Mammalian trans-editing factor ProX is able to deacylate tRNAThr mischarged with alanine.

The precise coupling of tRNAs with their cognate amino acids, known as tRNA aminoacylation, is a stringently regulated process that governs translation fidelity. To ensure fidelity, organisms deploy multiple layers of editing mechanisms to correct mischarged tRNAs. Prior investigations have unveiled the propensity of eukaryotic AlaRS to erroneously attach alanine onto tRNACys and tRNAThr featuring the G4:U69 base pair. In light of this, and given ProXp-ala's capacity in deacylating Ala-tRNAPro, we embarked on exploring whether this trans-editing factor could extend its corrective function to encompass these mischarged tRNAs. Our in vitro deacylation assays demonstrate that murine ProXp-ala (mProXp-ala) is able to efficiently hydrolyze Ala-tRNAThr, while Ala-tRNACys remains unaffected. Subsequently, we determined the first structure of eukaryotic ProXp-ala, revealing a dynamic helix α2 involved in substrate binding. By integrating molecular dynamics simulations and biochemical assays, we pinpointed the pivotal interactions between mProXp-ala and Ala-tRNA, wherein the basic regions of mProXp-ala as well as the C3-G70 plays essential role in recognition. These observations collectively provide a cogent rationale for mProXp-ala's deacylation proficiency against Ala-tRNAThr. Our findings offer valuable insights into the translation quality control within higher eukaryotic organisms, where the fidelity of translation is safeguarded by the multi-functionality of extensively documented proteins.

Assessment of tRNAThr and tRNAGln Variants and Mitochondrial Functionality in Parkinson's Disease (PD) Patients of Tamil Nadu Population.

Parkinson's disease (PD) is speculated with genetic and environmental factors. At molecular level, the mitochondrial impact is stated to be one of the causative reasons for PD. In this study, we investigated the mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and adenosine triphosphate (ATP) levels along with mitochondrial tRNA alterations among three age categories of PD. By determining the genetic and organellar functionality using molecular techniques, the ROS levels were reported to be high with decreased MMP and ATP in the late-onset age group than in other two age categories. Likewise, the tRNA significancy in tRNAThr and tRNAGln was noticed with C4335T and G15927A mutations in late-onset and early-onset PD groups respectively. Therefore, from the findings, ageing has shown a disruption in tRNA metabolism leading to critical functioning of ATP synthesis and MMP, causing oxidative stress in PD patients. These physiological outcomes show that ageing has a keen role in the divergence of mitochondrial function, thereby proving a correlation with ageing and maintenance of mitochondrial homeostasis in PD.

Molecular basis for t6A modification in human mitochondria.

N 6-Threonylcarbamoyladenosine (t6A) is a universal tRNA modification essential for translational accuracy and fidelity. In human mitochondria, YrdC synthesises an l-threonylcarbamoyl adenylate (TC-AMP) intermediate, and OSGEPL1 transfers the TC-moiety to five tRNAs, including human mitochondrial tRNAThr (hmtRNAThr). Mutation of hmtRNAs, YrdC and OSGEPL1, affecting efficient t6A modification, has been implicated in various human diseases. However, little is known about the tRNA recognition mechanism in t6A formation in human mitochondria. Herein, we showed that OSGEPL1 is a monomer and is unique in utilising C34 as an anti-determinant by studying the contributions of individual bases in the anticodon loop of hmtRNAThr to t6A modification. OSGEPL1 activity was greatly enhanced by introducing G38A in hmtRNAIle or the A28:U42 base pair in a chimeric tRNA containing the anticodon stem of hmtRNASer(AGY), suggesting that sequences of specific hmtRNAs are fine-tuned for different modification levels. Moreover, using purified OSGEPL1, we identified multiple acetylation sites, and OSGEPL1 activity was readily affected by acetylation via multiple mechanisms in vitro and in vivo. Collectively, we systematically elucidated the nucleotide requirement in the anticodon loop of hmtRNAs, and revealed mechanisms involving tRNA sequence optimisation and post-translational protein modification that determine t6A modification levels.

