Mitochondrial DNA mutation m.5512A > G in the acceptor-stem of mitochondrial tRNATrp causing maternally inherited essential hypertension.

Essential hypertension (EH) is a common complex disorder with high heritability. Maternal inherited pattern was observed in some families with EH, which was known as maternally inherited essential hypertension (MIEH). Mitochondrial DNA (mtDNA) mutations were identified to account for some MIEH in previous studies. In the present study, we characterized clinical manifestations and the complete mitochondrial genome of a Chinese family with MIEH. Through analyzing the whole mtDNA genome of the proband, we identified a mutation m.5512A > G in the MT-TW gene that changed a highly conserved nucleotide and could potentially affect the function of tRNATrp. Furthermore, significantly exercise intolerance, left ventricular remodeling and increased arterial stiffness were observed in carriers with mutation m.5512A > G, which further supported the potentially pathogenic effect of m.5512A > G in MIEH.

A role for [Fe4S4] clusters in tRNA recognition--a theoretical study.

Over the past several years, structural studies have led to the unexpected discovery of iron-sulfur clusters in enzymes that are involved in DNA replication/repair and protein biosynthesis. Although these clusters are generally well-studied cofactors, their significance in the new contexts often remains elusive. One fascinating example is a tryptophanyl-tRNA synthetase from the thermophilic bacterium Thermotoga maritima, TmTrpRS, that has recently been structurally characterized. It represents an unprecedented connection among a primordial iron-sulfur cofactor, RNA and protein biosynthesis. Here, a possible role of the [Fe4S4] cluster in tRNA anticodon-loop recognition is investigated by means of density functional theory and comparison with the structure of a human tryptophanyl-tRNA synthetase/tRNA complex. It turns out that a cluster-coordinating cysteine residue, R224, and polar main chain atoms form a characteristic structural motif for recognizing a putative 5' cytosine or 5' 2-thiocytosine moiety in the anticodon loop of the tRNA molecule. This motif provides not only affinity but also specificity by creating a structural and energetical penalty for the binding of other bases, such as uracil.

Functional elucidation of a key contact between tRNA and the large ribosomal subunit rRNA during decoding.

The selection of cognate tRNAs during translation is specified by a kinetic discrimination mechanism driven by distinct structural states of the ribosome. While the biochemical steps that drive the tRNA selection process have been carefully documented, it remains unclear how recognition of matched codon:anticodon helices in the small subunit facilitate global rearrangements in the ribosome complex that efficiently promote tRNA decoding. Here we use an in vitro selection approach to isolate tRNA(Trp) miscoding variants that exhibit a globally perturbed tRNA tertiary structure. Interestingly, the most substantial distortions are positioned in the elbow region of the tRNA that closely approaches helix 69 (H69) of the large ribosomal subunit. The importance of these specific interactions to tRNA selection is underscored by our kinetic analysis of both tRNA and rRNA variants that perturb the integrity of this interaction.

Rational design of an orthogonal tryptophanyl nonsense suppressor tRNA.

While a number of aminoacyl tRNA synthetase (aaRS):tRNA pairs have been engineered to alter or expand the genetic code, only the Methanococcus jannaschii tyrosyl tRNA synthetase and tRNA have been used extensively in bacteria, limiting the types and numbers of unnatural amino acids that can be utilized at any one time to expand the genetic code. In order to expand the number and type of aaRS/tRNA pairs available for engineering bacterial genetic codes, we have developed an orthogonal tryptophanyl tRNA synthetase and tRNA pair, derived from Saccharomyces cerevisiae. In the process of developing an amber suppressor tRNA, we discovered that the Escherichia coli lysyl tRNA synthetase was responsible for misacylating the initial amber suppressor version of the yeast tryptophanyl tRNA. It was discovered that modification of the G:C content of the anticodon stem and therefore reducing the structural flexibility of this stem eliminated misacylation by the E. coli lysyl tRNA synthetase, and led to the development of a functional, orthogonal suppressor pair that should prove useful for the incorporation of bulky, unnatural amino acids into the genetic code. Our results provide insight into the role of tRNA flexibility in molecular recognition and the engineering and evolution of tRNA specificity.

