The Catalytic Domain of Topological Knot tRNA Methyltransferase (TrmH) Discriminates between Substrate tRNA and Nonsubstrate tRNA via an Induced-fit Process

A conserved guanosine at position 18 (G18) in the D-loop of tRNAs is often modified to 2′-O-methylguanosine (Gm). Formation of Gm18 in eubacterial tRNA is catalyzed by tRNA (Gm18) methyltransferase (TrmH). TrmH enzymes can be divided into two types based on their substrate tRNA specificity. Type I TrmH, including Thermus thermophilus TrmH, can modify all tRNA species, whereas type II TrmH, for example Escherichia coli TrmH, modifies only a subset of tRNA species. Our previous crystal study showed that T. thermophilus TrmH is a class IV S-adenosyl-l-methionine-dependent methyltransferase, which maintains a topological knot structure in the catalytic domain. Because TrmH enzymes have short stretches at the N and C termini instead of a clear RNA binding domain, these stretches are believed to be involved in tRNA recognition. In this study, we demonstrate by site-directed mutagenesis that both N- and C-terminal regions function in tRNA binding. However, in vitro and in vivo chimera protein studies, in which four chimeric proteins of type I and II TrmHs were used, demonstrated that the catalytic domain discriminates substrate tRNAs from nonsubstrate tRNAs. Thus, the N- and C-terminal regions do not function in the substrate tRNA discrimination process. Pre-steady state analysis of complex formation between mutant TrmH proteins and tRNA by stopped-flow fluorescence measurement revealed that the C-terminal region works in the initial binding process, in which nonsubstrate tRNA is not excluded, and that structural movement of the motif 2 region of the catalytic domain in an induced-fit process is involved in substrate tRNA discrimination.     

Frameshift Suppression in SACCHAROMYCES CEREVISIAE. V. Isolation and Genetic Properties of Nongroup-Specific Suppressors

Two classes of frameshift suppressors distributed at 22 different loci were identified in previous studies in the yeast Saccharomyces cerevisiae. These suppressors exhibited allele-specific suppression of +1 G:C insertion mutations in either glycine or proline codons, designated as group II and group III frameshift mutations, respectively. Genes corresponding to representative suppressors of each group have been shown to encode altered glycine or proline tRNAs containing four base anticodons.—This communication reports the existence of a third class of frameshift suppressor that exhibits a wider range in specificity of suppression. The suppressors map at three loci, suf12, suf13, and suf14, which are located on chromosomes IV, XV, and XIV, respectively. The phenotypes of these suppressors suggest that suppression may be mediated by genes other than those encoding the primary structure of glycine or proline tRNAs.     

Intrinsic Precision of Aminoacyl-tRNA Synthesis Enhanced through Parallel Systems of Ligands

MOST aminoacyl-tRNAs possessed by an organism must contain amino-acids matched to their correct anticodon so that the meaning of structural genetic information will be preserved. It is only recently, however, that we have begun to understand the underlying molecular mechanisms with regard to isoleucyl-tRNA of Escherichia coli B. Isoleucyl-tRNA synthetase, which is representative of many others in size and other properties¹, is quite selective among tRNAs, in that it binds strongly only to the cognate tRNA^Ile species². However, a weaker, but still significant affinity for non-cognate tRNAs from E. coli can be detected³. In addition, non-cognates are isoleucylated (ref. 3 and unpublished work), albeit at a maximum rate considerably slower than tRNA^Ile. Two of these reactions, the binding and isoleucylation of tRNA^phe (E. coli)³ and of tRNA_f^Met (E. coli), have been studied in detail. The generality of this phenomenon could prove important. First, the tRNA concentrations in E. coli are high (they can be no less than about 0.5 × 10^?5 M^4,5 for individual species) compared with those usual in vitro and thus even weak binding could be significant. Second, many incorrect interactions are possible. Assuming that there are about sixty molecular species of tRNA and twenty species of aminoacyl-tRNA synthetase, there are 1,200 possible interacting pairs and only about sixty of these (or 5%) are cognates. Since it is presumably desirable that misacylated tRNAs be held to a very small fraction of the total, misacylation could be significant, even if it is always a slow reaction. I have, as of writing, examined five species of purified tRNA, of which the two already mentioned give misacylations which are sufficiently facile to be easily studied under usual in vitro conditions. The other three are much less easily isoleucylated, but also give indications of reaction (my unpublished data). Thus, this limited survey emphasizes that these reactions may be common and that rejection of non-cognate tRNAs by the aminoacyl-tRNA synthetase may not be the only mechanism by which the correctness of the aminoacyl-tRNAs is assured. In fact, I have already reported^3,6 that Ile-tRNA^phe, synthesized by isoleucyl-tRNA synthetase, is rapidly destroyed by phenylal-anyl-tRNA synthetase and have suggested⁶ that the aminoacyl-tRNA synthetases may have a function in addition to synthesis of aminoacyl-tRNAs; that of destruction of misacylated cognate tRNAs.     