Maternally inherited coronary heart disease is associated with a novel mitochondrial tRNA mutation.

BACKGROUND: Coronary heart disease (CHD) is the most common cause of mortality globally, yet mitochondrial genetic mutations associated with CHD development remain incompletely understood. METHODS: The subjects from three Chinese families with LHON underwent clinical, genetic, molecular, and biochemical evaluations. Biochemical characterizations included measuring the effects of the15910C > T mutation on tRNAThr levels, enzymatic activity of electron transport chain complexes, membrane permeability, and the mitochondria-mediated generation of both reactive oxygen species (ROS) and adenosine triphosphate (ATP). RESULTS: We characterize mitochondrial genetic mutations in a three-generation Chinese family exhibiting signs of maternally inherited CHD. Of the 24 different family members in this pedigree we assessed, CHD was detected in 6, with variable severity and age of first appearance. When we sequenced the mitochondrial genomes of these individuals, we found a tRNAThr 15910C > T mutation of the Eastern Asian haplogroup M7b'c. This mutation is predicted to destabilize the strongly conserved (24C-10G) base-pairing, thereby disrupting tRNAThr functionality. When we performed Northern blotting, we detected we observed a 37.5% reduction in tRNAThr levels at baseline in cybrid cell lines bearing the 15910C > T mutation. When we conducted western blot analysis, we detected a ~ 24.96% decrease in mitochondrial translation rates in these same cells. CONCLUSIONS: In the present report, Together these findings suggest a possible link between this 15910C > T tRNAThr mutation and CHD, potentially offering new avenues for future disease intervention.

RNAase III-Type Enzyme Dicer Regulates Mitochondrial Fatty Acid Oxidative Metabolism in Cardiac Mesenchymal Stem Cells.

Cardiac mesenchymal stem cells (C-MSC) play a key role in maintaining normal cardiac function under physiological and pathological conditions. Glycolysis and mitochondrial oxidative phosphorylation predominately account for energy production in C-MSC. Dicer, a ribonuclease III endoribonuclease, plays a critical role in the control of microRNA maturation in C-MSC, but its role in regulating C-MSC energy metabolism is largely unknown. In this study, we found that Dicer knockout led to concurrent increase in both cell proliferation and apoptosis in C-MSC compared to Dicer floxed C-MSC. We analyzed mitochondrial oxidative phosphorylation by quantifying cellular oxygen consumption rate (OCR), and glycolysis by quantifying the extracellular acidification rate (ECAR), in C-MSC with/without Dicer gene deletion. Dicer gene deletion significantly reduced mitochondrial oxidative phosphorylation while increasing glycolysis in C-MSC. Additionally, Dicer gene deletion selectively reduced the expression of ß-oxidation genes without affecting the expression of genes involved in the tricarboxylic acid (TCA) cycle or electron transport chain (ETC). Finally, Dicer gene deletion reduced the copy number of mitochondrially encoded 1,4-Dihydronicotinamide adenine dinucleotide (NADH): ubiquinone oxidoreductase core subunit 6 (MT-ND6), a mitochondrial-encoded gene, in C-MSC. In conclusion, Dicer gene deletion induced a metabolic shift from oxidative metabolism to aerobic glycolysis in C-MSC, suggesting that Dicer functions as a metabolic switch in C-MSC, which in turn may regulate proliferation and environmental adaptation.

A coronary artery disease-associated tRNAThr mutation altered mitochondrial function, apoptosis and angiogenesis.