An exposed cysteine residue of human angiostatic mini tryptophanyl-tRNA synthetase.

Human tryptophanyl-tRNA synthetase (TrpRS) catalyzes the aminoacylation of tRNA(Trp). Human TrpRS exists in two forms: a major form that is the full-length protein and a truncated form (mini TrpRS) in which most of the N-terminal extension is absent. Human mini, but not full-length, TrpRS has angiostatic activity. Because the full-length protein, which lacks angiostatic activity, has all of the amino acid determinants of the mini form, which has activity, I searched for conformational differences between the two proteins. Using a disulfide cross-linking assay, I showed that the molecular environment around Cys62 is significantly different between the two proteins. This difference can be explained by inspection of the three-dimensional structure of the full-length protein. These results give a clear demonstration of a significant difference, around a specific residue (Cys62), between a potent angiostatic and nonangiostatic version of human TrpRS.

A functionally dominant mitochondrial DNA mutation.

Mutations in mitochondrial DNA (mtDNA) tRNA genes can be considered functionally recessive because they result in a clinical or biochemical phenotype only when the percentage of mutant molecules exceeds a critical threshold value, in the range of 70-90%. We report a novel mtDNA mutation that contradicts this rule, since it caused a severe multisystem disorder and respiratory chain (RC) deficiency even at low levels of heteroplasmy. We studied a 13-year-old boy with clinical, radiological and biochemical evidence of a mitochondrial disorder. We detected a novel heteroplasmic C>T mutation at nucleotide 5545 of mtDNA, which was present at unusually low levels (<25%) in affected tissues. The pathogenic threshold for the mutation in cybrids was between 4 and 8%, implying a dominant mechanism of action. The mutation affects the central base of the anticodon triplet of tRNA(Trp) and it may alter the codon specificity of the affected tRNA. These findings introduce the concept of dominance in mitochondrial genetics and pose new diagnostic challenges, because such mutations may easily escape detection. Moreover, similar mutations arising stochastically and accumulating in a minority of mtDNA molecules during the aging process may severely impair RC function in cells.

Probing protein folding using site-specifically encoded unnatural amino acids as FRET donors with tryptophan.

The experimental study of protein folding is enhanced by the use of nonintrusive probes that are sensitive to local conformational changes in the protein structure. Here, we report the selection of an aminoacyl-tRNA synthetase/tRNA pair for the cotranslational, site-specific incorporation of two unnatural amino acids that can function as fluorescence resonance energy transfer (FRET) donors with Trp to probe the disruption of the hydrophobic core upon protein unfolding. l-4-Cyanophenylalanine (pCNPhe) and 4-ethynylphenylalanine (pENPhe) were incorporated into the hydrophobic core of the 171-residue protein, T4 lysozyme. The FRET donor ability of pCNPhe and pENPhe is evident by the overlap of the emission spectra of pCNPhe and pENPhe with the absorbance spectrum of Trp. The incorporation of both unnatural amino acids in place of a phenylalanine in the hydrophobic core of T4 lysozyme was well tolerated by the protein, due in part to the small size of the cyano and ethynyl groups. The hydrophobic nature of the ethynyl group of pENPhe suggests that this unnatural amino acid is a more conservative substitution into the hydrophobic core of the protein compared to pCNPhe. The urea-induced disruption of the hydrophobic core of the protein was probed by the change in FRET efficiency between either pCNPhe or pENPhe and the Trp residues in T4 lysozyme. The methodology for the study of protein conformational changes using FRET presented here is of general applicability to the study of protein structural changes, since the incorporation of the unnatural amino acids is not inherently limited by the size of the protein.

Two new mutations in the MT-TW gene leading to the disruption of the secondary structure of the tRNA(Trp) in patients with Leigh syndrome.

