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Small noncoding RNAs (sncRNAs) mediate a variety of cellular functions in animals and plants. Deep sequencing has made it possible to obtain highly detailed information on the types and abundance of sncRNAs in biological specimens, leading to the discovery that sncRNAs circulate in the blood of humans and mammals. The most abundant types of circulating sncRNAs are microRNAs (miRNAs), 5′ transfer RNA (tRNA) halves, and YRNA fragments, with minute amounts of other types that may nevertheless be significant. Of the more abundant circulating sncRNAs only miRNAs have well described functions, but characteristics of the others suggest specific processing and secretion as complexes that protect the RNA from degradation. The properties of circulating sncRNAs are consistent with their serving as signaling molecules, and investigations of circulating miRNAs support the view that they can enter cells and regulate cellular functions. The serum levels of specific sncRNAs change markedly with age, and these changes can be mitigated by calorie restriction (CR), indicating that levels are under physiologic control. The ability of circulating sncRNAs to transmit functions between cells and to regulate a broad spectrum of cellular functions, and the changes in their levels with age, implicate them in the manifestations of aging. Our understanding of the functions of circulating sncRNA, particularly in relation to aging, is currently at a very early stage; results to date suggest that more extensive investigation will yield important insights into mechanisms of aging. [ABSTRACT FROM AUTHOR]

The article discusses research reporting on ribosome stalling and neurodegeneration resulting from the loss of a novel binding partner of the ribosome recycling protein Pelota, called guanosine triphosphatase binding protein 2 (GTPBP2), in mice with a mutation in a transfer RNA (tRNA) gene expressed in the central nervous system. The disease potential of mutations in nuclear-encoded tRNA genes and the impact of those mutations on genetic translation and cellular homeostasis are discussed.

Methanogenic archaea lack cysteinyl-tRNA synthetase; they synthesize Cys-tRNA and cysteine in a tRNA-dependent manner. Two enzymes are required: Phosphoseryl-tRNA synthetase (SepRS) forms phosphoseryl-tRNACys (Sep-tRNACys), which is converted to Cys-tRNA5 by Sep-tRNA:Cys-tRNA synthase (SepCysS). This represents the ancestral pathway of Cys biosynthesis and coding in archaea. Here we report a translation factor, SepCysE, essential for metha-nococcal Cys biosynthesis; its deletion in Methanococcus maripaludis causes Cys auxotrophy. SepCysE acts as a scaffold for SepRS and SepCysS to form a stable high-affinity complex for tRNACys causing a 14-fold increase in the initial rate of Cys-tRNACys formation. Based on our crystal structure (2.8-A resolution) of a SepCysSSepCysE complex, a SepRSSepCysESepCysS structure model suggests that this ternary complex enables substrate channeling of Sep-tRNACys. A phylogenetic analysis suggests coevolution of SepCysE with SepRS and SepCysS in the last universal common ancestral state. Our findings suggest that the tRNA-dependent Cys biosynthesis proceeds in a multienzyme complex without release of the intermediate and this mechanism may have facilitated the addition of Cys to the genetic code. [ABSTRACT FROM AUTHOR]

Mollicutes is a class of parasitic bacteria that have evolved from a common Firmicutes ancestor mostly by massive genome reduction. With genomes under 1 Mbp in size, most Mollicutes species retain the capacity to replicate and grow autonomously. The major goal of this work was to identify the minimal set of proteins that can sustain ribosome biogenesis and translation of the genetic code in these bacteria. Using the experimentally validated genes from the model bacteria Escherichia coli and Bacillus subtilis as input, genes encoding proteins of the core translation machinery were predicted in 39 distinct Mollicutes species, 33 of which are culturable. The set of 260 input genes encodes proteins involved in ribosome biogenesis, tRNA maturation and aminoacylation, as well as proteins cofactors required for mRNA translation and RNA decay. A core set of 104 of these proteins is found in all species analyzed. Genes encoding proteins involved in post-translational modifications of ribosomal proteins and translation cofactors, post-transcriptional modifications of t+rRNA, in ribosome assembly and RNA degradation are the most frequently lost. As expected, genes coding for aminoacyl-tRNA synthetases, ribosomal proteins and initiation, elongation and termination factors are the most persistent (i.e. conserved in a majority of genomes). Enzymes introducing nucleotides modifications in the anticodon loop of tRNA, in helix 44 of 16S rRNA and in helices 69 and 80 of 23S rRNA, all essential for decoding and facilitating peptidyl transfer, are maintained in all species. Reconstruction of genome evolution in Mollicutes revealed that, beside many gene losses, occasional gains by horizontal gene transfer also occurred. This analysis not only showed that slightly different solutions for preserving a functional, albeit minimal, protein synthetizing machinery have emerged in these successive rounds of reductive evolution but also has broad implications in guiding the reconstruction of a minimal cell by synthetic biology approaches. [ABSTRACT FROM AUTHOR]

The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α, from yeast to mammals. The Gcn2 kinase domain (KD) is inherently inactive and requires allosteric stimulation by adjoining regulatory domains. Gcn2 contains a pseudokinase domain (YKD) required for high-level eIF2α phosphorylation in amino acid starved yeast cells; however, the role of the YKD in KD activation was unknown. We isolated substitutions of evolutionarily conserved YKD amino acids that impair Gcn2 activation without reducing binding of the activating ligand, uncharged tRNA, to the histidyl-tRNA synthetase-related domain of Gcn2. Several such Gcn− substitutions cluster in predicted helices E and I (αE and αI) of the YKD. We also identified Gcd− substitutions, evoking constitutive activation of Gcn2, mapping in αI of the YKD. Interestingly, αI Gcd− substitutions enhance YKD-KD interactions in vitro, whereas Gcn− substitutions in αE and αI suppress both this effect and the constitutive activation of Gcn2 conferred by YKD Gcd− substitutions. These findings indicate that the YKD interacts directly with the KD for activation of kinase function and identify likely sites of direct YKD-KD contact. We propose that tRNA binding to the HisRS domain evokes a conformational change that increases access of the YKD to sites of allosteric activation in the adjoining KD. [ABSTRACT FROM AUTHOR]