The tissue specificity of mitochondrial tRNA mutations remains largely elusive. In this study, we demonstrated the deleterious effects of tRNAThr 15927G>A mutation that contributed to pathogenesis of coronary artery disease. The m.15927G>A mutation abolished the highly conserved base-pairing (28C-42G) of anticodon stem of tRNAThr. Using molecular dynamics simulations, we showed that the m.15927G>A mutation caused unstable tRNAThr structure, supported by decreased melting temperature and slower electrophoretic mobility of mutated tRNA. Using cybrids constructed by transferring mitochondria from a Chinese family carrying the m.15927G>A mutation and a control into mitochondrial DNA (mtDNA)-less human umbilical vein endothelial cells, we demonstrated that the m.15927G>A mutation caused significantly decreased efficiency in aminoacylation and steady-state levels of tRNAThr. The aberrant tRNAThr metabolism yielded variable decreases in mtDNA-encoded polypeptides, respiratory deficiency, diminished membrane potential and increased the production of reactive oxygen species. The m.15927G>A mutation promoted the apoptosis, evidenced by elevated release of cytochrome c into cytosol and increased levels of apoptosis-activated proteins: caspases 3, 7, 9 and PARP. Moreover, the lower wound healing cells and perturbed tube formation were observed in mutant cybrids, indicating altered angiogenesis. Our findings provide new insights into the pathophysiology of coronary artery disease, which is manifested by tRNAThr mutation-induced alterations.

Detection of a Subset of Posttranscriptional Transfer RNA Modifications in Vivo with a Restriction Fragment Length Polymorphism-Based Method.

Transfer RNAs (tRNAs) are among the most heavily modified RNA species. Posttranscriptional tRNA modifications (ptRMs) play fundamental roles in modulating tRNA structure and function and are being increasingly linked to human physiology and disease. Detection of ptRMs is often challenging, expensive, and laborious. Restriction fragment length polymorphism (RFLP) analyses study the patterns of DNA cleavage after restriction enzyme treatment and have been used for the qualitative detection of modified bases on mRNAs. It is known that some ptRMs induce specific and reproducible base "mutations" when tRNAs are reverse transcribed. For example, inosine, which derives from the deamination of adenosine, is detected as a guanosine when an inosine-containing tRNA is reverse transcribed, amplified via polymerase chain reaction (PCR), and sequenced. ptRM-dependent base changes on reverse transcription PCR amplicons generated as a consequence of the reverse transcription reaction might create or abolish endonuclease restriction sites. The suitability of RFLP for the detection and/or quantification of ptRMs has not been studied thus far. Here we show that different ptRMs can be detected at specific sites of different tRNA types by RFLP. For the examples studied, we show that this approach can reliably estimate the modification status of the sample, a feature that can be useful in the study of the regulatory role of tRNA modifications in gene expression.

Assessing the effects of threonyl-tRNA synthetase on angiogenesis-related responses.

Several recent reports have found a connection between specific aminoacyl-tRNA synthetases and the regulation of angiogenesis. As this new area of research is explored, it is important to have reliable assays to assess the specific angiogenesis functions of these enzymes. This review provides information about specific in vitro and in vivo methods that were used to assess the angiogenic functions of threonyl-tRNA synthetase including endothelial cell migration and tube assays as well as chorioallantoic membrane and tumor vascularization assays. The theory and discussion include best methods of analysis and quantification along with the advantages and limitations of each type of assay.

Sequence-specific and Shape-selective RNA Recognition by the Human RNA 5-Methylcytosine Methyltransferase NSun6.

Human NSun6 is an RNA methyltransferase that catalyzes the transfer of the methyl group from S-adenosyl-l-methionine (SAM) to C72 of tRNAThr and tRNACys In the current study, we used mass spectrometry to demonstrate that human NSun6 indeed introduces 5-methylcytosine (m5C) into tRNA, as expected. To further reveal the tRNA recognition mechanism of human NSun6, we measured the methylation activity of human NSun6 and its kinetic parameters for different tRNA substrates and their mutants. We showed that human NSun6 requires a well folded, full-length tRNA as its substrate. In the acceptor region, the CCA terminus, the target site C72, the discriminator base U73, and the second and third base pairs (2:71 and 3:70) of the acceptor stem are all important RNA recognition elements for human NSun6. In addition, two specific base pairs (11:24 and 12:23) in the D-stem of the tRNA substrate are involved in interacting with human NSun6. Together, our findings suggest that human NSun6 relies on a delicate network for RNA recognition, which involves both the primary sequence and tertiary structure of tRNA substrates.