Leigh syndrome is a progressive neurodegenerative disorder occurring in infancy and childhood characterized in most cases by a psychomotor retardation, optic atrophy, ataxia, dystonia, failure to thrive, seizures and respiratory failure. In this study, we performed a systematic sequence analysis of mitochondrial genes associated with LS in Tunisian patients. We sequenced the encoded complex I units: ND2, ND3, ND4, ND5 and ND6 genes and the mitochondrial ATPase 6, tRNA(Val), tRNA(Leu(UUR)), tRNA(Trp) and tRNA(Lys) genes in 10 unrelated patients with Leigh syndrome. We revealed the presence of 34 reported polymorphisms, nine novel nucleotide variants and two new mutations (T5523G and A5559G) in the tested patients. These two mutations were localized in two conserved regions of the tRNA(Trp) and affect, respectively, the D-stem and the T-stem of the mitochondrial tRNA leading to a disruption of the secondary structure of this tRNA. SSP-PCR analysis showed that the T5523G and A5559G mutations were present with respective heteroplasmic rates of 66% and 43 %. We report here the first mutational screening of mitochondrial mutations in Tunisian patients with Leigh syndrome which described two novel mutations associated with this disorder.

Box C/D RNA-guided 2'-O methylations and the intron of tRNATrp are not essential for the viability of Haloferax volcanii.

Deleting the box C/D RNA-containing intron in the Haloferax volcanii tRNATrp gene abolishes RNA-guided 2'-O methylations of C34 and U39 residues of tRNATrp. However, this deletion does not affect growth under standard conditions.

Cladogenesis and phylogeography of the lizard Phrynocephalus vlangalii (Agamidae) on the Tibetan plateau.

Phrynocephalus vlangalii is restricted to dry sand or Gobi desert highlands between major mountain ranges in the Qinghai (Tibetan) Plateau. Mitochondrial DNA (mtDNA) sequence (partial ND2, tRNA(Trp) and partial tRNA(Ala)) was obtained from 293 Phrynocephalus sampled from 34 sites across the plateau. Partitioned Bayesian and maximum parsimony phylogenetic analyses revealed that P. vlangalii and two other proposed species (P. erythrus and P. putjatia) together form a monophyletic mtDNA clade which, in contrast with previous studies, does not include P. theobaldi and P. zetangensis. The main P. vlangalli clade comprises seven well-supported lineages that correspond to distinct geographical areas with little or no overlap, and share a most recent common ancestor at 5.06 +/- 0.68 million years ago (mya). This is much older than intraspecific lineages in other Tibetan animal groups. Analyses of molecular variance indicated that most of the observed genetic variation occurred among populations/regions implying long-term interruption of maternal gene flow. A combined approach based on tests of population expansion, estimation of node dates, and significance tests on clade areas indicated that phylogeographical structuring has been primarily shaped by three main periods of plateau uplift during the Pliocene and Pleistocene, specifically 3.4 mya, 2.5 mya and 1.7 mya. These periods corresponded to the appearance of several mountain ranges that formed physical barriers between lineages. Populations from the Qaidam Basin are shown to have undergone major demographic and range expansions in the early Pleistocene, consistent with colonization of areas previously covered by the huge Qaidam palaeolake, which desiccated at this time. The study represents one of the most detailed phylogeographical analyses of the Qinghai Plateau to date and shows how geological events have shaped current patterns of diversity.

Postglacial range expansion from northern refugia by the wood frog, Rana sylvatica.

Although the range dynamics of North American amphibians during the last glacial cycle are increasingly better understood, the recolonization history of the most northern regions and the impact of southern refugia on patterns of intraspecific genetic diversity and phenotypic variation in these regions are not well reconstructed. Here we present the phylogeographic history of a widespread and primarily northern frog, Rana sylvatica. We surveyed 551 individuals from 116 localities across the species' range for a 650-bp region of the NADH dehydrogenase subunit 2 and tRNA(TRP) mitochondrial genes. Our phylogenetic analyses revealed two distinct clades corresponding to eastern and western populations, as well as a Maritime subclade within the eastern lineage. Patterns of genetic diversity support multiple refugia. However, high-latitude refugia in the Appalachian highlands and modern-day Wisconsin appear to have had the biggest impact on northern populations. Clustering analyses based on morphology further support a distinction between eastern and western wood frogs and suggest that postglacial migration has played an important role in generating broad-scale patterns of phenotypic variation in this species.