Increased activity of rat liver N2-guanine tRNA methyltransferase II in response to liver damage

Alterations in rat liver transfer RNA (tRNA) methyltransferase activities have been observed after liver damage by various chemicals or by partial hepatectomy. The qualitative and quantitative nature of these activity changes and the time course for their induction have been studied. Since homologous tRNAs are essentially fully modified in vivo, E. coli tRNAs were used as in vitro substrates for the rat liver enzymes in these studies. Each of the liver-damaging agents tested rapidly caused increases in activities of the enzyme(s) catalyzing methyl group transfer to tRNAs that have an unmodified guanine at position 26 from the 5′ end of the molecule. This group of tRNAs includes E. coli tRNA^Nfmet, tRNA^Ala₁, tRNA^Leu₁, or ^Leu₂, and tRNA^Ser₃ (Group 1). In each case N²-methylguanine and N²,N²-dimethylguanine represented 90% or more of the products of these in vitro methylations. The product and substrate specificity observed are characteristic of N²-guanine methyltransferase II ($\text{S-}\text{adenosyl}\text{-}\text{L}\text{-}\text{methionine:tRNA}$ (guanine-2)-methyltransferase, EC 2.1.1.32). In crude and partially purified preparations derived from livers of both control and treated animals this enzyme activity was not diminished significantly by exposure to 50°C for 10 min. The same liver-damaging agents induced little or no change in the activities of enzymes that catalyze methyl group transfer to various other E. coli tRNAs that do not have guanine at position 26 (Group 2). The results of mixing experiments appear to rule out the likelihood that the observed enzyme activity changes are due to stimulatory or inhibitory materials present in the enzyme preperations from control or treated animals. Thus, our experiments indicate that liver damage by each of several different methods, including surgery or administration of chemicals that are strong carcinogens, hepatotoxins, or cancer-promoting substances, all produce changes in liver tRNA methyltransferase activity that represent a selective increase in activity of N²-guanine tRNA methyltransferase II. It is proposed that the specificity of this change is not fortuitous, but is the manifestation of an as yet unidentified regulatory process.     

Dual Targeting of a tRNAAsp Requires Two Different Aspartyl-tRNA Synthetases in Trypanosoma brucei