Human polypyrimidine tract-binding protein interacts with mitochondrial tRNA(Thr) in the cytosol.

Human polypyrimidine tract-binding protein PTB is a multifunctional RNA-binding protein with four RNA recognition motifs (RRM1 to RRM4). PTB is a nucleocytoplasmic shuttle protein that functions as a key regulator of alternative pre-mRNA splicing in the nucleoplasm and promotes internal ribosome entry site-mediated translation initiation of viral and cellular mRNAs in the cytoplasm. Here, we demonstrate that PTB and its paralogs, nPTB and ROD1, specifically interact with mitochondrial (mt) tRNA(Thr) both in human and mouse cells. In vivo and in vitro RNA-binding experiments demonstrate that PTB forms a direct interaction with the T-loop and the D-stem-loop of mt tRNA(Thr) using its N-terminal RRM1 and RRM2 motifs. RNA sequencing and cell fractionation experiments show that PTB associates with correctly processed and internally modified, mature mt tRNA(Thr) in the cytoplasm outside of mitochondria. Consistent with this, PTB activity is not required for mt tRNA(Thr) biogenesis or for correct mitochondrial protein synthesis. PTB association with mt tRNA(Thr) is largely increased upon induction of apoptosis, arguing for a potential role of the mt tRNA(Thr)/PTB complex in apoptosis. Our results lend strong support to the recently emerging conception that human mt tRNAs can participate in novel cytoplasmic processes independent from mitochondrial protein synthesis.

Discovery of the ß-barrel-type RNA methyltransferase responsible for N6-methylation of N6-threonylcarbamoyladenosine in tRNAs.

Methylation is a versatile reaction involved in the synthesis and modification of biologically active molecules, including RNAs. N(6)-methyl-threonylcarbamoyl adenosine (m(6)t(6)A) is a post-transcriptional modification found at position 37 of tRNAs from bacteria, insect, plants, and mammals. Here, we report that in Escherichia coli, yaeB (renamed as trmO) encodes a tRNA methyltransferase responsible for the N(6)-methyl group of m(6)t(6)A in tRNA(Thr) specific for ACY codons. TrmO has a unique single-sheeted ß-barrel structure and does not belong to any known classes of methyltransferases. Recombinant TrmO employs S-adenosyl-L-methionine (AdoMet) as a methyl donor to methylate t(6)A to form m(6)t(6)A in tRNA(Thr). Therefore, TrmO/YaeB represents a novel category of AdoMet-dependent methyltransferase (Class VIII). In a ΔtrmO strain, m(6)t(6)A was converted to cyclic t(6)A (ct(6)A), suggesting that t(6)A is a common precursor for both m(6)t(6)A and ct(6)A. Furthermore, N(6)-methylation of t(6)A enhanced the attenuation activity of the thr operon, suggesting that TrmO ensures efficient decoding of ACY. We also identified a human homolog, TRMO, indicating that m(6)t(6)A plays a general role in fine-tuning of decoding in organisms from bacteria to mammals.

Mechanism of oxidant-induced mistranslation by threonyl-tRNA synthetase.

Aminoacyl-tRNA synthetases maintain the fidelity during protein synthesis by selective activation of cognate amino acids at the aminoacylation site and hydrolysis of misformed aminoacyl-tRNAs at the editing site. Threonyl-tRNA synthetase (ThrRS) misactivates serine and utilizes an editing site cysteine (C182 in Escherichia coli) to hydrolyze Ser-tRNA(Thr). Hydrogen peroxide oxidizes C182, leading to Ser-tRNA(Thr) production and mistranslation of threonine codons as serine. The mechanism of C182 oxidation remains unclear. Here we used a chemical probe to demonstrate that C182 was oxidized to sulfenic acid by air, hydrogen peroxide and hypochlorite. Aminoacylation experiments in vitro showed that air oxidation increased the Ser-tRNA(Thr) level in the presence of elongation factor Tu. C182 forms a putative metal binding site with three conserved histidine residues (H73, H77 and H186). We showed that H73 and H186, but not H77, were critical for activating C182 for oxidation. Addition of zinc or nickel ions inhibited C182 oxidation by hydrogen peroxide. These results led us to propose a model for C182 oxidation, which could serve as a paradigm for the poorly understood activation mechanisms of protein cysteine residues. Our work also suggests that bacteria may use ThrRS editing to sense the oxidant levels in the environment.