To date, more than 90 modified nucleosides have been found in tRNA and the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s). Recent studies of the biosynthetic pathways have demonstrated that the availability of methyl group donors for the methylation in tRNA is important for correct and efficient protein synthesis. In this review, I focus on the methylated nucleosides and tRNA methyltransferases. The primary functions of tRNA methylations are linked to the different steps of protein synthesis, such as the stabilization of tRNA structure, reinforcement of the codon-anticodon interaction, regulation of wobble base pairing, and prevention of frameshift errors. However, beyond these basic functions, recent studies have demonstrated that tRNA methylations are also involved in the RNA quality control system and regulation of tRNA localization in the cell. In a thermophilic eubacterium, tRNA modifications and the modification enzymes form a network that responses to temperature changes. Furthermore, several modifications are involved in genetic diseases, infections, and the immune response. Moreover, structural, biochemical, and bioinformatics studies of tRNA methyltransferases have been clarifying the details of tRNA methyltransferases and have enabled these enzymes to be classified. In the final section, the evolution of modification enzymes is discussed. [ABSTRACT FROM AUTHOR]

The article reflects the interaction of transfer RNA (tRNA) with the ribosome. Efficient role of tRNA in genetics as well as protein synthesis, its effective participation in the genetic codes and translation process of the proteins and role of tRNA in ribosomal recognition, editing, and proofreading mechanisms are discussed. Other topics discussed include evolution of the tRNA molecules and the study of its structure.

Mollicutes is a class of parasitic bacteria that have evolved from a common Firmicutes ancestor mostly by massive genome reduction. With genomes under 1 Mbp in size, most Mollicutes species retain the capacity to replicate and grow autonomously. The major goal of this work was to identify the minimal set of proteins that can sustain ribosome biogenesis and translation of the genetic code in these bacteria. Using the experimentally validated genes from the model bacteria Escherichia coli and Bacillus subtilis as input, genes encoding proteins of the core translation machinery were predicted in 39 distinct Mollicutes species, 33 of which are culturable. The set of 260 input genes encodes proteins involved in ribosome biogenesis, tRNA maturation and aminoacylation, as well as proteins cofactors required for mRNA translation and RNA decay. A core set of 104 of these proteins is found in all species analyzed. Genes encoding proteins involved in post-translational modifications of ribosomal proteins and translation cofactors, post-transcriptional modifications of t+rRNA, in ribosome assembly and RNA degradation are the most frequently lost. As expected, genes coding for aminoacyl-tRNA synthetases, ribosomal proteins and initiation, elongation and termination factors are the most persistent (i.e. conserved in a majority of genomes). Enzymes introducing nucleotides modifications in the anticodon loop of tRNA, in helix 44 of 16S rRNA and in helices 69 and 80 of 23S rRNA, all essential for decoding and facilitating peptidyl transfer, are maintained in all species. Reconstruction of genome evolution in Mollicutes revealed that, beside many gene losses, occasional gains by horizontal gene transfer also occurred. This analysis not only showed that slightly different solutions for preserving a functional, albeit minimal, protein synthetizing machinery have emerged in these successive rounds of reductive evolution but also has broad implications in guiding the reconstruction of a minimal cell by synthetic biology approaches. [ABSTRACT FROM AUTHOR]

Protein translation is one of the most important processes in cell life, but despite being well-understood biochemically, the implications of its intrinsic stochastic nature have not been fully elucidated. In this paper we develop a microscopic and stochastic model which describes a crucial step in protein translation, namely the binding of the tRNA to the ribosome. Our model explicitly takes into consideration tRNA recharging dynamics, spatial inhomogeneity, and stochastic fluctuations in the number of charged tRNAs around the ribosome. By analyzing this nonequilibrium system we are able to derive the statistical distribution of the times needed by the tRNAs to bind to the ribosome, and to show that it deviates from an exponential due to the coupling between the fluctuations of charged and uncharged populations of tRNA. [ABSTRACT FROM AUTHOR]

Deciphering the architecture of the tRNA pool is a prime challenge in translation research, as tRNAs govern the efficiency and accuracy of the process. Towards this challenge, we created a systematic tRNA deletion library in Saccharomyces cerevisiae, aimed at dissecting the specific contribution of each tRNA gene to the tRNA pool and to the cell's fitness. By harnessing this resource, we observed that the majority of tRNA deletions show no appreciable phenotype in rich medium, yet under more challenging conditions, additional phenotypes were observed. Robustness to tRNA gene deletion was often facilitated through extensive backup compensation within and between tRNA families. Interestingly, we found that within tRNA families, genes carrying identical anti-codons can contribute differently to the cellular fitness, suggesting the importance of the genomic surrounding to tRNA expression. Characterization of the transcriptome response to deletions of tRNA genes exposed two disparate patterns: in single-copy families, deletions elicited a stress response; in deletions of genes from multi-copy families, expression of the translation machinery increased. Our results uncover the complex architecture of the tRNA pool and pave the way towards complete understanding of their role in cell physiology. [ABSTRACT FROM AUTHOR]

Odontamblyopus rubicundus is a species of gobiid fishes, inhabits muddy-bottomed coastal waters. In this paper, the first complete mitochondrial genome sequence of O. rubicundus is reported. The complete mitochondrial genome sequence is 17119 bp in length and contains 13 protein-coding genes, two rRNA genes, 22 tRNA genes, a control region and an L-strand origin as in other teleosts. Most mitochondrial genes are encoded on H-strand except for ND6 and seven tRNA genes. Some overlaps occur in protein-coding genes and tRNAs ranging from 1 to 7 bp. The possibly nonfunctional L-strand origin folded into a typical stem-loop secondary structure and a conserved motif (5-GCCGG-3) was found at the base of the stem within the tRNA gene. The TAS, CSB-2 and CSB-3 could be detected in the control region. However, in contrast to most of other fishes, the central conserved sequence block domain and the CSB-1 could not be recognized in O. rubicundus, which is consistent with Acanthogobius hasta (Gobiidae). In addition, phylogenetic analyses based on different sequences of species of Gobiidae and different methods showed that the classification of O. rubicundus into Odontamblyopus due to morphology is debatable. [ABSTRACT FROM AUTHOR]

