A novel nucleotide incorporation activity implicated in the editing of mitochondrial transfer RNAs in Acanthamoeba castellanii. (1/292)

In Acanthamoeba castellanii, most of the mtDNA-encoded tRNAs are edited by a process that replaces one or more of the first three nucleotides at their 5' ends. As a result, base pairing potential is restored at acceptor stem positions (1:72, 2:71, and/or 3:70, in standard tRNA nomenclature) that are mismatched according to the corresponding tRNA gene sequence. Here we describe a novel nucleotide incorporation activity, partially purified from A. castellanii mitochondria, that has properties implicating it in mitochondrial tRNA editing in this organism. This activity is able to replace nucleotides at the first three positions of a tRNA (positions 1, 2, and 3), matching the newly incorporated residues through canonical base pairing to the respective partner nucleotide in the 3' half of the acceptor stem. Labeling experiments with natural (Escherichia coli tRNATyr) and synthetic (run-off transcripts corresponding to A. castellanii mitochondrial tRNALeu1) substrates suggest that the nucleotide incorporation activity consists of at least two components, a 5' exonuclease or endonuclease and a template-directed 3'-to-5' nucleotidyltransferase. The nucleotidyltransferase component displays an ATP requirement and generates 5' pppN... termini in vitro. The development of an accurate and efficient in vitro system opens the way for detailed studies of the biochemical properties of this novel activity and its relationship to mitochondrial tRNA editing in A. castellanii. In addition, the system will allow delineation of the structural features in a tRNA that identify it as a substrate for the labeling activity.  (+info)

A model of whole-body protein turnover based on leucine kinetics in rodents. (2/292)

The measurement of fractional synthesis rate is based on the following assumptions: amino acids for protein synthesis are supplied by an intracellular pool; amino acids from protein degradation are not recycled preferentially to protein synthesis; and proteins turn over at a homogeneous rate. To test these assumptions, a mechanistic, theoretical model of protein turnover for a nongrowing 26-g mouse was developed on the basis of data from the literature. The model consisted of three protein pools turning over at fast (102 micromol Leu, t1/2= 11.5 h), medium (212 micromol Leu, t1/2 = 16.6 h) or slow (536 micromol Leu, t1/2 = 71.5 h) rates and extracellular (1.69 micromol Leu), leucyl-tRNA (0.0226 micromol Leu) and intracellular (5.72 micromol Leu) amino acid pools that exchanged amino acids. The flow of amino acids from the protein pools to the leucyl-tRNA pool determined the amount of recycling. The flow of amino acids from the extracellular pool to aminoacyl tRNA determined the amount of channeling. Two flooding dose data sets were used to evaluate specific radioactivity changes predicted by the model. Predictions of specific radioactivities using flooding dose, pulse dose or continuous infusion methods indicated that the model can be a useful tool in estimating the rates of channeling and recycling. However, it was found that use of data from flooding dose experiments might cause inaccurate predictions of certain fluxes.  (+info)

Recycling, channeling and heterogeneous protein turnover estimation using a model of whole-body protein turnover based on leucine kinetics in rodents. (3/292)

In the companion paper, a whole-body, mechanistic model of protein turnover in a rodent was described and evaluated with independent data sets that used the flooding dose method. On the basis of fitted fluxes, the model was able to predict specific radioactivity changes in the protein and free leucine pools and whole-body protein fractional synthesis rate (FSR). In this paper, results of model simulations of specific radioactivity changes in the flooding dose, pulse dose and continuous infusion methods were compared and the influence of recycling, channeling and multiple protein pools on model behavior were analyzed. For all methods, the percentage of channeling must be estimated to determine whether the extracellular or intracellular pool specific radioactivities better approximate the aminoacyl tRNA pool specific radioactivity. Recycling also affects the specific radioactivity of the aminoacyl-tRNA pool and therefore must be estimated. An analysis of fits of the flooding dose data indicated that 100% channeling was occurring, but the percentage of recycling could not be determined. Multiple protein pools turning over at different rates overestimated FSR by 2-3% at early time points (5 min) and underestimated FSR by 3-6% at 60 min in the flooding dose method. For the pulse dose method, FSR was underestimated by 40-50% at 5 min and underestimated by 9-10% at 60 min. An increase in time to measure FSR caused a decrease in the estimate of FSR (18% over 3 h) for the flooding dose method and an increase in the estimate of FSR (144% over 3 h) for the pulse dose method.  (+info)

Rapid progression of cardiomyopathy in mitochondrial diabetes. (4/292)

Cardiac involvement and its clinical course in a diabetic patient with a mitochondrial tRNA(Leu)(UUR) mutation at position 3243 is reported in a 54-year-old man with no history of hypertension. At age 46, an electrocardiogram showed just T wave abnormalities. At age 49, it fulfilled SV1 + RV5 or 6>35 mm with strain pattern. At age 52, echocardiography revealed definite left ventricular (LV) hypertrophy, and abnormally increased mitochondria were shown in biopsied endomyocardial specimens. He was diagnosed as having developed hypertrophic cardiomyopathy associated with the mutation. However, at age 54, SV1 and RV5,6 voltages were decreased, and echocardiography showed diffuse decreased LV wall motion and LV dilatation. Because he had mitochondrial diabetes, the patient's heart rapidly developed hypertrophic cardiomyopathy, and then it seemed to be changing to a dilated LV with systolic dysfunction. Rapid progression of cardiomyopathy can occur in mitochondrial diabetes.  (+info)