The mitochondrion of the parasitic protozoon Trypanosoma brucei does not encode any tRNAs. This deficiency is compensated for by partial import of nearly all of its cytosolic tRNAs. Most trypanosomal aminoacyl-tRNA synthetases are encoded by single copy genes, suggesting the use of the same enzyme in the cytosol and in the mitochondrion. However, the T. brucei genome encodes two distinct genes for eukaryotic aspartyl-tRNA synthetase (AspRS), although the cell has a single tRNA^Asp isoacceptor only. Phylogenetic analysis showed that the two T. brucei AspRSs evolved from a duplication early in kinetoplastid evolution and also revealed that eight other major duplications of AspRS occurred in the eukaryotic domain. RNA interference analysis established that both Tb-AspRS1 and Tb-AspRS2 are essential for growth and required for cytosolic and mitochondrial Asp-tRNA^Asp formation, respectively. In vitro charging assays demonstrated that the mitochondrial Tb-AspRS2 aminoacylates both cytosolic and mitochondrial tRNA^Asp, whereas the cytosolic Tb-AspRS1 selectively recognizes cytosolic but not mitochondrial tRNA^Asp. This indicates that cytosolic and mitochondrial tRNA^Asp, although derived from the same nuclear gene, are physically different, most likely due to a mitochondria-specific nucleotide modification. Mitochondrial Tb-AspRS2 defines a novel group of eukaryotic AspRSs with an expanded substrate specificity that are restricted to trypanosomatids and therefore may be exploited as a novel drug target.In most animal and fungal mitochondria, the total set of tRNAs required for translation is encoded on the mitochondrial genome and thus of bacterial evolutionary origin. The aminoacyl-tRNA synthetases (aaRSs)² responsible for charging of mitochondrial tRNAs are always nuclear encoded and need to be imported into mitochondria. We therefore expect to find two sets of aaRSs, one for cytosolic aminoacyl-tRNA synthesis and a second one, of bacterial evolutionary origin, for aminoacylation of mitochondrial tRNAs (1, 2).In most cells, however, some aaRSs are targeted to both the cytosol as well as to mitochondria (3). In Saccharomyces cerevisiae, for example, four aaRSs are double-targeted to both compartments, indicating that they are able to aminoacylate tRNAs of both eukaryotic and bacterial evolutionary origin (4–6). In plants, the situation is more complex, since protein synthesis occurs in three compartments: the cytosol, the mitochondria, and the plastids. A recent analysis in Arabidopsis has shown that, rather than having three unique sets of aaRSs specific for the three translation systems, more than 15 aaRSs were dually targeted to the mitochondria and the plastid (7). Moreover, there is at least one aaRS that is shared between all three compartments. In summary, these examples indicate that the overlap between the different sets of aaRSs used in the various translation systems is variable and can be extensive.Most eukaryotes, except many animals and fungi, lack a variable number of mitochondrial tRNA genes. Mitochondrial translation in these organisms depends on import of a small fraction of the corresponding nucleus-encoded cytosolic tRNAs (8–10). As a consequence, imported tRNAs are always of eukaryotic evolutionary origin. An intriguing situation is found in trypanosomatids (such as Trypanosoma brucei and Leishmania spp.), where all mitochondrial tRNA genes have apparently been lost and all mitochondrial tRNAs are imported from the cytosol. In these organisms, all mitochondrial tRNAs derive from cytosolic tRNAs (11). It is therefore reasonable to assume that trypanosomal aaRSs are dually targeted to the cytosol and the mitochondrion. For the T. brucei glutaminyl-tRNA synthetase (GlnRS) and the glutamyl-tRNA synthetase, the dual localization has been shown experimentally (12). Moreover, dual targeting of essentially all aaRSs is suggested by the fact that the genome of T. brucei and other trypanosomatids encodes only 23 distinct aaRSs, fewer than any other eukaryote that has a mitochondrial translation system (13). Unexpectedly, two distinct genes were found for the tryptophanyl-tRNA synthetase (TrpRS), the lysyl-tRNA synthetase and the aspartyl-tRNA synthetase (AspRS). A recent study has shown that the two trypanosomal TrpRSs are required for cytosolic and mitochondrial tryptophanyl-tRNA formation (14). Trypanosomal tRNA^Trp is imported to the mitochondria, where it undergoes C to U editing at the wobble nucleotide and is thiolated at position 33. The RNA editing is required to decode the reassigned mitochondrial tryptophan codon UGA (14–16). Both nucleotide modifications are antideterminants for the cytosolic TrpRS (14). As we concluded previously (14), the presence of a second TrpRS with expanded substrate specificity is required to efficiently aminoacylate imported, mature tRNA^Trp in trypanosomal mitochondria.The present study focuses on the characterization and functional analysis of another pair of duplicated trypanosomal aaRSs, the AspRSs. We show that the two enzymes are individually essential for normal growth of insect stage T. brucei. We also demonstrate that the two trypanosomal AspRSs are of eukaryotic evolutionary origin and that the aminoacylation of the cytosolic and mitochondrial tRNA^Asp species requires these two distinct AspRSs.     

Specific binding of bovine immunoglobulins to Sepharose and Sephadex

Bacteriophage T5 BglII/HindIII DNA fragment (803 basepairs), containing the genes for 2 tRNAs and 2 RNAs with unknown functions, was cloned in the plasmid pBR322. The analysis of DNA sequence indicates that tRNA genes code isoacceptor tRNAs^Ser (tRNA^Ser₁ and tRNA^Ser₂) with anticodons UGA and GGA, respectively. The main unusual structural feature of these tRNAs is the presence of extra non-basepaired nucleotides in the joinings of stem ‘b’ with stems ‘a’ and ‘c’.     