Structure of human tryptophanyl-tRNA synthetase in complex with tRNATrp reveals the molecular basis of tRNA recognition and specificity.

Aminoacyl-tRNA synthetases (aaRSs) are a family of enzymes responsible for the covalent link of amino acids to their cognate tRNAs. The selectivity and species-specificity in the recognitions of both amino acid and tRNA by aaRSs play a vital role in maintaining the fidelity of protein synthesis. We report here the first crystal structure of human tryptophanyl-tRNA synthetase (hTrpRS) in complex with tRNA(Trp) and Trp which, together with biochemical data, reveals the molecular basis of a novel tRNA binding and recognition mechanism. hTrpRS recognizes the tRNA acceptor arm from the major groove; however, the 3' end CCA of the tRNA makes a sharp turn to bind at the active site with a deformed conformation. The discriminator base A73 is specifically recognized by an alpha-helix of the unique N-terminal domain and the anticodon loop by an alpha-helix insertion of the C-terminal domain. The N-terminal domain appears to be involved in Trp activation, but not essential for tRNA binding and acylation. Structural and sequence comparisons suggest that this novel tRNA binding and recognition mechanism is very likely shared by other archaeal and eukaryotic TrpRSs, but not by bacterial TrpRSs. Our findings provide insights into the molecular basis of tRNA specificity and species-specificity.

An evolutionary 'intermediate state' of mitochondrial translation systems found in Trichinella species of parasitic nematodes: co-evolution of tRNA and EF-Tu.

EF-Tu delivers aminoacyl-tRNAs to ribosomes in the translation system. However, unusual truncations found in some animal mitochondrial tRNAs seem to prevent recognition by a canonical EF-Tu. We showed previously that the chromadorean nematode has two distinct EF-Tus, one of which (EF-Tu1) binds only to T-armless aminoacyl-tRNAs and the other (EF-Tu2) binds to D-armless Ser-tRNAs. Neither of the EF-Tus can bind to canonical cloverleaf tRNAs. In this study, by analyzing the translation system of enoplean nematode Trichinella species, we address how EF-Tus and tRNAs have evolved from the canonical structures toward those of the chromadorean translation system. Trichinella mitochondria possess three types of tRNAs: cloverleaf tRNAs, which do not exist in chromadorean nematode mitochondria; T-armless tRNAs; and D-armless tRNAs. We found two mitochondrial EF-Tu species, EF-Tu1 and EF-Tu2, in Trichinella britovi. T.britovi EF-Tu2 could bind to only D-armless Ser-tRNA, as Caenorhabditis elegans EF-Tu2 does. In contrast to the case of C.elegans EF-Tu1, however, T.britovi EF-Tu1 bound to all three types of tRNA present in Trichinella mitochondria. These results suggest that Trichinella mitochondrial translation system, and particularly the tRNA-binding specificity of EF-Tu1, could be an intermediate state between the canonical system and the chromadorean nematode mitochondrial system.

Characterization of an unusual tRNA-like sequence found inserted in a Neurospora retroplasmid.

We characterized an unusual tRNA-like sequence that had been found inserted in suppressive variants of the mitochondrial retroplasmid of Neurospora intermedia strain Varkud. We previously identified two forms of the tRNA-like sequence, one of 64 nt (TRL-64)and the other of 78 nt (TRL-78) containing a 14-nt internal insertion in the anticodon stem at a position expected for a nuclear tRNA intron. Here, we show that TRL-78 is encoded in Varkud mitochondrial (mt)DNA within a 7 kb sequence that is not present in Neurospora crassa wild-type 74A mtDNA. This 7-kb insertion also contains a perfectly duplicated tRNA(Trp)gene, segments of several mitochondrial plasmids and numerous GC-rich pallindromic sequences that are repeated elsewhere in the mtDNA. The mtDNA-encoded copy of TRL-78 is transcribed and apparently undergoes 5'- and 3'-end processing and 3' nucleotide addition by tRNA nucleotidyl transferase to yield a discrete tRNA-sized molecule. However, the 14 nt intron-like sequence in TRL-78, which is missing in the TRL-64 form, is not spliced detectably in vivo or in vitro. Our results show that TRL-78 is an unusual tRNA-like species that could be incorporated into suppressive retroplasmids by the same reverse transcription mechanism used to incorporate mt tRNAs. The tRNA-like sequence may have been derived from an intron-containing nuclear tRNA gene or it may serve some function, like tmRNA. Our results suggest that mtRNAs or tRNA-like species may be integrated into mtDNA via reverse transcription, analogous to SINE elements in animal cells.

