminoacylation; antideterminants impede it. Their combined effect ensures that a particular tRNA is a substrate for its cognate synthetase and not a substrate for all the other synthetases present in a cell (McClain 1993;Nureki et al 1994;Giegé et al 1998;McClain et al 1998;Beuning and Musier-Forsyth 1999;Fender et al 2004). An updated list of determinants based on a compilation by Giegé et al (1998) is shown in Supplemental Table S1.…”

A new assay for tRNA aminoacylation kinetics

“…Here we demonstrate the feasibility of an assay that directly measures the fraction of aminoacylated tRNA by its altered mobility on an acidic, denaturing polyacrylamide gel+ Such gels have been used to estimate the levels of aminoacylated tRNA in vivo (Varshney et al+, 1991;McClain et al+, 1998), and to detect selfaminoacylation by a ribozyme (Illangasekare et al+, 1997)+ However, initial experiments revealed that the resolution of these gels was not sufficient to obtain accurate kinetic data when intact tRNA was used+ Thus, we employed a tRNA made of two RNA fragments with a nick at a position near the 39 terminus that does not affect aminoacylation kinetics (Liu & Musier-Forsyth, 1994)+ Because this assay uses high-specific activity [ 32 P]-labeled tRNA and measures aminoacylation directly, it overcomes the major shortcomings of the tritiated amino acid assay and should be valuable for structure-function studies+…”