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.     

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.     

Mapping contacts between Escherichia coli alanyl tRNA synthetase and 2' hydroxyls using a complete tRNA molecule

A dual-specific derivative of yeast tRNA(Phe) is described whose features facilitate structure-function studies of tRNAs. This tRNA has been made in three different bimolecular forms that allow modifications to be easily introduced into any position within the molecule. A set of deoxynucleotide substituted versions of this tRNA has been created and used to examine contacts between tRNA and Escherichia coli alanyl-tRNA synthetase, an enzyme previously shown to interact with 2'-hydroxyls in the acceptor stem of the tRNA. Because the present experiments used a full-length tRNA, several contacts were identified that had not been previously found using microhelix substrates. Contacts at similar sites in the T-loop are seen in the cocrystal structure of tRNA(Ser) and Thermus thermophilus seryl-tRNA synthetase.     

Variations in mitochondrial tRNA gene organization of reptiles as phylogenetic markers

Amplification and sequencing of mitochondrial DNA regions corresponding to three major clusters of transfer RNA genes from a variety of species representing major groups of birds and reptiles revealed some new variations in tRNA gene organization. First, a gene rearrangement from tRNA(His)-tRNA(Ser)(AGY)-tRNA(Leu)(CUN) to tRNA(Ser)(AGY)- tRNA(His)tRNA(Leu)(CUN) occurs in all three crocodilians examined (alligator, caiman, and crocodile). In addition an exceptionally long spacer region between the genes for NADH dehydrogenase subunit 4 and tRNA(Ser)(AGY) is found in caiman. Second, in congruence with a recent finding by Seutin et al., a characteristic stem-and-loop structure for the putative light-strand replication origin located between tRNA(Asn) and tRNA(Cys) genes is absent for all the birds and crocodilians. This stem-and-loop structure is absent in an additional species, the Texas blind snake, whereas the stem-and-loop structure is present in other snakes, lizards, turtles, mammals, and a frog. The disappearance of the stem-and-loop structure in the blind snake most likely occurred independently of that on the lineage leading to birds and crocodilians. Finally, the blind snake has a novel type of tRNA gene arrangement in which the tRNA(Gln) gene moved from one tRNA cluster to another. Sequence substitution rates for the tRNA genes appeared to be somewhat higher in crocodialians than in birds and mammals. As regards the controversial phylogenetic relationship among the Aves, Crocodilia, and Mammalia, a sister group relationship of birds and crocodilians relative to mammals, as suggested from the common loss of the stem-and- loop structure, was supported with statistical significance by molecular phylogenetic analyses using the tRNA gene sequence data.      

The long extra arms of human tRNA((Ser)Sec) and tRNA(Ser) function as major identify elements for serylation in an orientation-dependent, but not sequence-specific manner.

Selenocysteine tRNA [tRNA((Ser)Sec)] is charged with serine by the same seryl-tRNA synthetase (SerRS) as the canonical serine tRNAs. Using site-directed mutagenesis, we have introduced a series of mutations into human tRNA((Ser)Sec) and tRNA(Ser) in order to study the identity elements of tRNA((Ser)Sec) for serylation and the effect of the orientation of the extra arm. Our results show that the long extra arm is one of the major identity elements for both tRNA(Ser) and tRNA((Ser)Sec) and gel retardation assays reveal that it appears to be a prerequisite for binding to the cognate synthetase. The long extra arm functions in an orientation-dependent, but not in a sequence-specific manner. The discriminator base G73 is another important identity element of tRNA((Ser)Sec), whereas the T- and D-arms play a minor role for the serylation efficiency.     

Superposition of a tRNASer acceptor stem microhelix into the seryl-tRNA synthetase complex

Aminoacyl-tRNA synthetases catalyze the formation of aminoacyl-tRNAs. Seryl-tRNA synthetase is a class II synthetase, which depends on rather few and simple identity elements in tRNA(Ser) to determine the amino acid specificity. tRNA(Ser) acceptor stem microhelices can be aminoacylated with serine, which makes this part of the tRNA a valuable tool for investigating the structural motifs in a tRNA(Ser)-seryl-tRNA synthetase complex. A 1.8A-resolution tRNA(Ser) acceptor stem crystal structure was superimposed to a 2.9A-resolution crystal structure of a tRNA(Ser)-seryl-tRNA synthetase complex for a visualization of the binding environment of the tRNA(Ser) microhelix.     

