Location alters tRNA identity: Trypanosoma brucei's cytosolic elongator tRNA^Met is both the initiator and elongator in mitochondria

As a rule, protein synthesis is initiated with methionine or formyl methionine from the first AUG codon in an ORF and that AUG is recognized by an initiator tRNA^Met-i. Internal AUG codons are then translated by a second tRNA, the elongator tRNA^Met-e (1). In eubacteria, mitochondria, and chloroplasts, the tRNA^Met-i is charged with methionine, and subsequently the methionine is formylated by a methionyl-tRNA transformylase, whereas in the eukaryotic cytosol and archea, the methionine on the tRNA^Met-i is not formylated (2). Until now, the one known exception to the tRNA^Met-i/tRNA^Met-e paradigm occurred in animal mitochondria where a single mtDNA-coded tRNA is thought to serve for both initiation and elongation (3). The work of Tan et al. (4) in this issue of PNAS provides a second exception with several different twists as they show that the function of a tRNA in initiation and/or elongation depends on its location.

Although all mtDNAs code for rRNAs necessary to support mitochondrial protein synthesis, the number of tRNAs coded by mtDNA varies widely. Some mtDNAs code for a complete set of tRNAs needed for protein synthesis, and some have an incomplete set and depend on import of certain cytosolic tRNAs (5). Still others do not contain any tRNA genes and must import a full complement of tRNAs from the cytosol. Trypanosoma brucei belongs to the latter class of organisms, and when Tan et al. (4) analyzed its nearly complete genome sequence (supplied by The Institute for Genomic Research (Rockville, MD) and the Sanger Institute (Cambridge, U.K.) for methionine tRNA genes, as expected, they found genes coding for distinct initiator-like and elongator-like tRNAs. One has an A1:U72 base pair and three consecutive GC base pairs at the end of the anticodon stem, A54 and A60, and lacks the TΨC sequence in loop IV, characteristics common in eukaryotic tRNA^Met-i so it has been assigned to the initiator tRNA class. The second gene codes for a tRNA with a loop IV sequence characteristic of tRNA^Met-e and has been assigned to the elongator tRNA class.

The work of Tan et al. shows that the function of a tRNA in initiation and/or elongation depends on its location.

Tan et al. (4) went on to examine the tRNA^Met population in T. brucei and demonstrated both genes are expressed. They sought to identify any additional tRNA^Mets by direct labeling but found only the two tRNAs predicted from the genome sequencing project. As expected, an in organello incubation with ¹⁴C formate yielded only one labeled tRNA. The surprising and unexpected result, however, was that the labeled tRNA corresponded to the cytosolic tRNA^Met-e, not the cytosolic tRNA^Met-i. Of the two tRNAs, the latter shares more characteristics with the Escherichia coli initiator tRNA that are important recognition elements for the E. coli formylase (1). Animal mitochondrial tRNA shares the same characteristics but in contrast to E. coli, the purine-pyrimidine pair at position 11–24 and the methionine are the two most important mitochondrial formylase recognition features (3). Unlike animal mitochondrial tRNA, the T. brucei tRNA^Met-e does not retain the 11–24 purine-pyrimidine pair.

In organello labeling with ³⁵S methionine demonstrated that only the tRNA^Met-e is imported. Thus the T. brucei mitochondrial protein synthetic system, like that of animals, uses a single tRNA^Met for initiation and elongation even though a priori both tRNA^Met-i and tRNA^Met-e should be available for import into the organelle. Formyl methionine is the initiating amino acid and a portion of the single tRNA^Met, as in animal mitochondria (3), is formylated by a mitochondrial-specific methionyl-tRNA transformylase for use in initiation. In mitochondria from other organisms, however, mitochondrial-encoded tRNA^Met-i and tRNA^Met-e are found (6).

