Dual mode recognition of two isoacceptor tRNAs by mammalian mitochondrial seryl-tRNA synthetase

Animal mitochondrial translation systems contain two serine tRNAs, corresponding to the codons AGY (Y = U and C) and UCN (N = U, C, A, and G), each possessing an unusual secondary structure; tRNA(GCU)(Ser) (for AGY) lacks the entire D arm, whereas tRNA(UGA)(Ser) (for UCN) has an unusual cloverleaf configuration. We previously demonstrated that a single bovine mitochondrial seryl-tRNA synthetase (mt SerRS) recognizes these topologically distinct isoacceptors having no common sequence or structure. Recombinant mt SerRS clearly footprinted at the TPsiC loop of each isoacceptor, and kinetic studies revealed that mt SerRS specifically recognized the TPsiC loop sequence in each isoacceptor. However, in the case of tRNA(UGA)(Ser), TPsiC loop-D loop interaction was further required for recognition, suggesting that mt SerRS recognizes the two substrates by distinct mechanisms. mt SerRS could slightly but significantly misacylate mitochondrial tRNA(Gln), which has the same TPsiC loop sequence as tRNA(UGA)(Ser), implying that the fidelity of mitochondrial translation is maintained by kinetic discrimination of tRNAs in the network of aminoacyl-tRNA synthetases.     

Comparative study of the reactability of phosphoric acid residues in tRNA(Ser) and tRNA(Leu) from Thermus thermophilus

The reactivity of phosphates in the Thermus thermophilus tRNA(Ser) (GCU) and tRNA(Leu) (CAG) was studied using the ethylnitrosourea modification. It was shown that phosphates of nucleotides 58-60 (T loop), 20-22 (D loop), and 48 (at the junction of the variable and T stems) were poorly modified in both tRNAs. The most pronounced differences in the reactivity were observed for phosphates at the junctions of the variable stem with T-stem (47q, 49) and anticodon stem (45). This indicates differences in orientations of the long variable arm relative to the backbone in the tRNAs studied.     

tRNA leucine identity and recognition sets

Transfer RNAs (tRNAs) are grouped into two classes based on the structure of their variable loop. In Escherichia coli, tRNAs from three isoaccepting groups are classified as type II. Leucine tRNAs comprise one such group. We used both in vivo and in vitro approaches to determine the nucleotides that are required for tRNA(Leu) function. In addition, to investigate the role of the tRNA fold, we compared the in vivo and in vitro characteristics of type I tRNA(Leu) variants with their type II counterparts.A minimum of six conserved tRNA(Leu) nucleotides were required to change the amino acid identity and recognition of a type II tRNA(Ser) amber suppressor from a serine to a leucine residue. Five of these nucleotides affect tRNA tertiary structure; the G15-C48 tertiary "Levitt base-pair" in tRNA(Ser) was changed to A15-U48; the number of nucleotides in the alpha and beta regions of the D-loop was changed to achieve the positioning of G18 and G19 that is found in all tRNA(Leu); a base was inserted at position 47n between the base-paired extra stem and the T-stem; in addition the G73 "discriminator" base of tRNA(Ser) was changed to A73. This minimally altered tRNA(Ser) exclusively inserted leucine residues and was an excellent in vitro substrate for LeuRS. In a parallel experiment, nucleotide substitutions were made in a glutamine-inserting type I tRNA (RNA(SerDelta); an amber suppressor in which the tRNA(Ser) type II extra-stem-loop is replaced by a consensus type I loop). This "type I" swap experiment was successful both in vivo and in vitro but required more nucleotide substitutions than did the type II swap. The type I and II swaps revealed differences in the contributions of the tRNA(Leu) acceptor stem base-pairs to tRNA(Leu) function: in the type I, but not the type II fold, leucine specificity was contingent on the presence of the tRNA(Leu) acceptor stem sequence. The type I and II tRNAs used in this study differed only in the sequence and structure of the variable loop. By altering this loop, and thereby possibly introducing subtle changes into the overall tRNA fold, it became possible to detect otherwise cryptic contributions of the acceptor stem sequence to recognition by LeuRS. Possible reasons for this effect are discussed.     

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.     