Mitochondrial tRNAs as light strand replication origins: Similarity between anticodon loops and the loop of the light strand replication origin predicts initiation of DNA replication

Stem-loop hairpins formed by mitochondrial light strand replication origins (OL) and by heavy strand DNA coding for tRNAs that form OL-like structures initiate mitochondrial replication. The loops are recognized by one of the two active sites of the vertebrate mitochondrial gamma polymerase, which are homologuous to the active sites of class II amino-acyl tRNA synthetases. Therefore, the polymerase site recognizing the OL loop could recognize tRNA anticodon loops and sequence similarity between anticodon and OL loops should predict initiation of DNA replication at tRNAs. Strengths of genome-wide deamination gradients starting at tRNA genes estimate extents by which replication starts at that tRNA. Deaminations (A→G and C→T) occur proportionally to time spent single stranded by heavy strand DNA during mitochondrial light strand replication. Results show that deamination gradients starting at tRNAs are proportional to sequence similarity between OL and tRNA loops: most for anticodon-, least D-, intermediate for TψC-loops, paralleling tRNA synthetase recognition interactions with these tRNA loops. Structural and sequence similarities with regular OLs predict OL function, loop similarity is dominant in most tRNAs. Analyses of sequence similarity and structure independently substantiate that DNA sequences coding for mitochondrial tRNAs sometimes function as alternative OLs. Pathogenic mutations in anticodon loops increase similarity with the human OL loop, non-pathogenic polymorphisms do not. Similarity/homology alignment hypotheses are experimentally testable in this system.     

Rapid Shifts in Mitochondrial tRNA Import in a Plant Lineage with Extensive Mitochondrial tRNA Gene Loss

In most eukaryotes, transfer RNAs (tRNAs) are one of the very few classes of genes remaining in the mitochondrial genome, but some mitochondria have lost these vestiges of their prokaryotic ancestry. Sequencing of mitogenomes from the flowering plant genus Silene previously revealed a large range in tRNA gene content, suggesting rapid and ongoing gene loss/replacement. Here, we use this system to test longstanding hypotheses about how mitochondrial tRNA genes are replaced by importing nuclear-encoded tRNAs. We traced the evolutionary history of these gene loss events by sequencing mitochondrial genomes from key outgroups (Agrostemma githago and Silene [=Lychnis] chalcedonica). We then performed the first global sequencing of purified plant mitochondrial tRNA populations to characterize the expression of mitochondrial-encoded tRNAs and the identity of imported nuclear-encoded tRNAs. We also confirmed the utility of high-throughput sequencing methods for the detection of tRNA import by sequencing mitochondrial tRNA populations in a species (Solanum tuberosum) with known tRNA trafficking patterns. Mitochondrial tRNA sequencing in Silene revealed substantial shifts in the abundance of some nuclear-encoded tRNAs in conjunction with their recent history of mt-tRNA gene loss and surprising cases where tRNAs with anticodons still encoded in the mitochondrial genome also appeared to be imported. These data suggest that nuclear-encoded counterparts are likely replacing mitochondrial tRNAs even in systems with recent mitochondrial tRNA gene loss, and the redundant import of a nuclear-encoded tRNA may provide a mechanism for functional replacement between translation systems separated by billions of years of evolutionary divergence.     

Misacylation of tRNA with methionine in Saccharomyces cerevisiae

Accurate transfer RNA (tRNA) aminoacylation by aminoacyl-tRNA synthetases controls translational fidelity. Although tRNA synthetases are generally highly accurate, recent results show that the methionyl-tRNA synthetase (MetRS) is an exception. MetRS readily misacylates non-methionyl tRNAs at frequencies of up to 10% in mammalian cells; such mismethionylation may serve a beneficial role for cells to protect their own proteins against oxidative damage. The Escherichia coli MetRS mismethionylates two E. coli tRNA species in vitro, and these two tRNAs contain identity elements for mismethionylation. Here we investigate tRNA mismethionylation in Saccharomyces cerevisiae. tRNA mismethionylation occurs at a similar extent in vivo as in mammalian cells. Both cognate and mismethionylated tRNAs have similar turnover kinetics upon cycloheximide treatment. We identify specific arginine/lysine to methionine-substituted peptides in proteomic mass spectrometry, indicating that mismethionylated tRNAs are used in translation. The yeast MetRS is part of a complex containing the anchoring protein Arc1p and the glutamyl-tRNA synthetase (GluRS). The recombinant Arc1p–MetRS–GluRS complex binds and mismethionylates many tRNA species in vitro. Our results indicate that the yeast MetRS is responsible for extensive misacylation of non-methionyl tRNAs, and mismethionylation also occurs in this evolutionary branch.     