Mitochondrial dysfunction in human breast cancer cells and their transmitochondrial cybrids.

Somatic mitochondrial DNA alterations have been found in all types of cancer. To better understand the role of mitochondria and their involvement in the pathogenic mechanisms of cancer development, the effects of cancer mitochondria were investigated in a defined nuclear background using a transmitochondrial cybrid system. Our results demonstrated that cancer mitochondria confer a significant reduction in cell growth when cells are metabolically stressed in a galactose medium. Activities of the respiratory chain complexes, cellular oxygen consumption, and ATP synthesis rates were found to be much lower in breast cancer cells, than those in normal breast epithelial cells of MCF-10A (10A). These results suggest that there is reduced mitochondrial function in the studied breast cancer cell lines. Similarly reduced mitochondrial function was observed in cybrids containing cancer mitochondria. Novel tRNA mutations were also identified in two breast cancer cell lines, possibly responsible for the observed mitochondrial dysfunction. We conclude that altered mitochondria in cancer cells may play a crucial role in tumor development.

The crystal structure of the ribosome bound to EF-Tu and aminoacyl-tRNA.

The ribosome selects a correct transfer RNA (tRNA) for each amino acid added to the polypeptide chain, as directed by messenger RNA. Aminoacyl-tRNA is delivered to the ribosome by elongation factor Tu (EF-Tu), which hydrolyzes guanosine triphosphate (GTP) and releases tRNA in response to codon recognition. The signaling pathway that leads to GTP hydrolysis upon codon recognition is critical to accurate decoding. Here we present the crystal structure of the ribosome complexed with EF-Tu and aminoacyl-tRNA, refined to 3.6 angstrom resolution. The structure reveals details of the tRNA distortion that allows aminoacyl-tRNA to interact simultaneously with the decoding center of the 30S subunit and EF-Tu at the factor binding site. A series of conformational changes in EF-Tu and aminoacyl-tRNA suggests a communication pathway between the decoding center and the guanosine triphosphatase center of EF-Tu.

The universal YrdC/Sua5 family is required for the formation of threonylcarbamoyladenosine in tRNA.

Threonylcarbamoyladenosine (t(6)A) is a universal modification found at position 37 of ANN decoding tRNAs, which imparts a unique structure to the anticodon loop enhancing its binding to ribosomes in vitro. Using a combination of bioinformatic, genetic, structural and biochemical approaches, the universal protein family YrdC/Sua5 (COG0009) was shown to be involved in the biosynthesis of this hypermodified base. Contradictory reports on the essentiality of both the yrdC wild-type gene of Escherichia coli and the SUA5 wild-type gene of Saccharomyces cerevisiae led us to reconstruct null alleles for both genes and prove that yrdC is essential in E. coli, whereas SUA5 is dispensable in yeast but results in severe growth phenotypes. Structural and biochemical analyses revealed that the E. coli YrdC protein binds ATP and preferentially binds RNA(Thr) lacking only the t(6)A modification. This work lays the foundation for elucidating the function of a protein family found in every sequenced genome to date and understanding the role of t(6)A in vivo.

Mitochondrial ND5 T12338C, tRNA(Cys) T5802C, and tRNA(Thr) G15927A variants may have a modifying role in the phenotypic manifestation of deafness-associated 12S rRNA A1555G mutation in three Han Chinese pedigrees.