In the fruit fly Drosophila melanogaster, RNA polymerase III transcription was found to be dependent not upon the canonical TATA box-binding protein (TBP) but instead upon the TBP-related factor 1 (TRF1) (Takada, S., Lis, J. T., Zhou, S., and Tjian, R. (2000) Cell 101, 459-469). Here we confirm that transcription of fly tRNA genes requires TRF1. However, we unexpectedly find that U6 snRNA gene promoters are occupied primarily by TBP in cells and that knockdown of TBP, but not TRF1, inhibits U6 transcription in cells. Moreover, U6 transcription in vitro effectively utilizes TBP, whereas TBP cannot substitute for TRF1 to promote tRNA transcription in vitro. Thus, in fruit flies, different classes of RNA polymerase III promoters differentially utilize TBP and TRF1 for the initiation of transcription. [ABSTRACT FROM AUTHOR]

Streptococcus pneumoniae is a causative agent of nosocomial infections such as pneumonia, meningitis, and septicemia. Penicillin resistance in S. pneumoniae depends in part upon MurM, an aminoacyl-tRNA ligase that attaches L-serine or L-alanine to the stem peptide lysine of Lipid II in cell wall peptidoglycan. To investigate the exact substrates the translation machinery provides MurM, quality control by alanyl-tRNA synthetase (AlaRS) was investigated. AlaRS mischarged serine and glycine to tRNAAla, as observed in other bacteria, and also transferred ala-nine, serine, and glycine to tRNAPhe. S. pneumoniae tRNAPhe has an unusual U4:C69 mismatch in its acceptor stem that prevents editing by phenylalanyl-tRNA synthetase (PheRS), leading to the accumulation of misaminoacylated tRNAs that could serve as substrates for translation or for MurM. Although the peptidoglycan layer of S. pneumoniae tolerates a combination of both branched and linear muropeptides, deletion of MurM results in a reversion to penicillin sensitivity in strains that were previously resistant. However, because MurM is not required for cell viability, the reason for its functional conservation across all strains of S. pneumoniae has remained elusive. We now show that MurM can directly function in translation quality control by acting as a broad specificity lipid-independent trans editing factor that deacylates tRNA. This activity of MurM does not require the presence of its second substrate, Lipid II, and can functionally substitute for the activity of widely conserved editing domain homologues of AlaRS, termed AlaXPs proteins, which are themselves absent from S. pneumoniae. [ABSTRACT FROM AUTHOR]

The yeast Saccharomyces cerevisiae possesses two distinct glycyl-tRNA synthetase (GlyRS) genes: GRS1 and GRS2. GRS1 is dually functional, encoding both cytoplasmic and mitochondrial activities, while GRS2 is dysfunctional and not required for growth. The protein products of these two genes, GlyRS1 and GlyRS2, are much alike but are distinguished by an insertion peptide of GlyRS1, which is absent from GlyRS2 and other eukaryotic homologues. We show that deletion or mutation of the insertion peptide modestly impaired the enzyme's catalytic efficiency in vitro (with a 2- to 3-fold increase in Km and a 5- to 8-fold decrease in kcat). Consistently, GRS2 can be conveniently converted to a functional gene via codon optimization, and the insertion peptide is dispensable for protein stability and the rescue activity of GRS1 at 30°C in vivo. A phylogenetic analysis further showed that GRS1 and GRS2 are paralogues that arose from a gene duplication event relatively recently, with GRS1 being the predecessor. These results indicate that GlyRS2 is an active enzyme essentially resembling the insertion peptide-deleted form of GlyRS1. Our study suggests that the insertion peptide represents a novel auxiliary domain, which facilitates both productive docking and catalysis of cognate tRNAs. [ABSTRACT FROM AUTHOR]

Recognition strategies for tRNA aminoacylation are ancient and highly conserved, having been selected very early in the evolution of the genetic code. In most cases, the trinucleotide anticodons of tRNA are important identity determinants for aminoacylation by cognate aminoacyl-tRNA synthetases. However, a degree of ambiguity exists in the recognition of certain tRNAIle isoacceptors that are initially transcribed with the methioninespecifying CAU anticodon. In most organisms, the C34 wobble position in these tRNAIle precursors is rapidly modified to lysidine to prevent recognition by methionyl-tRNA synthetase (MRS) and production of a chimeric Met-tRNAIle that would compromise translational fidelity. In certain bacteria, however, lysidine modification is not required for MRS rejection, indicating that this recognition strategy is not universally conserved and may be relatively recent. To explore the actual distribution of lysidine-dependent tRNAIle rejection by MRS, we have investigated the ability of bacterial MRSs from different clades to differentiate cognate tRNACAU Met from near-cognate tRNACAU Ile. Discrimination abilities vary greatly and appear unrelated to phylogenetic or structural features of the enzymes or sequence determinants of the tRNA. Our data indicate that tRNAIle identity elements were established late and independently in different bacterial groups. We propose that the observed variation in MRS discrimination ability reflects differences in the evolution of genetic code machineries of emerging bacterial clades. [ABSTRACT FROM AUTHOR]

The Elongator complex, including the histone acetyl transferase Sin3/Elp3, was isolated as an RNA polymerase II-interacting complex, and cells deficient in Elongator subunits display transcriptional defects. However, it has also been shown that Elongator mediates the modification of some tRNAs, modulating translation efficiency. We show here that the fission yeast Sin3/Elp3 is important for oxidative stress survival. The stress transcriptional program, governed by the Sty1-Atf1-Pcr1 pathway, is affected in mutant cells, but not severely. On the contrary, cells lacking Sin3/Elp3 cannot modify the uridine wobble nucleoside of certain tRNAs, and other tRNA modifying activities such as Ctu1-Ctu2 are also essential for normal tolerance to H2O2. In particular, a plasmid over-expressing the tRNALysUUU complements the stress-related phenotypes of Sin3/Elp3 mutant cells. We have determined that the main H2O2-dependent genes, including those coding for the transcription factors Atf1 and Pcr1, are highly expressed mRNAs containing a biased number of lysine-coding codons AAA versus AAG. Thus, their mRNAs are poorly translated after stress in cells lacking Sin3/Elp3 or Ctu2, whereas a mutated atf1 transcript with AAA-to-AAG lysine codons is efficiently translated in all strain backgrounds. Our study demonstrates that the lack of a functional Elongator complex results in stress phenotypes due to its contribution to tRNA modification and subsequent translation inefficiency of certain stress-induced, highly expressed mRNAs. These results suggest that the transcriptional defects of these strain backgrounds may be a secondary consequence of the deficient expression of a transcription factor, Atf1-Pcr1, and other components of the transcriptional machinery. [ABSTRACT FROM AUTHOR]