Characterization of an unusual tRNA-like sequence found inserted in a Neurospora retroplasmid.

We characterized an unusual tRNA-like sequence that had been found inserted in suppressive variants of the mitochondrial retroplasmid of Neurospora intermedia strain Varkud. We previously identified two forms of the tRNA-like sequence, one of 64 nt (TRL-64) and the other of 78 nt (TRL-78) containing a 14-nt internal insertion in the anticodon stem at a position expected for a nuclear tRNA intron. Here, we show that TRL-78 is encoded in Varkud mitochondrial (mt)DNA within a 7 kb sequence that is not present in Neurospora crassa wild-type 74 A mtDNA. This 7-kb insertion also contains a perfectly duplicated tRNA(Trp)gene, segments of several mitochondrial plasmids and numerous GC-rich palindromic sequences that are repeated elsewhere in the mtDNA. The mtDNA-encoded copy of TRL-78 is transcribed and apparently undergoes 5'- and 3'-end processing and 3' nucleotide addition by tRNA nucleotidyl transferase to yield a discrete tRNA-sized molecule. However, the 14 nt intron-like sequence in TRL-78, which is missing in the TRL-64 form, is not spliced detectably in vivo or in vitro. Our results show that TRL-78 is an unusual tRNA-like species that could be incorporated into suppressive retroplasmids by the same reverse transcription mechanism used to incorporate mt tRNAs. The tRNA-like sequence may have been derived from an intron-containing nuclear tRNA gene or it may serve some function, like mtRNA. Our results suggest that mt tRNAs or tRNA-like species may be integrated into mtDNA via reverse transcription, analogous to SINE elements in animal cells.

Transfer RNA identity contributes to transition state stabilization during aminoacyl-tRNA synthesis.

Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs ensure both accurate RNA recognition and the efficient catalysis of aminoacylation. The effects of tRNA(Trp)variants on the aminoacylation reaction catalyzed by wild-type Escherichia coli tryptophanyl-tRNA synthe-tase (TrpRS) have now been investigated by stopped-flow fluorimetry, which allowed a pre-steady-state analysis to be undertaken. This showed that tRNA(Trp)identity has some effect on the ability of tRNA to bind the reaction intermediate TrpRS-tryptophanyl-adenylate, but predominantly affects the rate at which trypto-phan is transferred from TrpRS-tryptophanyl adenylate to tRNA. Use of the binding ( K (tRNA)) and rate constants ( k (4)) to determine the energetic levels of the various species in the aminoacylation reaction showed a difference of approximately 2 kcal mol(-1)in the barrier to transition state formation compared to wild-type for both tRNA(Trp)A-->C73 and. These results directly show that tRNA identity contributes to the degree of complementarity to the transition state for tRNA charging in the active site of an aminoacyl-tRNA synthetase:aminoacyl-adenylate:tRNA complex.

tRNA structure and ribosomal function. I. tRNA nucleotide 27-43 mutations enhance first position wobble.