We report here on the clinical, genetic, and molecular characterization of three Han Chinese pedigrees with aminoglycoside-induced and nonsyndromic hearing loss. Clinical evaluation revealed the variable phenotype of hearing impairment including severity, age-at-onset, audiometric configuration in these subjects. The penetrance of hearing loss in WZD8, WZD9, and WZD10 pedigrees were 46%, 46%, and 50%, respectively, when aminoglycoside-induced deafness was included. When the effect of aminoglycosides was excluded, the penetrance of hearing loss in these pedigrees were 23%, 31%, and 37.5%, respectively. Mutational analysis of the complete mitochondrial genomes showed the homoplasmic A1555G mutation and distinct sets of mitochondrial DNA variants belonging to haplogroups D4b2b, B5b1, and F2, respectively. Of these, the tRNA(Cys) T5802C, tRNA(Thr) A15924C, and ND5 T12338C variants are of special interest as these variants occur at positions which are highly evolutionarily conserved nucleotides of tRNAs or amino acid of polypeptide. These homoplasmic mtDNA variants were absent among 156 unrelated Chinese controls. The T5802C and G15927A variants disrupted a highly conserved A-U or C-G base-pairing at the anticodon-stem of tRNA(Cys) or tRNA(Thr), while the ND5 T12338C mutation resulted in the replacement of the translation-initiating methionine with a threonine, and also located in two nucleotides adjacent to the 3' end of the tRNA(Leu(CUN)). Thus, mitochondrial dysfunctions, caused by the A1555G mutation, would be worsened by these mtDNA variants. Therefore, these mtDNA mutations may have a potential modifier role in increasing the penetrance and expressivity of the deafness-associated 12S rRNA A1555G mutation in those Chinese pedigrees.

Further pitfalls in the diagnosis of mtDNA mutations: homoplasmic mt-tRNA mutations.

BACKGROUND: Mitochondrial DNA (mtDNA) mutations are important causes of human genetic disease, with mutations in tRNA genes particularly prevalent. In many patients, mutations are heteroplasmic, affecting a population of mtDNA molecules. Establishing the pathogenicity of homoplasmic mitochondrial tRNA (mt-tRNA) mutations, in which the mutation is present in every mtDNA molecule, is extremely difficult. These mutations must conform to specific pathogenic criteria, documenting unequivocally a functional defect of the mutant mt-tRNA. AIMS: To investigate the pathogenic nature of two homoplasmic mt-tRNA(Thr) deletions, m.15940delT (previously reported as pathogenic) and m.15937delA, by assessing the steady state levels of the mutant mt-tRNA in tissue and cell-line samples from six unrelated families, in which affected individuals were thoroughly investigated for mitochondrial DNA disease on the basis of clinical presentations. Rates of de novo mitochondrial protein synthesis were also examined in control and m.15937delA mutant fibroblasts. RESULTS: Our data strongly suggest that both single nucleotide deletions are neutral polymorphisms; no obvious defects were apparent in either steady state mt-tRNA(Thr) levels or rates of mitochondrial protein synthesis. CONCLUSIONS: These findings have important implications for the investigation of other families with suspected mtDNA disease, in particular the requirement to fulfil strict and established pathogenic criteria in order to avoid misattribution of pathogenicity to mt-tRNA variants.

Achieving error-free translation; the mechanism of proofreading of threonyl-tRNA synthetase at atomic resolution.

The fidelity of aminoacylation of tRNA(Thr) by the threonyl-tRNA synthetase (ThrRS) requires the discrimination of the cognate substrate threonine from the noncognate serine. Misacylation by serine is corrected in a proofreading or editing step. An editing site has been located 39 A away from the aminoacylation site. We report the crystal structures of this editing domain in its apo form and in complex with the serine product, and with two nonhydrolyzable analogs of potential substrates: the terminal tRNA adenosine charged with serine, and seryl adenylate. The structures show how serine is recognized, and threonine rejected, and provide the structural basis for the editing mechanism, a water-mediated hydrolysis of the mischarged tRNA. When the adenylate analog binds in the editing site, a phosphate oxygen takes the place of one of the catalytic water molecules, thereby blocking the reaction. This rules out a correction mechanism that would occur before the binding of the amino acid on the tRNA.