The length of the aminoacyl-acceptor stem of the selenocysteine-specific tRNA(Sec) of Escherichia coli is the determinant for binding to elongation factors SELB or Tu

Mutations in selC, which reduce the 8-base pair aminoacyl-acceptor helix to the canonical 7-base pair length (tRNA(Sec)(delAc] or which replace the extra arm of tRNA(Sec) by that of a serine acceptor tRNA species (tRNA(Sec)(ExS), block the function in selenoprotein synthesis in vivo (Baron, C., Heider, J., and B?ck, A. (1990) Nucleic Acids Res. 18, 6761-6766). tRNA(Sec), tRNA(Sec)(delAc), and tRNA(Sec)(ExS) were purified and analyzed for their interaction with purified seryl-tRNA synthetase, selenocysteine synthase and translation factors SELB and EF-Tu. It was found that seryl-tRNA synthetase displays 10-fold impaired Km and Kcat values for tRNA(Sec) in comparison to tRNA(Ser), decreasing the overall charging efficiency (Kcat/Km) of tRNA(Sec) to 1% of that characteristic for tRNA(Ser). tRNA(Sec)(ExS) was a less efficient substrate for the enzyme (Kcat/Km 0.2% of the tRNA(Ser) value) whereas the tRNA(Ser)(delAc) variant was charged with an approximately 2-3-fold improved rate compared to wild-type tRNA(Sec). Both mutant tRNA variants, when charged with L-serine, were able to interact with selenocysteine synthase to give rise to selenocysteyl-tRNA with tRNA(Sec)(ExS) being as efficient as wild-type tRNA(Sec). Seryl-tRNA(Sec)(delAc), on the other hand, was selenylated very slowly. Reduction of the length of the aminoacyl-acceptor stem to 7 base pairs prevented the interaction with translation factor SELB but allowed binding to EF-Tu, irrespective of whether tRNA(Sec)(delAc) was charged with serine or selenocysteine. The aminoacyl-acceptor helix of tRNA(Sec), therefore, is a major determinant directing binding to SELB and precluding interaction with EF-Tu.     

Adaptation of aminoacylation identity rules to mammalian mitochondria

Many mammalian mitochondrial aminoacyl-tRNA synthetases are of bacterial-type and share structural domains with homologous bacterial enzymes of the same specificity. Despite this high similarity, synthetases from bacteria are known for their inability to aminoacylate mitochondrial tRNAs, while mitochondrial enzymes do aminoacylate bacterial tRNAs. Here, the reasons for non-aminoacylation by a bacterial enzyme of a mitochondrial tRNA have been explored. A mutagenic analysis performed on in vitro transcribed human mitochondrial tRNA^Asp variants tested for their ability to become aspartylated by Escherichia coli aspartyl-tRNA synthetase, reveals that full conversion cannot be achieved on the basis of the currently established tRNA/synthetase recognition rules. Integration of the full set of aspartylation identity elements and stabilization of the structural tRNA scaffold by restoration of D- and T-loop interactions, enable only a partial gain in aspartylation efficiency. The sequence context and high structural instability of the mitochondrial tRNA are additional features hindering optimal adaptation of the tRNA to the bacterial enzyme. Our data support the hypothesis that non-aminoacylation of mitochondrial tRNAs by bacterial synthetases is linked to the large sequence and structural relaxation of the organelle encoded tRNAs, itself a consequence of the high rate of mitochondrial genome divergence.     

A model for the tertiary structure of mammalian mitochondrial transfer RNAs lacking the entire 'dihydrouridine' loop and stem

The mammalian mitochondrial tRNA(AGY)Ser is unique in lacking the entire dihydrouridine arm. This reduces its secondary structure to a 'truncated cloverleaf'. Experimental evidence on the tertiary structure has been obtained by chemically probing the conformation of both the bovine and human species in their native conformation and at various stages of denaturation. A structural model of the bovine tRNA is presented based on the results of this chemical probing, on a comparison between nine homologous 'truncated cloverleaf' secondary structures and on analogies with the crystal structure of yeast phenylalanine tRNA. The proposed structure is very similar in shape to that of yeast tRNA(Phe) but is slightly smaller in size. It is defined by a unique set of tertiary interactions. Structural considerations suggest that other mammalian mitochondrial tRNAs have smaller dimensions as well.     

A study of the interaction between tRNASer and seryl-tRNA synthetase from bovine liver

Study by chemical modification of Ser, Arg, His residues and sulfhydryl groups on bovine seryl-tRNA synthetase showed that Ser residues appeared to be unnecessary for the recognition mechanism, but Arg and His residues were essential. It was considered that different sulfhydryl groups related with each recognition of tRNA and ATP. Poly-arginine inhibited the interaction between serine tRNA and SerRS. The CD spectra of a mixture of serine tRNA and poly-arginine indicated that higher-order structure of tRNA changed. Furthermore, the Km and Vmax values of bovine serine isoacceptor, yeast serine tRNA and E. coli serine tRNA for bovine SerRS examined and it was discussed the differences of those base sequences.     

Nucleotide sequences of three tRNA(Ser) from Drosophila melanogaster reading the six serine codons

The nucleotide sequences of three serine tRNAs from Drosophila melanogaster, together capable of decoding the six serine codons, were determined. tRNA(Ser)2b has the anticodon GCU, tRNA(Ser)4 has CGA and tRNA(Ser)7 has IGA. tRNA(Ser)2b differs from the last two by about 25%. However, tRNA(Ser)4 and tRNA(Ser)7 are 96% homologous, differing only at the first position of the anticodon and two other sites. This unusual sequence relationship suggests, together with similar pairs in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, that eukaryotic tRNA(Ser)UCN may be undergoing concerted evolution.