Initiator-Elongator Discrimination in Vertebrate tRNAs for Protein Synthesis

Abstract

Initiator tRNAs are used exclusively for initiation of protein synthesis and not for the elongation step. We show, in vivo and in vitro, that the primary sequence feature that prevents the human initiator tRNA from acting in the elongation step is the nature of base pairs 50:64 and 51:63 in the T⌿C stem of the initiator tRNA. Various considerations suggest that this is due to sequence-dependent perturbation of the sugar phosphate backbone in the T⌿C stem of initiator tRNA, which most likely blocks binding of the elongation factor to the tRNA. Because the sequences of all vertebrate initiator tRNAs are identical, our findings with the human initiator tRNA are likely to be valid for all vertebrate systems. We have developed reporter systems that can be used to monitor, in mammalian cells, the activity in elongation of mutant human initiator tRNAs carrying anticodon sequence mutations from CAU to CCU (the C35 mutant) or to CUA (the U35A36 mutant). Combination of the anticodon sequence mutation with mutations in base pairs 50:64 and 51:63 yielded tRNAs that act as elongators in mammalian cells. Further mutation of the A1:U72 base pair, which is conserved in virtually all eukaryotic initiator tRNAs, to G1:C72 in the C35 mutant background yielded tRNAs that were even more active in elongation. In addition, in a rabbit reticulocyte in vitro protein-synthesizing system, a tRNA carrying the T⌿C stem and the A1:U72-to-G1:C72 mutations was almost as active in elongation as the elongator methionine tRNA. The combination of mutant initiator tRNA with the CCU anticodon and the reporter system developed here provides the first example of missense suppression in mammalian cells.A special methionine tRNA is used for the initiation of protein synthesis in all organisms that have been studied. Of the two classes of methionine tRNAs found universally, the initiator is used exclusively for initiation and the elongator is used for insertion of methionine into internal peptidic linkages (33,48). Eubacteria, mitochondria, and chloroplasts use formylmethionine-tRNA (41) for initiation (10, 33), whereas the cytoplasmic protein synthesis system of eukaryotes uses methionyltRNA (Met-tRNA) without formylation (25, 59).Because of their unique function, initiator tRNAs from eubacteria and eukaryotes possess a number of special properties distinct from those of elongator tRNAs. For eukaryotic cytoplasmic initiator tRNAs, these properties are (i) the formation of a specific complex among the initiator Met-tRNA, eukaryotic initiation factor 2 (eIF2), and GTP (42); (ii) the binding of the initiator Met-tRNA to the ribosomal P site; and (iii) the exclusion of the initiator Met-tRNA from the ribosomal A site. In contrast, elongator aminoacyl-tRNAs form a ternary complex with the eukaryotic elongation factor 1 (eEF1) and GTP and bind to the ribosomal A site.Along with their special properties, eukaryotic initiator tRNAs also possess a number of unique sequence and structural features not found in elongator tRNAs (49, 57). These include (i) an A1:U72...

Twenty-first aminoacyl-tRNA synthetase–suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria

“…The 21st synthetase-tRNA pairs that we have developed for use in eukaryotes and in eubacteria consist, in both cases, of a heterologous aaRS and a suppressor tRNA derived from the initiator tRNA of the same organism or a related organism (11,12). Besides providing the only example of a 21st synthetasetRNA pair for use in a eukaryotic system, our approach differs somewhat from that of Schultz and coworkers, in which both the aaRS and the suppressor tRNA are imported from a heterologous organism (13,14), and it offers certain advantages.…”

“…Results 21st Synthetase-tRNA Pair for Use in Yeast. We reported previously on a mutant human initiator tRNA that can act as an amber suppressor in mammalian COS1 cells (12). This tRNA has the U35A36 mutations in the anticodon sequence, which allows it to read the UAG codon and the U50G51:C63A64 mutations in the T⌿C stem, which allows it to participate in elongation.…”

“…The gene encoding the human initiator tRNA is not transcribed by yeast RNA polymerase III in the context of its own 5Ј-flanking sequence (29). Therefore, for expression in yeast, the mutant human initiator tRNA coding sequence was cloned downstream of the 5Ј-flanking sequence of the yeast tRNA Arg gene (20).…”

“…The yeast system represents the only 21st synthetase-tRNA pair for use in a eukaryote. The suppressor tRNAs (11,12) are derived from a tRNA of another eukaryotic organism (human initiator tRNA for use in yeast) or from a tRNA of the same organism (E. coli initiator tRNA 2 fMet for use in E. coli). The 21st synthetases are E. coli GlnRS for use in yeast and mutants of yeast TyrRS for use in E. coli.…”

Making sense out of nonsense

“…The approach is based on their previous studies comparing the sequences and properties of elongator and initiator tRNA Met (31,32) and uses tRNA from one organism and a synthetase from another to create new cognate pairs. The E. coli system uses an engineered E. coli nonsense-suppressing initiator tRNA Met that participates in protein chain elongation rather than initiation, that is not aminoacylated by any of the E. coli synthetases, but that is aminoacylated by yeast tyrosyl-tRNA synthetase (TyrRS).…”