A conserved motif in group IC3 introns is a new class of GNRA receptor. (5/292)

Terminal tetraloops consisting of GNRA sequences are often found in biologically active large RNAs. The loops appear to contribute towards the organization of higher order RNA structures by forming specific tertiary interactions with their receptors. Group IC3 introns which possess a GAAA loop in the L2 region often have a phylogenetically conserved motif in their P8 domains. In this report, we show that this conserved motif stands as a new class of receptor that distinguishes the sequences of GNRA loops less stringently than previously known receptors. The motif can functionally substitute an 11 nt motif receptor in the Tetrahymena ribozyme. Its structural and functional similarity to one class of synthetic receptors obtained from in vitro selection is observed.  (+info)

Gene shifting: a novel therapy for mitochondrial myopathy. (6/292)

Mutations in mitochondrial DNA (mtDNA) are the most frequent causes of mitochondrial myopathy in adults. In the majority of cases mutant and wild-type mtDNAs coexist, a condition referred to as mtDNA heteroplasmy; however, the relative frequency of each species varies widely in different cells and tissues. Nearly complete segregation of mutant and wild-type mtDNAs has been observed in the skeletal muscle of many patients. In such patients mutant mtDNAs pre-dominate in mature myofibers but are rare or undetectable in skeletal muscle satellite cells cultured in vitro. This pattern is thought to result from positive selection for the mutant mtDNA in post-mitotic myofibers and loss of the mutant by genetic drift in satellite cells. Satellite cells are dormant myoblasts that can be stimulated to re-enter the cell cycle and fuse with existing myofibers in response to signals for muscle growth or repair. We tested whether we could normalize the mtDNA genotype in mature myofibers in a patient with mitochondrial myopathy by enhancing the incorporation of satellite cells through regeneration following injury or muscle hypertrophy, induced by either eccentric or concentric resistance exercise training. We show a remarkable increase in the ratio of wild-type to mutant mtDNAs, in the proportion of muscle fibers with normal respiratory chain activity and in muscle fiber cross-sectional area after a short period of concentric exercise training. These data show that it is possible to reverse the molecular events that led to expression of metabolic myopathy and demonstrate the effectiveness of this form of 'gene shifting' therapy.  (+info)

Complex evolutionary patterns of tRNA Leu(UAA) group I introns in the cyanobacterial radiation [corrected]. (7/292)

Based on the findings that plastids and cyanobacteria have similar group I introns inserted into tRNAUAALeu genes, these introns have been suggested to be immobile and of ancient origin. In contrast, recent evidence suggests lateral transfer of cyanobacterial group I introns located in tRNAUAALeu genes. In light of these new findings, we have readdressed the evolution and lateral transfer of tRNAUAALeu group I introns in cyanobacteral radiation. We determined the presence of introns in 38 different strains, representing the major cyanobacterial lineages, and characterized the introns in 22 of the strains. Notably, two of these strains have two tRNAUAALeu genes, with each of these genes interrupted by introns, while three of the strains have both interrupted and uninterrupted genes. Two evolutionary distinct clusters of tRNA genes, with the genes interrupted by introns belonging to two distinct intron clusters, were identified. We also compared 16S rDNA and intron evolution for both closely and distantly related strains. The distribution of the introns in the clustered groups, as defined from 16S rDNA analysis, indicates relatively recent gain and/or loss of the introns in some of these lineages. The comparative analysis also suggests differences in the phylogenetic trees for 16S rDNA and the tRNAUAALeu group I introns. Taken together, our results show that the evolution of the intron is considerably more complex than previous studies found to be the case. We discuss, based on our results, evolutionary models involving lateral intron transfer and models involving differential loss of the intron.  (+info)

Activation of Escherichia coli leuV transcription by FIS. (8/292)

The transcription factor FIS has been implicated in the regulation of several stable RNA promoters, including that for the major tRNALeu species in Escherichia coli, tRNA1Leu. However, no evidence for direct involvement of FIS in tRNA1Leu expression has been reported. We show here that FIS binds to a site upstream of the leuV promoter (centered at -71) and that it directly stimulates leuV transcription in vitro. A mutation in the FIS binding site reduces transcription from a leuV promoter in strains containing FIS but has no effect on transcription in strains lacking FIS, indicating that FIS contributes to leuV expression in vivo. We also find that RNA polymerase forms an unusual heparin-sensitive complex with the leuV promoter, having a downstream protection boundary of approximately -7, and that the first two nucleotides of the transcript, GTP and UTP, are required for formation of a heparin-stable complex that extends downstream of the transcription start site. These studies have implications for the regulation of leuV transcription.  (+info)