The contacts of yeast tRNA(Ser) with seryl-tRNA synthetase studied by footprinting experiments

Yeast tRNA(Ser) is a member of the class II tRNAs, whose characteristic is the presence of an extended variable loop. This additional structural feature raises questions about the recognition of these class II tRNAs by their cognate synthetase and the possibility of the involvement of the extra arm in the recognition process. A footprinting study of yeast tRNA(Ser) complexed with its cognate synthetase, yeast seryl-tRNA synthetase (an alpha 2 dimer), was undertaken. Chemical (ethylnitrosourea) and enzymatic (nucleases S1 and V1) probes were used in the experiments. A map of the contact points between the tRNA and the synthetase was established and results were analyzed with respect to a three-dimensional model of yeast tRNA(Ser). Regions in close vicinity with the synthetase are clustered on one face of tRNA. The extra arm, which is strongly protected from chemical modifications, appears as an essential part of the contact area. The anticodon triplet and a large part of the anticodon arm are, in contrast, still accessible to the probes when the complex is formed. These results are discussed in the context of the recognition of tRNAs in the aminoacylation reaction.     

Intracardiac injection of a capsid-modified Ad5/35 results in decreased heart toxicity when compared to standard Ad5

Background

Human immunodeficiency virus type 1 (HIV-1) Nef-encoded protein plays key functions at almost all stages of the viral life cycle, but its role in translation is largely unknown.

Methods

To determine the effect of Nef on translation we used an in vitro translation assay. The detection of Nef/RPS10 complexes and the presence of 18S rRNA and tRNAs in the complexes were performed by coimmunoprecipitation and RT-PCR assay.

Results

We observed that the HIV-1 Nef protein specifically impaired translation in vitro. We observed the interaction of Nef with RPS10 by coimmunoprecipitation assay. In addition 18S rRNA and tRNAs were present in the Nef/RPS10 complexes.

Conclusions

Our results are consistent with a model in which the Nef protein by binding to two components of the 40S small ribosomal subunit, RPS10 and 18S rRNA, and to a lesser extent to tRNAs, could lead to decreased protein synthesis.     

What does the homology between E. coli tRNAs and RNAs controlling ColE1 plasmid replication mean?

Nucleotide sequences of E. coli tRNAs and RNA I or RNA II (controlling replication of ColE1 plasmids) were compared using the computer. The homology between some of these molecules is over 60%. The distribution of homologous nucleotides among the functional elements (stems and loops) of either RNA I or RNA II and the tRNAs molecules was studied. It was found that the homologous domains are located mainly in the loop regions of RNA I or RNA II. A consensus sequence, the nonanucleotide AGUUGGUAG, was discovered in loop II of RNA I and in the dihydrouridylic loop of tRNAs showing homology with RNA I. Based on this observation, a hypothesis was drawn for a possible role of the tRNAs in the regulation of plasmid DNA replication.     

Evolution of transfer RNA

Evolution by gene duplication and subsequent divergence is indicated by similarities common to 43 different transfer RNAs. Pairwise comparisons of these tRNAs reveal additional similarity, greatest for certain pairs of tRNAs for the same amino acid in the same organism, and also occurring in certain pairs of tRNAs for different amino acids in the same organism. Although tRNAs functionally interact with several other molecules, there have been surprisingly few restrictions on the divergence of their primary structures. This divergence has proceeded so far that clear phylogenetic separations are absent in most cases: it it impossible to construct a coherent phylogeny for most of the 43. Selection and stochastic processes have both been active in the evolution of tRNA. Selection has favored moderate change more than expected and has reduced radical change below that expected from stochastic processes alone. Two obvious effects of selection are nine invariant loci, another five that are always purines and five others that are always pyrimidines, in the tRNAs involved in protein synthesis. In addition to these constraints in the primary nucleotide sequence, the method of “identical site equivalents”, introduced here, demonstrates that further constraints exist equivalent to about 12 additional invariant loci. These “invisible” restraints reflect disperse chemical forces maintaining the tertiary structure and reducing evolutionary divergence to an extent quantitatively comparable to that of the nine observable invariant loci. The average divergence (49·4%) for pairs of tRNAs for different amino acids involved in protein synthesis represents an equilibrium between natural selection and stochastic processes. These tRNAs have had time to diverge nearly to the 75% maximum expected from stochastic process alone; this is shown by comparing the two glycine tRNAs involved in peptidoglycan synthesis with tRNAs for different amino acids participating in polypeptide synthesis. The rates of nucleotide replacements in genes coding for the tRNAs and the cytochromes c are about the same: 2 × 10 ^?10 replacements per nucleotide site per year.     