Background: Methylaminomethyl modification of uridine or 2-thiouridine (mnm5U34 or mnm5s2U34) at the wobble position of tRNAs specific for glutamate, lysine and arginine are observed in Escherichia coli and allow for specific recognition of codons ending in A or G. In the biosynthetic pathway responsible for this posttranscriptional modification, the bifunctional enzyme MnmC catalyzes the conversion of its hypermodified substrate carboxymethylaminomethyl uridine (cmnm5U34) to mnm5U34. MnmC catalyzes the flavin adenine dinucleotide (FAD)-dependent oxidative cleavage of carboxymethyl group from cmnm5U34 via an imine intermediate to generate aminomethyl uridine (nm5U34), which is subsequently methylated by S-adenosyl-L-methionine (SAM) to yield methylaminomethyl uridine (mnm5U34). Results: The X-ray crystal structures of SAM/FAD-bound bifunctional MnmC from Escherichia coli and Yersinia pestis, and FAD-bound bifunctional MnmC from Yersinia pestis were determined and the catalytic functions verified in an in vitro assay. Conclusion: The crystal structures of MnmC from two Gram negative bacteria reveal the overall architecture of the enzyme and the relative disposition of the two independent catalytic domains: a Rossmann-fold domain containing the SAM binding site and an FAD containing domain structurally homologous to glycine oxidase from Bacillus subtilis. The structures of MnmC also reveal the detailed atomic interactions at the interdomain interface and provide spatial restraints relevant to the overall catalytic mechanism. [ABSTRACT FROM AUTHOR]

The complete sequences of three mitochondrial genomes from the land snail Cornu aspersum were determined. The mitogenome has a length of 14050 bp, and it encodes 13 protein-coding genes, 22 transfer RNA genes and two ribosomal RNA genes. It also includes nine small intergene spacers, and a large AT-rich intergenic spacer. The intra-specific divergence analysis revealed that COX1 has the lower genetic differentiation, while the most divergent genes were NADH1, NADH3 and NADH4. With the exception of Euhadra herklotsi, the structural comparisons showed the same gene order within the family Helicidae, and nearly identical gene organization to that found in order Pulmonata. Phylogenetic reconstruction recovered Basommatophora as polyphyletic group, whereas Eupulmonata and Pulmonata as paraphyletic groups. Bayesian and Maximum Likelihood analyses showed that C. aspersum is a close relative of Cepaea nemoralis, and with the other Helicidae species form a sister group of Albinaria caerulea, supporting the monophyly of the Stylommatophora clade. [ABSTRACT FROM AUTHOR]

Mouse Ikbkap gene encodes IKAP—one of the core subunits of Elongator—and is thought to be involved in transcription. However, the biological function of IKAP, particularly within the context of an animal model, remains poorly characterized. We used a loss-of-function approach in mice to demonstrate that Ikbkap is essential for meiosis during spermatogenesis. Absence of Ikbkap results in defects in synapsis and meiotic recombination, both of which result in increased apoptosis and complete arrest of gametogenesis. In Ikbkap-mutant testes, a few meiotic genes are down-regulated, suggesting IKAP's role in transcriptional regulation. In addition, Ikbkap-mutant testes exhibit defects in wobble uridine tRNA modification, supporting a conserved tRNA modification function from yeast to mammals. Thus, our study not only reveals a novel function of IKAP in meiosis, but also suggests that IKAP contributes to this process partly by exerting its effect on transcription and tRNA modification. [ABSTRACT FROM AUTHOR]

Endonuclease‐mediated tRNA fragmentation has been observed in many species suggesting functional importance for tRNA fragments. The size distribution of tRNA‐derived fragments indicates the existence of mechanisms that protect tRNAs and their fragments from total degradation by exonucleases. Could post‐transcriptional modifications be important for the controlled processing of tRNAs? [ABSTRACT FROM AUTHOR]

The ribosomal DNA (rDNA) of eukaryotes is organized as large tandem arrays. Here, we compare the genomic locations of rDNA among yeast species and show that, despite its huge size (>1 Mb), the rDNA array has moved around the genome several times within the family Saccharomycetaceae. We identify an ancestral, nontelomeric, rDNA site that is conserved across many species including Saccharomyces cerevisiae. Within the genus Lachancea, however, the rDNA apparently transposed from the ancestral site to a new site internal to a different chromosome, becoming inserted into a short intergenic region beside a tRNA gene. In at least four other yeast lineages, the rDNA moved from the ancestral site to telomeric locations. Remarkably, both the ancestral rDNA site and the new site in Lachancea are adjacent to protein-coding genes whose products maintain the specialized chromatin structure of rDNA (HMO1 and CDC14, respectively). In almost every case where the rDNA was lost from the ancestral site, the entire array disappeared without any other rearrangements in the region, leaving just an intergenic spacer of less than 2 kb. The mechanism by which this large and complex locus moves around the genome is unknown, but we speculate that it may involve the formation of double-strand DNA breaks by Fob1 protein or the formation of extrachromosomal rDNA circles. [ABSTRACT FROM AUTHOR]

Abstract: The monophyletic and polyphyletic origins of tRNAs are compared. The monophyletic hypothesis of the origin of the tRNA molecule would turn out to be strictly true only if a universal tRNA, possessing a number of anticodons with which to read primitive mRNAs, was present at the Last Universal Common Ancestor (LUCA) stage. On the other hand, whether a tRNA precursor less complex than it – for instance a hairpin – was present at the LUCA stage, then one should imperatively predict a phase of evolutionary convergence towards the cloverleaf structure typical of tRNAs, equivalent to a polyphyletic origin, also in presence of a real monophyletic origin. Thus, the monophyletic hypothesis is not able to eliminate the main trouble inherent in the evolutionary convergence, for which it is usually invoked. Arguments opposed to the existence of a universal tRNA and in favour of less complex precursors to tRNA, combined with the existence of two different classes of tRNAs, and, in particular, tyrosine tRNA being a class II tRNA in the bacterial domain but a class I tRNA in the eukaryal and archaeal domains, support a polyphyletic origin of tRNA molecules. It might have been the main path for the evolution of tRNAs, also because a polyphyletic origin of tRNA genes has been corroborated. [Copyright &y& Elsevier]