Mitochondrial RNA import in Leishmania tropica: aptamers homologous to multiple tRNA domains that interact cooperatively or antagonistically at the inner membrane

A large number of cytoplasmic tRNAs are imported into the kinetoplast-mitochondrion of Leishmania by a receptor-mediated process. To identify the sequences recognized by import receptors, mitochondria were incubated with a combinatorial RNA library. Repeated cycles of amplification of the imported sequences (SELEX) resulted in rapid selection of several import aptamers containing sequence motifs present in the anticodon arm, the D arm, the V-T region, and acceptor stem of known tRNAs, confirming or suggesting the presence of import signals in these domains. As predicted, truncated derivatives of tRNA(Ile)(UAU) containing the D arm or the V-T region were imported in vitro. Four aptamers were studied in detail. All were imported in vitro as well as in transiently transfected cells, using the same pathway as tRNA, but their individual import efficiencies were different. Two types of aptamers were discernible: the A arm and D arm homologues (type I), which were efficiently transferred across the inner mitochondrial membrane, and the V-T homologues (type II), which were not. Remarkably, subnanomolar concentrations of type I RNAs stimulated the entry of type II RNAs into the matrix, whereas type II RNAs inhibited inner membrane transfer of type I RNAs. Moreover, tRNA(Tyr)(GUA) and tRNA(Ile)(UAU) interacted with one another as type I and type II, respectively. Such cooperative and antagonistic interactions may allow the use of a limited number of receptors to recognize a large number of tRNAs of variable affinity and enable the maintenance of a properly balanced tRNA pool for mitochondrial translation.     

Dual-mode recognition of noncanonical tRNAs(Ser) by seryl-tRNA synthetase in mammalian mitochondria

The secondary structures of metazoan mitochondrial (mt) tRNAs(Ser) deviate markedly from the paradigm of the canonical cloverleaf structure; particularly, tRNA(Ser)(GCU) corresponding to the AGY codon (Y=U and C) is highly truncated and intrinsically missing the entire dihydrouridine arm. None of the mt serine isoacceptors possesses the elongated variable arm, which is the universal landmark for recognition by seryl-tRNA synthetase (SerRS). Here, we report the crystal structure of mammalian mt SerRS from Bos taurus in complex with seryl adenylate at an atomic resolution of 1.65 A. Coupling structural information with a tRNA-docking model and the mutagenesis studies, we have unraveled the key elements that establish tRNA binding specificity, differ from all other known bacterial and eukaryotic systems, are the characteristic extensions in both extremities, as well as a few basic residues residing in the amino-terminal helical arm of mt SerRS. Our data further uncover an unprecedented mechanism of a dual-mode recognition employed to discriminate two distinct 'bizarre' mt tRNAs(Ser) by alternative combination of interaction sites.     

Differences in the interaction of Escherichia coli RNase P RNA with tRNAs containing a short or a long extra arm.

The phosphorothioate footprinting technique was applied to the investigation of phosphate moieties in tRNA substrates involved in interactions with M1 RNA, the catalytic subunit of Escherichia coli RNase P. In general agreement with previous data, all affected sites were localized in acceptor stem and T arm. But the analyzed examples for class I (Saccharomyces cerevisiae pre-tRNA(Phe) with short variable arm) and class II tRNAs (E. coli pre-tRNA(Tyr) with large variable arm) revealed substantial differences. In the complex with pre-tRNA(Phe), protection was observed at U55, C56, and G57, along the top of the T loop in the tertiary structure, whereas in pre-tRNA(Tyr), the protected positions were G57, A58, and A59, at the bottom of the T loop. These differences suggest that the size of the variable arm affects the spatial arrangement of the T arm, providing a possible explanation for the discrepancy in reports about the D arm requirement in truncated tRNA substrates for eukaryotic RNase P enzymes. Enhanced reactivities were found near the junction of acceptor and T stem (U6, 7, 8 in pre-tRNA(Phe) and G7, U63, U64 in pre-tRNA(Tyr)). This indicates a partial unfolding of the tRNA structure upon complex formation with RNase P RNA.     

Nucleotide sequence of Neurospora crassa cytoplasmic initiator tRNA.

Initiator methionine tRNA from the cytoplasm of Neurospora crassa has been purified and sequenced. The sequence is: pAGCUGCAUm1GGCGCAGCGGAAGCGCM22GCY*GGGCUCAUt6AACCCGGAGm7GU (or D) - CACUCGAUCGm1AAACGAG*UUGCAGCUACCAOH. Similar to initiator tRNAs from the cytoplasm of other eukaryotes, this tRNA also contains the sequence -AUCG- instead of the usual -TphiCG (or A)- found in loop IV of other tRNAs. The sequence of the N. crassa cytoplasmic initiator tRNA is quite different from that of the corresponding mitochondrial initiator tRNA. Comparison of the sequence of N. crassa cytoplasmic initiator tRNA to those of yeast, wheat germ and vertebrate cytoplasmic initiator tRNA indicates that the sequences of the two fungal tRNAs are no more similar to each other than they are to those of other initiator tRNAs.     

