Structural studies on tRNA acceptor stem microhelices: exchange of the discriminator base A73 for G in human tRNALeu switches the acceptor specificity from leucine to serine possibly by decreasing the stability of the terminal G1-C72 base pair

Abstract

Correct recognition of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases (aaRS) is crucial to the maintenance of translational fidelity. The discriminator base A73 in human tRNALeuis critical for its specific recognition by the aaRS. Exchanging A73 for G abolishes leucine acceptance and converts it into a serine acceptor in vitro . Two RNA microhelices of 24 nt length that correspond to the tRNALeuacceptor stem and differ only in the discriminator base were synthesized: a wild-type tRNALeumicrohelix, where nt 21 corresponds to the discriminator base position 73, and an A21G mutant microhelix. To investigate whether different identities of both tRNAs are caused by conformational differences, NMR and UV melting experiments were performed on both microhelices. Two-dimentional NOESY spectra showed both microhelices to exhibit the same overall conformation at their 3'-CCA ends. Thermodynamic analysis and melting behaviour of the base-paired imino protons observed by NMR spectroscopy suggest that the A21G (A73G in tRNA) exchange results in a decrease of melting transition cooperativity and a destabilization of the terminal G1-C20 (G1-C72 in tRNA) base pair. Furthermore, the fact that this 3'-terminal imino proton is more solvent-exposed at physiological temperature might be another indication for the importance of the stability of the terminal base pair for specific tRNA recognition.

Leucyl-tRNA Synthetase from the Hyperthermophilic Bacterium Aquifex aeolicus Recognizes Minihelices

“…As reported previously, E. coli and H. sapiens cytoplasmic LeuRSs cannot charge minihelix or microhelix tRNA Leu (13,14). For E. coli tRNA Leu , minimal RNA is efficiently aminoacylated when the anticodon stem loop and variable arm stem loop (but not D-arm or T-arm) are deleted (13).…”

“…Previous attempts to charge the minihelix and microhelix of tRNA Leu have been unsuccessful (13,14). In this investigation, we show for the first time that ␣␤-LeuRS charges minihelices.…”

“…Attempts to charge the minihelix or microhelix tRNA Leu (13,14). However, some studies show that structurally similar ValRS and IleRS charge minihelices (20,29).…”

Trypanosoma Seryl-tRNA Synthetase Is a Metazoan-like Enzyme with High Affinity for tRNASec

“…The crystallographic structures of Thermus thermophilus SerRS complexed with tRNA Ser , confirm these observations (15,16). A second type of recognition mode, seen in archaeal and human SerRS, is characterized by the crucial role of the discriminator base (17)(18)(19). In these cases, the sequence of the variable loop is not important, but its size and orientation are fundamental for the interaction with the enzyme (20,21).…”

“…This sensitivity to the discriminator base sequence is reminiscent of the recognition mechanisms described for H. sapiens and archaeal SerRSs (17)(18)(19).…”