Crucial role of conserved lysine 277 in the fidelity of tRNA aminoacylation by Escherichia coli valyl-tRNA synthetase.

Valyl-tRNA synthetase (ValRS) from Escherichia coli undergoes covalent valylation by a donor valyl adenylate synthesized by the enzyme itself. ValRS could also be modified, although to a lesser extent, by the noncognate isosteric substrate L-threonine from a donor threonyl adenylate synthesized by the synthetase itself, or by the nonsubstrate methionine from methionyl adenylate produced by catalytic amounts of methionyl-tRNA synthetase. MALDI mass spectrometry analysis designated lysines 154, 162, 170, 533, 554, 593, 894, 930, and 940 of ValRS as the target residues for the attachment of valine. Following autothreonylation, lysines 162, 170, 178, 277, 291, 554, 580, 593, 861, 894, and 930 were found to be modified. Finally, L-Met-labeled residues were lysines 118, 162, 170, 178, 277, and 938. Alignment of the available ValRS amino acid sequences showed that lysines 277 and 554 are strictly conserved (with the exception concerning replacement of Lys-277 with a methionine or a tyrosine in archaebacteria), suggesting that these residues might be functionally significant. Indeed, lysine 554 of ValRS is the first lysine of the Lys-Met-Ser-Lys-Ser signature of the catalytic site of class I aminoacyl-tRNA synthetases. Lys-277 which is labeled by L-threonine or L-methionine, and not by L-valine, is located at or near the editing site, in the three-dimensional structure of ValRS. The role of lysine 277 was evaluated by site-directed mutagenesis. The Lys277Ala mutant (K277A) exhibited a posttransfer Thr-tRNA(Val) editing rate that was significantly lower than that observed for the wild-type enzyme. In addition, the K277A substitution altered amino acid discrimination in the editing site, resulting in hydrolysis of the correctly charged cognate Val-tRNA(Val). Finally, significant amounts of mischarged Thr-tRNA(Val) were produced by the K277A mutant, and not by wild-type ValRS. Altogether, our results designate Lys-277 as a likely candidate for nucleophilic attack of misacylated tRNA in the editing site of ValRS.

Molecular phylogeny of a circum-global, diverse gastropod superfamily (Cerithioidea: Mollusca: Caenogastropoda): pushing the deepest phylogenetic limits of mitochondrial LSU rDNA sequences.

The Cerithioidea is a very diverse group of gastropods with ca. 14 extant families and more than 200 genera occupying, and often dominating, marine, estuarine, and freshwater habitats. While the composition of Cerithioidea is now better understood due to recent anatomical and ultrastructural studies, the phylogenetic relationships among families remain chaotic. Morphology-based studies have provided conflicting views of relationships among families. We generated a phylogeny of cerithioideans based on mitochondrial large subunit rRNA and flanking tRNA gene sequences (total aligned data set 1873 bp). Nucleotide evidence and the presence of a unique pair of tRNA genes (i.e., threonine + glycine) between valine-mtLSU and the mtSSU rRNA gene support conclusions based on ultrastructural data that Vermetidae and Campanilidae are not Cerithioidea, certain anatomical similarities being due to convergent evolution. The molecular phylogeny shows support for the monophyly of the marine families Cerithiidae [corrected], Turritellidae, Batillariidae, Potamididae, and Scaliolidae as currently recognized. The phylogenetic data reveal that freshwater taxa evolved on three separate occasions; however, all three recognized freshwater families (Pleuroceridae, Melanopsidae, and Thiaridae) are polyphyletic. Mitochondrial rDNA sequences provide valuable data for testing the monophyly of cerithioidean [corrected] families and relationships within families, but fail to provide strong evidence for resolving relationships among families. It appears that the deepest phylogenetic limits for resolving caenogastropod relationships is less than about 245--241 mya, based on estimates of divergence derived from the fossil record.