28 The study of tRNA modifications by molecular dynamics

Modified nucleosides are prevalent in tRNA. Experimental studies reveal that modifications play an important role in tuning tRNA activity. In this study, molecular dynamics (MD) simulations were used to investigate how modifications alter tRNA structure and dynamics. The X-ray crystal structures of tRNA-Asp, tRNA-Phe, and tRNA-iMet, both with and without modifications, were used as initial structures for 333-ns time-scale MD trajectories with AMBER. For each tRNA molecule, three independent trajectory calculations were performed. Force field parameters were built using the RESP procedure of Cieplak et al. for 17 nonstandard tRNA residues. The global root-mean-square deviations (RMSDs) of atomic positions show that modifications only introduce significant rigidity to tRNA-Phe’s global structure. Interestingly, regional RMSDs of anticodon stem-loop suggest that modified tRNA has more rigid structure compared to the unmodified tRNA in this domain. The anticodon RMSDs of the modified tRNAs, however, are higher than those of corresponding unmodified tRNAs. These findings suggest that rigidity of the anticodon arm is essential for tRNA translocation in the ribosome complex, and, on the other hand, flexibility of anticodon might be critical for anticodon–codon recognition. We also measure the angle between the 3D L-shaped arms of tRNA; backbone atoms of acceptor stem and TψC stem loop are selected to indicate one vector, and backbone atoms of anticodon stem and D stem loop are selected to indicate the other vector. By measuring the angle between two vectors, we find that the initiator tRNA has a narrower range of hinge motion compared to tRNA-Asp and tRNA-Phe, which are elongator tRNA. This suggests that elongator tRNAs, which might require significant flexibility in this hinge to transition from the A–to-P site in the ribosome, have evolved to specifically accommodate this need.     

The translational efficiency of tRNA is a property of the anticodon arm

We have reciprocally transplanted the anticodon arm sequences of a set of amber suppressor tRNA genes, using recombinant DNA techniques. By this means, a very efficient suppressor may be converted to a poor one, and the poorest tRNA to the efficiency of the best one. In tRNA molecules of normal 2 degrees and 3 degrees structure, the suppressor efficiencies of different composite tRNAs having the same anticodon arm sequence are approximately the same. Large numbers of simultaneous changes throughout the rest of the molecule do not affect the efficiency. Selective nucleotide modification as a result of varied anticodon arm sequences cannot explain these efficiencies. Efficiencies are also unlikely to differ because of selective aminoacylation. Measurement of in vivo tRNA shows, however, that tRNA levels do vary if the anticodon arm sequence is changed. If tRNA levels are normalized, the anticodon arm effect on the translational efficiency remains. Therefore, different anticodon arms, all of normal secondary structure, are not equivalent in translation. The most efficient sequences in this series resemble those found in natural tRNAs associated with similar anticodons, as is proposed in the extended anticodon theory (Yarus, M. (1982) Science 218, 646-652). These molecules also provide some information on the specificity of nucleotide modification enzymes and on determinants of the steady-state tRNA level.     

Identification of yeast tRNA Um(44) 2'-O-methyltransferase (Trm44) and demonstration of a Trm44 role in sustaining levels of specific tRNA(Ser) species

A characteristic feature of tRNAs is the numerous modifications found throughout their sequences, which are highly conserved and often have important roles. Um(44) is highly conserved among eukaryotic cytoplasmic tRNAs with a long variable loop and unique to tRNA(Ser) in yeast. We show here that the yeast ORF YPL030w (now named TRM44) encodes tRNA(Ser) Um(44) 2'-O-methyltransferase. Trm44 was identified by screening a yeast genomic library of affinity purified proteins for activity and verified by showing that a trm44-delta strain lacks 2'-O-methyltransferase activity and has undetectable levels of Um(44) in its tRNA(Ser) and by showing that Trm44 purified from Escherichia coli 2'-O-methylates U(44) of tRNA(Ser) in vitro. Trm44 is conserved among metazoans and fungi, consistent with the conservation of Um(44) in eukaryotic tRNAs, but surprisingly, Trm44 is not found in plants. Although trm44-delta mutants have no detectable growth defect, TRM44 is required for survival at 33 degrees C in a tan1-delta mutant strain, which lacks ac(4)C12 in tRNA(Ser) and tRNA(Leu). At nonpermissive temperature, a trm44-delta tan1-delta mutant strain has reduced levels of tRNA(Ser(CGA)) and tRNA(Ser(UGA)), but not other tRNA(Ser) or tRNA(Leu) species. The trm44-delta tan1-delta growth defect is suppressed by addition of multiple copies of tRNA(Ser(CGA)) and tRNA(Ser(UGA)), directly implicating these tRNA(Ser) species in this phenotype. The reduction of specific tRNA(Ser) species in a trm44-delta tan1-delta mutant underscores the importance of tRNA modifications in sustaining tRNA levels and further emphasizes that tRNAs undergo quality control.