The role of the anticodon and discriminator base intRNA identity, in vivo

Abstract

There are more than 40 different tRNAs in an E. coli cell, most of which have very similar three-dimensional structures. These facts raise an interesting question, namely, how is it that an aminoacyl-tRNA synthetase can distinguish its tRNA substrate from the large pool of non-cognate tRNAs to allow it to specifically aminoacylate only the correct substrate. To attempt to identify the structural features in a tRNA which allow for its specific aminoacylation, an in vivo assay based on the initiation of protein synthesis was developed.;The assay system exploits the fact that only the methionine initiator tRNA (tRNA{dollar}\sp{lcub}\rm fMet{rcub}{dollar}) is capable of commencing protein synthesis. By introducing mutations into the anticodon as well as other sites of tRNA{dollar}\sp{lcub}\rm fMet{rcub}{dollar}, the affect of these mutations on aminoacylation can be addressed by identifying the amino acid inserted into a reporter protein from a complementary initiation codon. Using this system we have found that introducing only a Cys, Gln, Ile, Phe, Trp, Tyr, or Val anticodon into the tRNA{dollar}\sp{lcub}\rm fMet{rcub}{dollar} body allows these tRNAs to be aminoacylated with the corresponding amino acids. In addition, introducing the discriminator base (nucleotide 73) of the Cys or Gly tRNA into tRNA{dollar}\sp{lcub}\rm fMet{rcub}{dollar} mutants with the corresponding anticodons allowed, or improved, aminoacylation with those amino acids. Optimal insertion of both Cys and Gly required the cognate anticodon however, since substitution of any three nucleotide of the Cys anticodon, or of the second two positions of the Gly anticodon resulted in tRNAs with reduced or abolished Cys and Gly acceptance. Additional work carried out with Phe, and Tyr accepting tRNA{dollar}\sp{lcub}\rm fMet{rcub}{dollar} derivatives indicated that these tRNAs also harbor identity elements at the discriminator base in addition to those in the anticodon.;In conclusion, these results indicate that the anticodon and discriminator base are perhaps the most important tRNA domains in allowing an aminoacyl-tRNA synthetase to choose the proper tRNA substrate for aminoacylation.