Anabaena sp. PCC 7120 is a filamentous cyanobacterium that bears a cluster of 26 t RNA genes and pseudogenes in the delta plasmid. The sequences of these t RNAs suggest that they have been acquired by horizontal gene transfer from another organism. The cluster is transcribed as a single transcript that is quickly processed to individual t RNAs. RNase P and RNase Z, in vitro, are able to process precursors containing some of these t RNAs. Deletion of the cluster causes no obvious phenotype or effect on growth under diverse culture conditions, indicating that the t RNAs encoded in the cluster are not required for growth under laboratory conditions, although they are aminoacylated in vivo. We have studied a possible t RNASer [t RNASer GCU(2)] present in the cluster with a sequence that deviates from consensus. This t RNA is processed in vitro by RNase P at the expected position. In addition, this t RNASer GCU is specifically aminoacylated with serine by an Anabaena sp. PCC 7120 crude extract. These data indicate that t RNASer GCU(2) is fully functional, despite its unusual structure. Similar clusters are found in other three cyanobacteria whose genomes have been sequenced. [ABSTRACT FROM AUTHOR]

The method developed for the total synthesis of a given DNA containing biologically specific sequences consists of the following. The DNA in the double-stranded form is carefully divided into short single-stranded segments with suitable overlaps in the complementary strands. All the segments are chemically synthesized starting with protected nucleosides and mono-nucleotides. The 5′-OH ends of the appropriate oligonucleotides are then phosphorylated with the use of [γ-P]ATP and polynucleotide kinase. A few to several neighboring oligonucleotides are then allowed to form bihelical complexes in aqueous solution, and the latter are joined end to end by polynucleotide ligase to form covalently linked duplexes. Subsequent head-to-tail joining of the short duplexes leads to the total DNA. The methods are described for the construction of a biologically functional suppressor transfer RNA gene. The total work involved (i) the synthesis of a 126-nucleotide-long bihelical DNA corresponding to a known precursor to the tyrosine suppressor transfer RNA, (ii) the sequencing of the promoter region and the distal region adjoining the C-C-A end, which contained a signal for the processing of the RNA transcript, (iii) total synthesis of the 207 base-pair-long DNA, which included the control elements, as well as the Eco R1 restriction endonu-clease specific sequences at the two ends, and (iv) full characterization by transcription in vitro and amber suppressor activity in vivo of the synthetic gene. [ABSTRACT FROM AUTHOR]

BACKGROUND AND PURPOSE MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) is a mitochondrial disease most usually caused by point mutations in tRNA genes encoded by mitochondrial DNA (mtDNA). Approximately 80% of cases of MELAS syndrome are associated with a m.3243A > G mutation in the MT-TL1 gene, which encodes the mitochondrial tRNALeu (UUR). Currently, no effective treatments are available for this chronic progressive disorder. Treatment strategies in MELAS and other mitochondrial diseases consist of several drugs that diminish the deleterious effects of the abnormal respiratory chain function, reduce the presence of toxic agents or correct deficiencies in essential cofactors. EXPERIMENTAL APPROACH We evaluated the effectiveness of some common pharmacological agents that have been utilized in the treatment of MELAS, in yeast, fibroblast and cybrid models of the disease. The yeast model harbouring the A14G mutation in the mitochondrial tRNALeu(UUR) gene, which is equivalent to the A3243G mutation in humans, was used in the initial screening. Next, the most effective drugs that were able to rescue the respiratory deficiency in MELAS yeast mutants were tested in fibroblasts and cybrid models of MELAS disease. KEY RESULTS According to our results, supplementation with riboflavin or coenzyme Q10 effectively reversed the respiratory defect in MELAS yeast and improved the pathologic alterations in MELAS fibroblast and cybrid cell models. CONCLUSIONS AND IMPLICATIONS Our results indicate that cell models have great potential for screening and validating the effects of novel drug candidates for MELAS treatment and presumably also for other diseases with mitochondrial impairment. [ABSTRACT FROM AUTHOR]

Abstract: To investigate the features of the control region (CR) and the gene rearrangement in the mitochondrial (mt) genome of Thysanoptera insects, we sequenced the whole mt genome of the western flower thrips Frankliniella occidentalis (Thysanoptera: Thripidae). The mt genome is a circular molecule with 14,889 nucleotides and an A+T content of 76.6%, and it has triplicate putative CRs. We propose that tandem duplication and deletion account for the evolution of the CR and the gene translocations. Intramitochondrial recombination is a plausible model for the gene inversions. We discuss the excessive duplicate CR sequences and the transcription of the rRNA genes, which are distant from one another and from the CR. Finally, we address the significance of the complicated mt genomes in Thysanoptera for the evolution of the CR and the gene arrangement of the mt genome. [Copyright &y& Elsevier]

Abstract: Aspergillus is a genus of mold fungi that includes more than 200 described species. Many members of the group are relevant pathogens and other species are economically important. Only one species has been analyzed for codon usage, and this was performed with a small number of genes. In this paper, we report the codon usage patterns of eight completely sequenced genomes which belong to this genus. The results suggest that selection for translational efficiency and accuracy are the major factors shaping codon usage in all of the species studied so far, and therefore they were active in the last common ancestor of the group. Composition and molecular distances analyses show that highly expressed genes evolve slower at synonymous sites. We identified a conserved core of translationally optimal codons and study the tRNA gene pool in each genome. We found that the great majority of preferred triplets match the respective cognate tRNA with more copies in the respective genome. We discuss the possible scenarios that can explain the observed differences among the species analyzed. Finally we highlight the biotechnological application of this research regarding heterologous protein expression. [Copyright &y& Elsevier]

