Mutants of Escherichia coli initiator tRNA that suppress amber codons in Saccharomyces cerevisiae and are aminoacylated with tyrosine by yeast extracts.

Abstract

We recently described mutants ofEschenchia coU initiator tRNA that suppress amber termination codons (UAG) in E. coUl. These mutants have changes in the anticodon sequence (CAU --CUA) that allow them to read the amber codon and changes in the acceptor stem that allow them to bind to the ribosomal aminoacyl (A) site. We show here that a subset of these mutants suppress amber codons in Saccharomyces cerevsiae and that they are aminoacylated with tyrosine by yeast extracts. Analysis of a number of mutants as substrates for yeast tyrosyl-tRNA synthetase has led to identification ofthe C1 G72 base pair and the discriminator base A73, conserved in all eukaryotic cytoplasmic and archaebacterial tyrosine tRNAs, as being important for recognition. Our results suggest that the C1G72 base pair and the discriminator base, in addition to the anticodon nucleotides previously identified the generation and characterization of mutants that suppress amber codons in E. coli (16,23). One of these mutants has a ClG72 base pair, at the end of the acceptor stem. This feature is otherwise found only in tyrosine tRNA of eukaryotic cells and archaebacteria and in proline tRNAs of eubacteria and bacterial viruses (24). We therefore investigated the possibility that this mutant might function as an amber suppressor in the yeast Saccharomyces cerevisiae. We show here that it does and that it is aminoacylated with tyrosine by yeast extracts. Results ofaminoacylation assays on several of the mutants suggest an absolute requirement for the C1lG72 base pair for aminoacylation with tyrosine. Comparison of kinetic parameters in aminoacylation suggests that one of the mutant E. coli initiator tRNAs is as good a substrate for yeast TyrRS as a yeast tyrosine-inserting amber suppressor tRNA studied by Bare and Uhlenbeck (21). We discuss the implications of these results for recognition of tRNAs by yeast TyrRS.The ability to introduce site-specific mutations into tRNA genes (1-4) and to study the properties of mutant tRNAs in vitro and in vivo has led to major advances in identification of sequence elements important in recognition of tRNAs by the corresponding aminoacyl-tRNA synthetases. These studies (reviewed in refs. 5-7) show that, in most cases, sequences important for specificity in recognition are located primarily in the anticodon sequence (8-10), the acceptor stem (11, 12), or both (13-16). However, there are several examples in which nucleotides important for recognition are more widely dispersed, including a combination of anticodon sequence, acceptor stem, dihydrouridine stem, dihydrouridine loop, variable loop, etc. (4, 17-19). Details of the nature of interactions between aminoacyl-tRNA synthetases and tRNAs are beginning to emerge from parallel work on x-ray crystallographic structure of the enzyme-tRNA complexes (13,15,20). In the two known structures of such complexes (15,20), the enzyme makes extensive contact with the tRNA substrate and there is substantial change in the local structure of the tRNA in the anticodon loop and...

Initiator transfer RNAs

“…These studies confirm the results of in vitro analyses and show that all the mutants that have a 1:72 base pair are active in elongation in vivo in E. coli and in Saccharomyces cerevisiae (10,19,29,35).…”

tRNA anticodon shifts in eukaryotic genomes

“…Two shifts involving tRNA-Lys and tRNA-Asn do not contradict any known identity determinants, since the posterior state tRNA-Lys identity is dependent mainly on the anticodon (Stello et al 1999;Francin and Mirande 2006). Out of the remaining six shifts, two have mutations that contradict the known identity determinants of one anticodon state: These are for tRNA-Tyr (G72) (Lee and RajBhandary 1991) and tRNA-Leu (A73 and A37) (Breitschopf and Gross 1994;Breitschopf et al 1995). For the remaining four, we found no relevant identity determinant data to support or contradict coevolution at these sites.…”

The many applications of acid urea polyacrylamide gel electrophoresis to studies of tRNAs and aminoacyl-tRNA synthetases

“…Otherwise, once the aminoacylated suppressor tRNA has inserted the unnatural amino acid at the designated site in the target protein, it will be re-aminoacylated with a natural amino acid and insert the natural amino acid instead of the analogue, thus generating a heterogeneous pool of target protein molecules. Many such orthogonal suppressor tRNAs have been established over the past years, including suppressor tRNAs derived from the eubacterial tRNA Tyr [31][32][33][34][35][36], archaeal tRNA Tyr [37], yeast tRNA Phe [38], bacterial tRNA Trp [39], E. coli tRNA Gln [15,18], and even human and E. coli initiator tRNAs [40,16,13]. The next requirement is the use of an orthogonal aminoacyl-tRNA synthetase that specifically recognizes the suppressor tRNA but no other tRNA in the cell ( Figure 3A) (reviewed in [41][42][43][44]).…”