Partition of tRNAGly isoacceptors between protein and cell-wall peptidoglycan synthesis in Staphylococcus aureus

The sequence of tRNAs is submitted to evolutionary constraints imposed by their multiple interactions with aminoacyl-tRNA synthetases, translation elongation factor Tu in complex with GTP (EF-Tu•GTP), and the ribosome, each being essential for accurate and effective decoding of messenger RNAs. In Staphylococcus aureus, an additional constraint is imposed by the participation of tRNA^Gly isoacceptors in the addition of a pentaglycine side chain to cell-wall peptidoglycan precursors by transferases FmhB, FemA and FemB. Three tRNA^Gly isoacceptors poorly interacting with EF-Tu•GTP and the ribosome were previously identified. Here, we show that these ‘non-proteogenic’ tRNAs are preferentially recognized by FmhB based on kinetic analyses and on synthesis of stable aminoacyl-tRNA analogues acting as inhibitors. Synthesis of chimeric tRNAs and of helices mimicking the tRNA acceptor arms revealed that this discrimination involves identity determinants exclusively present in the D and T stems and loops of non-proteogenic tRNAs, which belong to an evolutionary lineage only present in the staphylococci. EF-Tu•GTP competitively inhibited FmhB by sequestration of ‘proteogenic’ aminoacyl-tRNAs in vitro. Together, these results indicate that competition for the Gly-tRNA^Gly pool is restricted by both limited recognition of non-proteogenic tRNAs by EF-Tu•GTP and limited recognition of proteogenic tRNAs by FmhB.     

Changes in transfer ribonucleic acids of Bacillus subtilis during different growth phases

The transfer ribonucleic acids (tRNAs) of B. subtilis at different growth phases are examined for changes in the composition and the methylation of minor constituents. The composition of the tRNAs indicates about equal amounts of adenosine and uridine, and of guanosine and cytidine. About 3-4 residues are present as modified bases in the average tRNA molecule. The net composition of tRNAs appears to remain unaltered during different growth phases. In vitro methylation of tRNAs indicates lack of methyl groups in both exponentially growing cells and spores. In vivo methylation studies show tRNA methylation occurs during the stationary phase in the absence of net tRNA synthesis. Thus, both in vitro and in vivo methylation indicates that the tRNAs in exponentially growing cells do not contain their full complement of modified bases. More complete modification is noted in tRNAs from stationary cells or spores. Hence, tRNA modifications in general are preserved with fidelity even in the dormant spore but the possibility is left open that specific modifications of selected isoacceptors of tRNAs may occur.     

Allosteric regulation of tRNA import: interactions between tRNA domains at the inner membrane of Leishmania mitochondria

Import of nucleus-encoded tRNAs into the mitochondria of the kinetoplastid protozoon Leishmania involves recognition of specific import signals by the membrane-bound import machinery. Multiple signals on different tRNA domains may be present, and further, importable RNAs interact positively (Type I) or negatively (Type II) with one another at the inner membrane in vitro. By co-transfection assays, it is shown here that tRNA^Tyr (Type I) transiently stimulates the rate of entry of tRNA^Ile (Type II) into Leishmania mitochondria in transfected cells, and conversely, is inhibited by tRNA^Ile. Truncation and mutagenesis experiments led to the co-localization of the effector and import activities of tRNA^Tyr to the D domain, and those of tRNA^Ile to the variable region–T domain (V-T region), indicating that both activities originate from a single RNA–receptor interaction. A third tRNA, human tRNA^Lys, is imported into Leishmania mitochondria in vitro as well as in vivo. This tRNA has Type I and Type II motifs in the D domain and the V-T region, respectively, and shows both Type I and Type II effector activities. Such dual-type tRNAs may interact simultaneously with the Type I and Type II binding sites of the inner membrane import machinery.