Purpose: To understand the role of mitochondrial DNA (mtDNA) mutations in patients with polycystic ovary syndrome (PCOS). Methods: A total of 57 women with PCOS and 38 controls were recruited in this study, mutational analysis of mitochondrial genome was performed using polymerase chain reaction and under a direct sequence analysis. Results: Sequence characterization of mitochondrial genome showed a distinct set of polymorphisms mainly focused on oxidative phosphorylation (OXPHOS) complex, in addition, six variants in mitochondrial tRNA genes, including tRNA, tRNA, tRNA, tRNA, tRNA and tRNA were also identified in PCOS patients. Interestingly, these variants occurred at highly conserved nucleotides of corresponding tRNAs, which are important for tRNA stability level and biochemical function. Conclusions: Mutations in mtDNA, especially the OXPHOS complex and tRNAs, may be associated with PCOS patients, thus, our results shed new insight into the pathogenesis of PCOS. [ABSTRACT FROM AUTHOR]

While genomic erosion is common among intracellular symbionts, patterns of genome evolution in heritable extracellular endosymbionts remain elusive. We study vertically transmitted extracellular endosymbionts (Verminephrobacter, Betaproteobacteria) that form a beneficial, species-specific, and evolutionarily old (60–130 Myr) association with earthworms. We assembled a draft genome of Verminephrobacter aporrectodeae and compared it with the genomes of Verminephrobacter eiseniae and two nonsymbiotic close relatives (Acidovorax). Similar to V. eiseniae, the V. aporrectodeae genome was not markedly reduced in size and showed no A–T bias. We characterized the strength of purifying selection (ω = dN/dS) and codon usage bias in 876 orthologous genes. Symbiont genomes exhibited strong purifying selection (ω = 0.09 ± 0.07), although transition to symbiosis entailed relaxation of purifying selection as evidenced by 50% higher ω values and less codon usage bias in symbiont compared with reference genomes. Relaxation was not evenly distributed among functional gene categories but was overrepresented in genes involved in signal transduction and cell envelope biogenesis. The same gene categories also harbored instances of positive selection in the Verminephrobacter clade. In total, positive selection was detected in 89 genes, including also genes involved in DNA metabolism, tRNA modification, and TonB-dependent iron uptake, potentially highlighting functions important in symbiosis. Our results suggest that the transition to symbiosis was accompanied by molecular adaptation, while purifying selection was only moderately relaxed, despite the evolutionary age and stability of the host association. We hypothesize that biparental transmission of symbionts and rare genetic mixing during transmission can prevent genome erosion in heritable symbionts. [ABSTRACT FROM AUTHOR]

Abstract: Eukaryotic cells are characterized by their content of intracellular membrane-bound organelles, including mitochondria as well as nuclei. These two DNA-containing compartments employ two distinct strategies for storage and readout of genetic information. The diploid nuclei of human cells contain about 6billion base pairs encoding about 25,000 protein-encoding genes, averaging 120kB/gene, packaged in chromatin arranged as a regular nucleosomal array. In contrast, human cells contain hundreds to thousands of copies of a ca.16kB mtDNA genome tightly packed with 13 protein-coding genes along with rRNA and tRNA genes required for their expression. The mtDNAs are dispersed throughout the mitochondrial network as histone-free nucleoids containing single copies or small clusters of genomes. This review will summarize recent advances in understanding the microscopic structure and molecular composition of mtDNA nucleoids in higher eukaryotes. This article is part of a Special Issue entitled: Mitochondrial Gene Expression. [Copyright &y& Elsevier]

The nucleotide variation and structural patterns of mitochondrial RNA molecule have been proposed as useful tools in molecular systematics; however, their usefulness is always subject to a proper assessment of homology in the sequence alignment. The present study describes the secondary structure of mitochondrial tRNA for the amino acid serine (UCN) on 13 Euptychiina species and the evaluation of its potential use for evolutionary studies in this group of butterflies. The secondary structure of tRNAs showed variation among the included species except between Hermeuptychia sp1 and sp2. Variation was concentrated in the ribotimidina-pseudouridine-cystosine (TψC), dihydrouridine (DHU) and variable loops and in the DHU and TψC arms. These results suggest this region as a potential marker useful for taxonomic differentiation of species in this group and also confirm the importance of including information from the secondary structure of tRNA to optimize the alignments. [ABSTRACT FROM AUTHOR]

The elongator (ELP) complex consisting of Elp1-6p has been indicated to play roles in multiple cellular processes. In yeast, the ELP complex has been shown to genetically interact with Uba4p/Urm1p and Kti11-13p for a function in tRNA modification. Through a Caenorhabditis elegans genetic suppressor screen and positional cloning, we discovered that loss-of-function mutations of moc-3 and dph-3, orthologs of the yeast UBA4 and KTI11, respectively, effectively suppress the Multivulva (Muv) phenotype of the lin-1(e1275, R175Opal) mutation. These mutations do not suppress the Muv phenotype caused by other lin-1 alleles or by gain-of-function alleles of ras or raf that act upstream of lin-1. The suppression can also be reverted by RNA interference of lin-1. Furthermore, we showed that dph-3(lf) also suppressed the defect of lin-1(e1275) in promoting the expression of a downstream target (egl-17). These results indicate that suppression by the moc-3 and dph-3 mutations is due to the elevated activity of lin-1(e1275) itself rather than the altered activity of a factor downstream of lin-1. We further showed that loss-of-function mutations of urm-1 and elpc-1-4, the worm counterparts of URM1 and ELP complex components in yeast, also suppressed lin-1(e1275). We also confirmed that moc-3(lf) and dph-3(lf) have defects in tRNA modifications as do the mutants of their yeast orthologs. These results, together with the observation of a likely readthrough product from a lin-1(e1275)Tgfp fusion transgene indicate that the aberrant tRNA modification led to failed recognition of a premature stop codon in lin-1(e1275). Our genetic data suggest that the functional interaction of moc-3/urm-1 and dph-3 with the ELP complex is an evolutionarily conserved mechanism involved in tRNA functions that are important for accurate translation. [ABSTRACT FROM AUTHOR]

