Initiator-Elongator Discrimination in Vertebrate tRNAs for Protein Synthesis

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 methionyl-tRNA (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 base pair at the end of the acceptor stem, (ii) a sequence of three consecutive G:C base pairs (G29G30G31:C39C40C41) in the anticodon stem, and (iii) A54 and A60 in the TΨC loop instead of T54 and pyrimidine 60 found in virtually all elongator tRNAs. Interestingly, the first two of these unique features are also found in archaebacterial initiator tRNAs (60). In previous work, we have used in vitro functional studies on mutant human initiator tRNAs expressed in mammalian CV-1 cells or in yeasts to study the role of the above features in the activities of the mutant tRNAs in initiation of protein synthesis and in binding to eIF2 (13, 17). Here, we describe functional studies, in vivo and in vitro, to identify the determinants on the human initiator tRNA which prevent it from acting as an elongator in mammalian cells. Functional studies in vitro have shown that the primary determinant which blocks the participation of yeast and wheat germ initiator tRNAs in the elongation step of protein synthesis is a bulky 2′-O-phosphoribosyl modification of ribose at position 64 (11, 19, 20, 57). This is most likely due to protrusion of the bulky group into the minor groove of the TΨC stem helix, leading to a steric block in binding of these tRNAs to eEF1 (4, 19). Further support comes from isolation of mutants of the yeast Saccharomyces cerevisiae that are defective in modification of the ribose 64 in the initiator tRNA and from the demonstration that in this mutant strain, the initiator methionine tRNA can act as an elongator in vivo (1). In contrast to fungi and plants (20, 48, 57), vertebrate initiator tRNAs do not have a 2′-O-phosphoribosyl modification of the ribose at position 64 (21, 22, 46, 58, 60). This raises the question of how the vertebrate initiator tRNAs are prevented from acting as elongators in the corresponding protein synthesis systems. In this paper, using functional studies of mutant tRNAs in vivo in mammalian COS1 cells and in vitro in rabbit reticulocyte cell extracts, we show that the primary determinant preventing the human initiator tRNA from acting as an elongator is the nature of the base pairs 50:64 and 51:63 in the TΨC stem. A secondary determinant is the A1:U72 base pair at the end of the acceptor stem. Mutation of A1:U72 alone to G1:C72 endows the mutant tRNA with partial activity in elongation, but less so than mutations in the TΨC stem. A mutant human initiator tRNA carrying both the G1:C72 mutation and mutations in base pairs 50:64 and 51:63 in the TΨC stem is almost as active in elongation in vitro as the elongator methionine tRNA.