The rules that govern the import of tRNAs into mitochondria are poorly characterized so there is little basis for understanding why one T. brucei cytosolic tRNA^Met is imported and one is not. There are few experiments that address the determinants necessary for import of tRNA into mitochondria. The anticodon is important for mitochondrial import in Tetrahymena where there are three cytoplasmic glutamine tRNAs with three different anticodons. Only the tRNA with the UUG anticodon is imported, and swapping UUG for the anticodons of the other cytosolic glutamine tRNAs converts each from a solely cytosolic tRNA to a tRNA shared between the cytosol and mitochondria (7). In yeast, a tRNA^Lys is imported and the anticodon and the first base pair of the acceptor stem are important import determinants (8). The elements necessary to promote import of T. brucei cytosolic tRNAs into mitochondria are not yet clear but at least some appear to be imported as precursors (9). However, it is clear from the work of Tan et al. (4) that the anticodon, at least for these two methionine tRNAs, cannot be the only determinant as both have the same anticodon but only one methionine tRNA is imported.

T. brucei cytosolic and mitochondrial extracts charge both tRNAs with methionine but only the mitochondrial extracts contain the formylation activity. In vitro transcripts of the tRNA^Met-e gene can be aminoacylated but not formylated whereas tRNA^Met-e isolated from either compartment is formylated with similar efficiency. The difference in behavior between in vitro-synthesized tRNA and those tRNAs isolated from cells raises the possibility that a modified base is an important recognition element for the T. brucei transformylase. Because cytosolic and mitochondrial tRNAs both can be formylated, any modification must be made in the cytosol. Presumably sequestration of the transformylase in the mitochondria separates it from the cytosolic pool of tRNA^Met-e and prevents formylation of the tRNA^Met-e in the cytosol. The tRNA^Met-i is not a substrate for the transformylase but this is not unusual per se. Many, but not all, eukaryotic initiator tRNAs are substrates for prokaryotic and organelle transformylases (1).

Tan et al. (4) went on to identify a T. brucei gene homologous to bacterial and mitochondrial methionyl-tRNA transformylases, and using an RNA interference assay, proved that it codes for the comparable activity. Interestingly, although formylase activity was substantially reduced, there was no obvious effect on cell growth. This finding contrasts with results showing that elimination of transformylase activity in E. coli causes a severe growth defect (10) but is consistent with experiments in yeast where deletion of the gene coding for the mitochondrial methionyl transformylase had no obvious effect on cell growth (11). Either formylation is preferred, but not necessary, for efficient initiation of protein synthesis in mitochondria, or it is required and as-yet-unrecognized compensatory changes in the mitochondrial protein synthetic system in these mutant backgrounds in yeast and T. brucei compensate for the lack of formylation. Artificial manipulations of initiation factor 2 and overexpression of tRNA^Met-i can compensate for a lack of formylation activity in E. coli (12) and at least raise the possibility that similar changes could serve to compensate in mitochondria in vivo.

tRNA^Met-i and tRNA^Met-e have fundamentally different functions because the former must bind directly to the P site on the ribosome and must be excluded from binding to the A site. The latter must bind first to the A site and then translocate to the P site. In E. coli, the ability to be formylated and the ability to bind to the ribosomal P site are the two most important properties needed for a tRNA to function in initiation (13). In addition, formylated tRNA^Met-i binds poorly, if at all, to EF-Tu-GTP and thus cannot bind the A site. Nonetheless, RajBhandary and his colleagues (reviewed in ref. 1) have isolated and/or created a number of mutations in the E. coli initiator tRNA that allow it to act in both initiation and elongation and in the E. coli elongator tRNA that allows it to act as both elongator and initiator. Therefore, specific nucleotide changes can expand the ability of prokaryotic tRNAs to provide both initiation and elongation functions.

tRNAs can be made to function in both elongation and initiation regardless of whether their wild-type counterparts normally function in one role or the other.