The complete mitochondrial genome of theEpinephelus tukulawas presented in this study. The mitochondrial genome is 16,503 bp long and consists of 13 protein-coding genes, two rRNA genes, 22 tRNA genes and a control region. The gene order and composition ofEpinephelus tukulamitochondrial genome was similar to that of most other vertebrates. The nucleotide compositions of the light strand are 28.37% of A, 29.00% of C, 26.36% of T and 16.26% of G. With the exception of the NADH dehydrogenase subunit 6 (ND6) and seven tRNA genes, all other mitochondrial genes are encoded on the heavy strand. [ABSTRACT FROM AUTHOR]

The complete mitochondrial genome of the Critically Endangered southern purple-spotted gudgeon,Mogurnda adspersa, was determined for the first time using 1/8 of a 454 pyrosequencing plate. The mitogenome was assembled using the bioinformatic software MIRA. TheM. adspersagenome organization was very similar to most vertebrates, being 16,523 bp in length. It contained 13 protein-coding genes, 22 transfer RNA, 2 ribosomal RNA genes, and 1 non-coding control region. The current methodology was effective to assemble the whole mitogenome using 2008 mitochondrial reads representing 0.23% of total genomic reads produced and providing average mitogenome coverage of 17.4 reads per site. The whole mitogenome sequence provided here is deposited in NCBI with the accession number KJ130031 and may benefit systematics studies and conservation programs ofM. adspersa. [ABSTRACT FROM AUTHOR]

Blue-throated Macaw (Ara glaucogularis) is Critically Endangered species of parrot endemic to small Bolivian area. In this study, full mitochondrial genome of considered species was sequenced. It is 16,983 bp long and contains 13 protein-coding genes, 2 rRNAs, 22 tRNAs and a control region. It`s comparison with publishedAra macaomitogenome revealed their high degree of identity. On the other hand, analysis of both the genome compositions showed incompleteness ofAra macaoCYTB gene. Hence, mitogenome ofAra macaospecies occurred only partial sequence. In consequence,Ara glaucogularismitogenome is the first complete Macaw sequence, which will be indispensable to refine the phylogenetic relationships within the tribeAriniand will enrich the resource of markers for systematic, phylogenetic and population genetic studies. [ABSTRACT FROM AUTHOR]

In this study, we present the complete mitochondrial genome ofP. s. hainanus, which is spread over Hainan Island, southern China and northern Vietnam, containing 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA genes (srRNAandlrRNA) and 2 extensive heteroplasmic control regions. All protein-coding, rRNA genes have more than 99.0% nucleotide sequence similarity with the previously reported individual ofP. s. formosae, except forcox3and CRs. Notably, we found that the nucleotide sequence ofP. s. formosaemtDNA has more similarity withP. taivanus. [ABSTRACT FROM AUTHOR]

This work presents the coral-inhabiting barnacleNobia grandisSowerby, 1839 complete mitochondrial genome, which is the first report from the family Pyrgomatidae (Cirripedia: Sessilia). TheN. grandismitochondrial genome is 15,032 bp in length, containing a total of 469 bp of non-coding nucleotides spreading in 11 intergenic regions (with the largest region of 376 bp). Compared with the pancrustacean ground pattern, there are not less than seven tRNAs rearranged in theN. grandismitochondrial genome. Gene overlaps are founded in eight places. Nine PCGs (COX1-3, ATP6, ATP8, CYTB, ND2, ND3andND6) are encoded on the heavy strand while the remaining 4 PCGs and the two rRNAs are located on the light strand. As the first representative from the family Pyrgomatidae, theN. grandismitochondrial genome will help us to explore the evolutionary history and molecular evolution of coral barnacles and Sessilia in future studies. [ABSTRACT FROM AUTHOR]

In this study, we have sequenced the complete mitochondrial genome of theParastromateus niger. The mitochondrial genome is 16,561 bp long and contains 13 protein-coding genes, two rRNA genes, 22 tRNA genes and a control region. The gene order and composition ofParastromateus nigermitochondrial genome is similar to that of most other vertebrates. The nucleotide compositions of the light strand are 28.23% of A, 29.51% of C, 26.01% of T and 16.16% of G. With the exception of five tRNA genes, all other mitochondrial genes are encoded on the heavy strand. [ABSTRACT FROM AUTHOR]

In this study, the complete mitochondrial genome of the fat-nose goby Pseudogobius javanicus has been amplified. P. javanicus is widely contributed species in Southeast to East Asia. The complete mitochondrial genome is 16,493 base pairs (bp) in total length, had the typical vertebrate mitochondrial gene arrangement, including 13 protein-coding genes, 2 rRNAs, 22 tRNAs, 1 control region (CR) and 1 light-strand replication origin (OL). The overall base composition of P. javanicus is 27.1% for A, 27.3% for T, 28.6% for C, 17.0% for G, with higher AT content of 54.4%. This study will contribute for understanding the phylogenetic approach in genus Pseudogobius. [ABSTRACT FROM AUTHOR]

The Watson-Crick base pairs between the 3′-terminal end of tRNAs and ribosomal RNA in the peptidyl transferase center are universally conserved. Here, we report that the introduction of compensatory mutations to Escherichia coli RNAs in this site leads to an orthogonal system independent of the wild-type counterpart, as demonstrated via the production of two peptide sequences from a single mRNA. This work thus identifies a new way to reprogram the genetic code. [ABSTRACT FROM AUTHOR]

Zebrias zebrinus belongs in the family Soleidae of Pleuronectiformes. There are overlaps in the ranges of identification characters between Z. zebrinus and another striped sole Z. fasciatus. In this study, the complete mitochondrial genome of Z. zebrinus was first determined. The total length was 16,758 bp, including 13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes ( 12S and 16S), as well as a putative control region and a putative L-strand replication origin (OL). Gene contents, locations, and arrangements were identical to those of typical bony fishes. The overall base composition of the mitogenome was 28.7%, 30.0%, 15.2%, and 26.1% for A, C, G, and T, respectively, with an A+T content of 54.8%. This result would expect the contribution to the molecular identification of this species and further phylogenetic study of Soleidae and Pleuronectiformes. [ABSTRACT FROM AUTHOR]

