Role of acceptor stem conformation in tRNAVal recognition by its cognate synthetase

Abstract

Although the anticodon is the primary element in Escherichia coli tRNAValfor recognition by valyl-tRNA synthetase (ValRS), nucleotides in the acceptor stem and other parts of the tRNA modulate recognition. Study of the steady state aminoacylation kinetics of acceptor stem mutants of E.coli tRNAValdemonstrates that replacing any base pair in the acceptor helix with another Watson-Crick base pair has little effect on aminoacylation efficiency. The absence of essential recognition nucleotides in the acceptor helix was confirmed by converting E.coli tRNAAlaand yeast tRNAPhe, whose acceptor stem sequences differ significantly from that of tRNAVal, to efficient valine acceptors. This transformation requires, in addition to a valine anticodon, replacement of the G:U base pair in the acceptor stem of these tRNAs. Mutational analysis of tRNAValverifies that G:U base pairs in the acceptor helix act as negative determinants of synthetase recognition. Insertion of G:U in place of the conserved U4:A69 in tRNAValreduces the efficiency of aminoacylation, due largely to an increase in K m. A smaller but significant decline in aminoacylation efficiency occurs when G:U is located at position 3:70; lesser effects are observed for G:U at other positions in the acceptor helix. The negative effects of G:U base pairs are strongly correlated with changes in helix structure in the vicinity of position 4:69 as monitored by19F NMR spectroscopy of 5-fluorouracil-substituted tRNAVal. This suggests that maintaining regular A-type RNA helix geometry in the acceptor stem is important for proper recognition of tRNAValby valyl-tRNA synthetase.19F NMR also shows that formation of the tRNAVal-valyl-tRNA synthetase complex does not disrupt the first base pair in the acceptor stem, a result different from that reported for the tRNAGln-glutaminyl-tRNA synthetase complex.

Activation of microhelix charging by localized helix destabilization

“…On the other hand, any disturbance of the region around the 1⅐72 position could adversely affect the kinetic pathway for the specific conformational change at the 3Ј end that is promoted by interactions at the anticodon. In studies with tRNA Met (30,31,44,57,58). Any effects are typically not large and are consistent with the 1⅐72 position not being used for essential functional contacts by the enzyme.…”

The identity determinants required for the discrimination between tRNAGlu and tRNAAsp by glutamyl‐tRNA synthetase from Escherichia coli

“…Of course, we cannot exclude the possibility that the low activity of the 4G•69U variant is a result of incorrect ordering of the acceptor arm into the active site, due to the insertion of a wobble G•U base pair. It was found recently that inserting a G•U base pair at position 4•69 of tRNA Val causes the largest change in the acceptor helix structure amongst the seven acceptor stem base pairs [17], consistent with the slight decrease in the valylation kinetics of the 4G•69U variant [18]. However, based on the glutamylation kinetics with tRNA Glu variant transcripts and the interactions of the phosphate groups with GluRS, the present results highlight the importance of the local contact in the middle of the acceptor stem by GluRS.…”

Refining RNA solution structures with the integrative use of label-free paramagnetic relaxation enhancement NMR

“…To fold these two RNAs, the in vitro transcribed RNA samples were heated to 95°C for 5 min and were immediately put on ice for cooling. The tRNA Val was transcribed in E. coli using the recombinant plasmid, pVAL119-21, containing the wild-type E. coli tRNA Val gene downstream of a T7 promoter (Chu and Horowitz 1989;Mingsong Liu et al 1997). BL21 star cells were used for the tRNA expression, and were grown in LB medium (for unlabeled RNA) or in M9minimum medium with U-15 N-NH 4 Cl as the sole nitrogen source (for 15 N-labeled RNA).…”