Experiments using genetic manipulations demonstrated that at high gene dosage, the yeast cytosolic tRNA^Met-i could function in elongation. The wild-type tRNA^Met-e cannot, however, when overexpressed, function in initiation, but tRNA^Met-e with mutations at position 54 can (14). Also, Astrom and Bystrom (15) identified a gene called RIT1 that modifies the yeast cytosolic tRNA^Met-i at position 64 with a 2′-O-ribosyl phosphate. Rit1 mutants do not make this modification, and the unmodified tRNA^Met-i participates in both initiation and elongation. Indeed, incomplete modification of cytosolic tRNA^Met-i could explain why it can act in elongation when it is overproduced. These experiments demonstrate that nucleotide modifications (or the lack thereof) play an important role in determining the ability of tRNAs to function in either initiation or elongation. Experiments with vertebrate tRNAs revealed that the nature of base pairs 50:64 and 51:63 are important in preventing the human tRNA^Met-i from serving as an elongator. This is presumably because the wild-type TΨC stem structure blocks binding of the elongation factor that is a necessary prerequisite for A site binding. Again, mutations yield tRNAs that can act as elongators (15).

It is clear from the above brief descriptions of the experiments in E. coli, yeast, and vertebrates that tRNAs can be made to function in both elongation and initiation regardless of whether their wild-type counterparts normally function in one role or the other. There are several possible ways that an elongator tRNA in one compartment could function as both an initiator and elongator in another. Alterations to the tRNA in the mitochondria could create distinct tRNAs. First of all, given the robust RNA editing machinery required to turn incomplete T. brucei mtDNA-encoded ORFs into translatable mRNAs, one real possibility is that a portion of the imported tRNA^Met-e could be altered by this machinery upon import. For example, a mitochondrial-specific base change that could prevent formylation would assure a population of elongators. There is precedent for editing tRNAs imported from the cytoplasm as editing of an imported tRNA does occur in Leishmania (16). However, primer extension sequencing experiments (4) do not support the idea that the sequence of the imported tRNA^Met-e has been altered in T. brucei mitochondria. The second possibility is that a portion of the tRNA that is imported could be modified such that it becomes a poor formylation substrate and/or less likely to interact with initiation factors than elongation factors. In this case, because tRNAs from both compartments are substrates for the transformylase, the restricting modification would have to take place in mitochondria. Alternatively, the modification that seemingly promotes formylation on the imported tRNA^Met-e (see above) could, rather than just the formylation itself, be an important determining factor dictating a role in initiation. Comparisons of the actual sequences, including modified bases, of the mitochondrial tRNA and the cytosolic tRNA from T. brucei will be necessary to begin to evaluate these possibilities. A third possibility is that the tRNA^Met-e population in the cytosol and mitochondria of T. brucei is identical as it appears to be in animal mitochondria (3). In this case, an interplay of interactions between the formylase, elongation factors, and initiation factors in mitochondria would have to limit formylation so only a percent of the total tRNA substrate is channeled toward a role in initiation, reserving another portion for a role in elongation in vivo.

Most likely, the presence of formyl methionine, rather than a modification per se, is going to be the important determinant for the initiation role of tRNA^Met-e in T. brucei mitochondria. Indeed, the formyl-methionine moiety is clearly an important, if not the most important, determining factor in initiation in bacteria and other organelles studied to date. It also may be that the CCC/GGG sequence in the anticodon stem plays a role in P site binding in T. brucei as it does in E. coli. However, nothing is known about T. brucei tRNAs P site and/or A site binding, and it is not likely rules that apply in E. coli will apply to a protein synthetic system operating with unusual mitochondrial ribosomes containing the smallest known rRNAs (17).

Tan et al. (4) set out to consider the question of how mitochondrial translation initiation can function with imported eukaryotic-type tRNAs and found the unexpected result that only one methionyl tRNA is imported in T. brucei and the imported tRNA had sequence characteristics of tRNA^Met-e but was formylated and functioned in both initiation and elongation. Further work will be necessary to sort through the various possibilities that allow this single tRNA to fulfill both initiation and elongation functions. We can expect new insights into protein synthesis as a result.