The complete mitochondrial genome (mitogenome) of the swallowtail butterfly Papilio maackii has been completed. It is 15,357 bp in length, and contains the typical complement of 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA), and 2 ribosomal RNA (rRNA) genes. Two A+T-rich regions are included in this mitogenome. Gene order in P. maackii mitogenome is basically identical to that of the inferred ancestral insect genome, with the exception of translocations of trnM, which is shared by some Papilios. [ABSTRACT FROM AUTHOR]

In this study, we sequenced and annotated the complete mitochondrial genome of Schizothorax waltoni (Cypriniformes:Cyprinidae). The mitogenome is 16,587 bp in length, which contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and 2 non-coding regions: origin of light-strand replication (OL) and control region (D-loop). The overall nucleotide base composition of S. waltoni mtDNA is A 29.96%, C 27.07%, G 17.58%, and T 25.39%, with an A+T content of 55.35%. Except for ND6 gene and eight tRNA genes, all other mitochondrial genes were encoded on the heavy strand. The mitochondrial genome of S. waltoni can contribute to the studies on conservation genetics and stock evaluation of S. waltoni resource, as well as molecular phylogeny and species identification of Cyprinidae. [ABSTRACT FROM AUTHOR]

The complete mitochondrial genome of a stream-dwelling frog species Paramegophrys oshanensis was determined. This mitogenome was 17,747 bp in length, containing 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and a control region (D-loop). The base composition of the light strand was 28.77% A, 29.85% T, 26.26% C, and 15.11% G. Compared with most other vertebrates, this mitogenome has some different features: a non-tandem duplication of tRNA-Met gene (forming tRNA-Met1 and tRNA-Met2 genes), the replacement of tRNA-Trp gene by a 72-bp noncoding region translocated from the cluster of WANCY to the back of Cytb gene, the translocation of tRNA-Val gene from the front of 16S rRNA to the back of tRNA-Met1 gene, and the translocation of tRNA-Pro gene from the front of D-loop region to the front of tRNA-Met2 gene. Three kinds of tandem repeats with size ranging from 71 to 92 bp were detected within D-loop region. [ABSTRACT FROM AUTHOR]

The complete mitochondrial genome (mitogenome) of Dolycoris baccarum (Hemiptera: Pentatomidae) has been sequenced and annotated in this study. This mitogenome is 16,549 bp in length with an A+T content of 73.4%, and contains the typical 37 genes that are arranged in the same order as that of the putative ancestor of hexapods. All protein-coding genes (PCGs) start with ATN codons except for cox1 that uses TTG as the initial codon. Eleven PCGs stop with termination codon TAA, and cox1 and cox2 have single T as the incomplete stop codon. All the transfer RNA genes have the typical clover leaf secondary structure, except for trnS1 (AGN) that lacks the dihydrouridine arm as known in many other metazoa. The control region is located between rrnS and trnI, and is composed of 479 bp of non-repeat region and 1401 bp of repeat region. This is the third completely sequenced mitogenome from the family Pentatomidae of Hemiptera. [ABSTRACT FROM AUTHOR]

The long polymerase chain reaction and primer walking methods were employed for the complete mitochondrial genome (mitogenome) sequence of Japanese Konosirus punctatus. The mitogenome is a circular molecule 16,698 bp in length, including the typical structure of 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 2 non-coding regions (L-strand replication origin and control region), the gene contents of which are identical to those observed in most bony fishes. Within the control region, we identified the termination-associated sequence domain, the central conserved sequence block domains (CSB-F, CSB-E, CSB-D, CSB-C, and CSB-B), and the conserved sequence block domains (CSB-2 and CSB-3). [ABSTRACT FROM AUTHOR]

Lamprotula tortuosa is the endemic species of freshwater pearl mussel in China. The complete F-type mitochondrial genome of L. tortuosa was first determined. This circle genome (15,722 bp in size) comprises 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, except for COI, CYTB, ND6, and ND4L, with TTG, ATT, ATT, and ATA start codon, respectively; the remaining protein-coding genes initiated with the orthodox ATG start codon. There were 25 noncoding regions found throughout the mitogenome of L. tortuosa, ranging in size from 1 to 241 bp, the largest of which is between ND5 and tRNAGln (241 bp), but is shorter than the control region sequences(about 280 bp) of freshwater mussels from Poland and South Korea. [ABSTRACT FROM AUTHOR]

This paper reports the complete mitochondrial genome (mitogenome) sequence of Channa maculate (Taiwan snakehead). The mitogenome of Taiwan snakehead was determined to be 16,558 bp in length. It contains 13 typical vertebrate protein-coding genes, 22 tRNA genes, 2 rRNA genes, 1 OL (putative light-strand origin of replication), and 1 putative control region (D-loop). This is the first report on the complete mitogenome sequence of Channa maculata. [ABSTRACT FROM AUTHOR]

The complete mitochondrial genome sequence of the hybrid of Ctenopharyngodon idella (♀) ×  Squaliobarbus curriculus (♂) was determined using PCR-based method. The total length of the mitogenome is 16,609 bp. It contains the typical structure as that of most other vertebrates, including 2 ribosomal RNA genes, 13 protein-coding genes, 22 transfer RNA genes, and 1 non-coding control region (D-loop region). The overall composition of the mitogenome was estimated to be 31.86% for A, 26.08% for T, 26.38% for C, and 15.68% for G, respectively, indicating that an A+T (57.94%)-rich feature occurs in the hybrid mitogenome. Both the termination-associated sequence and three conserved sequence blocks (CSB1, CSB2, and CSB3) were also detected in the D-loop region. [ABSTRACT FROM AUTHOR]

The complete mitochondrial genome (mitogenome) of Cryptocentrus yatsui was first determined. The genome was 16,584 bp in length and consisted of 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes, and 2 main noncoding regions (the control region and the origin of the light strand replication), the gene composition and order of which were similar to most other vertebrates. The overall base composition values of the heavy strand are 26.0% for T, 29.9% for C, 26.4% for A, and 17.7% for G, with a slight A+T bias of 52.4%. To clarify the phylogenetic relationships of C. yatsui within the genus Cryptocentrus, 18 sequences, which were generated from COI region of six species, were used to construct the neighbor-joining phylogenetic tree. And we found that C. yatsui was rooted with the other Cryptocentrus species and has a distant relationship with others. [ABSTRACT FROM AUTHOR]