Transfer RNA su7 G36 is a derivative of tRNA(Trp) with a 3'GUC anticodon complementary to the glutamine codon CAG. This tRNA requires a normally forbidden G-U wobble at the first codon position to suppress a UAG (amber) termination codon. Measurement of amber suppression by mutated su7 G36 tRNAs and correction for tRNA levels and aminoacylation allowed calculation of KUAG, a linearized index of in vivo ribosomal function. Following saturating mutagenesis of the anticodon arm of su7 G36, screening for UAG suppression using a lacZ reporter yielded tRNAs with up to 40-fold increased first position G-U wobble, judged from KUAG. The parental anticodon helix has minimized this type of miscoding, and virtually all changes in the top base-pair of the anticodon helix, nucleotides (nt) 27-43, increased the error. Thus, misincorporation of amino acids due to aberrant first position wobble is apparently prevented by normal tRNA structure, which is specifically altered by substitution at nt 27-43, the top base-pair of the anticodon helix. All 16 permutations of nt 27-43, the hotspot for increased wobble, were subsequently constructed and compared. Comparison of values for tRNA coding function, tRNA level, and aminoacylation for the 16 suggest that a tRNA conformational change, specifically involving both nt 27-43, differentially affects all these tRNA functions. This conformational alteration, which presumably occurs normally on the ribosome, appears more complex than simple breakage of the normal 27-43 base-pair. We suggest that the change is in the angle and/or flexibility of the tRNA L-shape. Among these 16 tRNAs, efficient wobble is strongly and inversely correlated with good aminoacylation and high tRNA levels; this quality may have been selected. Constraints on the sequences of natural tRNAs suggest that nt 27-43 have effects on function in many tRNAs.

The functional analysis of nonsense suppressors derived from in vitro engineered Saccharomyces cerevisiae tRNA(Trp) genes.

Nonsense suppressors derived from Saccharomyces cerevisiae tRNA(Trp) genes have not been identified by classical genetic screens, although one can construct efficient amber (am) suppressors from them by making the appropriate anticodon mutation in vitro. Herein, a series of in vitro constructed putative suppressor genes was produced to test if pre-tRNA(Trp) processing difficulties could help to explain the lack of classical tRNA(Trp)-based suppressors. It is clear that inefficient processing of introns from precursor tRNA(Trp), or inaccurate overall processing, may explain why some of these constructs fail to promote nonsense suppression in vivo. However, deficient processing must be only one of the reasons why classical tRNA(Trp)-based suppressors have not been characterized, as suppression may still be extremely weak or absent in instances where the in vitro construct can lead to an accumulation of mature tRNA(Trp). Furthermore, suppression is also very weak in strains transformed with an intronless derivative of a putative tRNA(Trp) ochre (oc) suppressor gene, wherein intron removal cannot pose a problem.

Random-splitting of tRNA transcripts as an approach for studying tRNA-protein interactions.

Location of phosphodiester bonds essential for aminoacylation of bovine tRNA(Trp) was identified using a randomly cleaved transcript synthesized in vitro. It was found that cleavage of phosphodiester bonds after nucleotides in positions 21, 22, 36-38, 57-59, 62 and 64 were critical for aminoacylation capacity of tRNA(Trp)-transcript. These cleavage sites were located in the regions of tRNA molecule protected by the cognate synthetase against chemical modification and in the regions presumably outside the contact area as well. These results indicate that for maintenance of aminoacylation ability the intactness of the certain regions of the tRNA backbone structure is necessary. Random splitting of non-modified RNA with alkali followed by separation of active and inactive molecules and identification of cleavage sites developed in this work may become a general approach for studying the role of RNA covalent structure in its interaction with proteins.

Nucleotides U28-A42 and A37 in unmodified yeast tRNA(Trp) as negative identity elements for bovine tryptophanyl-tRNA synthetase.

Wild-type bovine and yeast tRNA(Trp) are efficiently aminoacylated by tryptophanyl-tRNA synthetase both from beef and from yeast. Upon loss of modified bases in the synthetic transcripts, mammalian tRNA(Trp) retains the double recognition by the two synthetases, while yeast tRNA(Trp) loses its substrate properties for the bovine enzyme and is recognised only by the cognate synthetase. By testing chimeric bovine-yeast transcripts with tryptophanyl-tRNA synthetase purified from beef pancreas, the nucleotides responsible for the loss of charging of the synthetic yeast transcript have been localised in the anticodon arm. A complete loss of charging akin to that observed with the yeast transcript requires substitution in the bovine backbone of G37 in the anticodon loop with yeast A37 and of C28-G42 in the anticodon stem with yeast U28-A42. Since A37 does not prevent aminoacylation of the wild-type yeast tRNA(Trp) by the beef enzyme, a negative combination apparently emerges in the synthetic transcript after unmasking of U28 by loss of pseudourydilation.