Rapid and accurate translation of the genetic code into protein is fundamental to life. Yet due to lack of a suitable assay, little is known about the accuracy-determining parameters and their ...correlation with translational speed. Here, we develop such an assay, based on Mg2+ concentration changes, to determine maximal accuracy limits for a complete set of single-mismatch codon–anticodon interactions. We found a simple, linear trade-off between efficiency of cognate codon reading and accuracy of tRNA selection. The maximal accuracy was highest for the second codon position and lowest for the third. The results rationalize the existence of proofreading in code reading and have implications for the understanding of tRNA modifications, as well as of translation error-modulating ribosomal mutations and antibiotics. Finally, the results bridge the gap between in vivo and in vitro translation and allow us to calibrate our test tube conditions to represent the environment inside the living cell.
Pompe disease is a severe form of muscular dystrophy due to glycogen accumulation in all tissues, especially striated muscle. Disease severity is directly related to the deficiency of acid ...α-glucosidase (GAA), which degrades glycogen in the lysosome. Respiratory dysfunction is a hallmark of the disease, muscle weakness has been viewed as the underlying cause, and the possibility of an associated neural contribution has not been evaluated previously. Therefore, we examined behavioral and neurophysiological aspects of breathing in 2 animal models of Pompe disease--the Gaa⁻/⁻ mouse and a transgenic line (MTP) expressing GAA only in skeletal muscle, as well as a detailed analysis of the CNS in a Pompe disease patient. Glycogen content was elevated in the Gaa⁻/⁻ mouse cervical spinal cord. Retrograde labeling of phrenic motoneurons showed significantly greater soma size in Gaa⁻/⁻ mice vs. isogenic controls, and glycogen was observed in Gaa⁻/⁻ phrenic motoneurons. Ventilation, assessed via plethysmography, was attenuated during quiet breathing and hypercapnic challenge in Gaa⁻/⁻ mice (6 to >21 months of age) vs. controls. We confirmed that MTP mice had normal diaphragmatic contractile properties; however, MTP mice had ventilation similar to the Gaa⁻/⁻ mice during quiet breathing. Neurophysiological recordings indicated that efferent phrenic nerve inspiratory burst amplitudes were substantially lower in Gaa⁻/⁻ and MTP mice vs. controls. In human samples, we demonstrated similar pathology in the cervical spinal cord and greater accumulation of glycogen in spinal cord compared with brain. We conclude that neural output to the diaphragm is deficient in Gaa⁻/⁻ mice, and therapies targeting muscle alone may be ineffective in Pompe disease.
Nanoscale Transistors—Just Around the Gate? Cress, Cory D.; Datta, Suman
Science (American Association for the Advancement of Science),
07/2013, Letnik:
341, Številka:
6142
Journal Article
Recenzirano
Advanced geometries for gate electrodes that reduce current leakage can decrease the size of high-performance transistors.
Further reduction in the size of the metaloxide semiconductor field-effect ...transistors (MOSFETs) used in computer chips will require more complex geometries to enhance the gate control of the current flow in the transistor channel (
1
). These advanced designs allow transistor scaling (maintaining performance as size decreases) and minimize the leakage of current when the device is in the off-state. The voltage of operation can be reduced without loss of performance, making the devices function with less power dissipation per operation. The optimal MOSFET geometry surrounds a cylindrical channel with the gate electrode (
2
). This “gate-all-around” (GAA) enhances electrostatic control of the entire channel surface (see the figure), and when used with superior charge transport materials, should deliver enhanced performance. Franklin
et al.
(
3
) now report on nanoscale complementary MOSFETs in which suspended single-walled carbon nanotubes (SWCNTs) form the channel with a GAA geometry. This work marks a milestone in moving SWCNT nanoelectronics from laboratory prototypes toward a manufacturable technology.
tRNA modifications are crucial to ensure translation efficiency and fidelity. In eukaryotes, the URM1 and ELP pathways increase cellular resistance to various stress conditions, such as nutrient ...starvation and oxidative agents, by promoting thiolation and methoxycarbonylmethylation, respectively, of the wobble uridine of cytoplasmic $tRNA_{Lys}^{UUU}$ (tKUUU), $tRNA_{G\ln }^{UUG}$ (tQUUG), and $tRNA_{Glu}^{UUC}$ (tEUUC). Although in vitro experiments have implicated these tRNA modifications in modulating wobbling capacity and translation efficiency, their exact in vivo biological roles remain largely unexplored. Using a combination of quantitative proteomics and codon-specific translation reporters, we find that translation of a specific gene subset enriched for AAA, CAA, and GAA codons is impaired in the absence of URM1-and ELP-dependent tRNA modifications. Moreover, in vitro experiments using native tRNAs demonstrate that both modifications enhance binding of tKUUU to the ribosomal A-site. Taken together, our data suggest that tRNA thiolation and methoxycarbonylmethylation regulate translation of genes with specific codon content.
Two-codon T-box riboswitch binding two tRNAs Saad, Nizar Y.; Stamatopoulou, Vassiliki; Brayé, Mélanie ...
Proceedings of the National Academy of Sciences - PNAS,
07/2013, Letnik:
110, Številka:
31
Journal Article
Recenzirano
Odprti dostop
T-box riboswitches control transcription of downstream genes through the tRNA-binding formation of terminator or antiterminator structures. Previously reported T-boxes were described as ...single-specificity riboswitches that can bind specific tRNA anticodons through codon–anticodon interactions with the nucleotide triplet of their specifier loop (SL). However, the possibility that T-boxes might exhibit specificity beyond a single tRNA had been overlooked. In Clostridium acetobutylicum , the T-box that regulates the operon for the essential tRNA-dependent transamidation pathway harbors a SL with two potential overlapping codon positions for tRNA ᴬˢⁿ and tRNA ᴳˡᵘ. To test its specificity, we performed extensive mutagenic, biochemical, and chemical probing analyses. Surprisingly, both tRNAs can efficiently bind the SL in vitro and in vivo. The dual specificity of the T-box is allowed by a single base shift on the SL from one overlapping codon to the next. This feature allows the riboswitch to sense two tRNAs and balance the biosynthesis of two amino acids. Detailed genomic comparisons support our observations and suggest that “flexible” T-box riboswitches are widespread among bacteria, and, moreover, their specificity is dictated by the metabolic interconnection of the pathways under control. Taken together, our results support the notion of a genome-dependent codon ambiguity of the SLs. Furthermore, the existence of two overlapping codons imposes a unique example of tRNA-dependent regulation at the transcriptional level.
cDNA microarray technology has been increasingly used to monitor global gene expression patterns in various tissues and cell types. However, applications to mammalian development have been hampered ...by the lack of appropriate cDNA collections, particularly for early developmental stages. To overcome this problem, a PCR-based cDNA library construction method was used to derive 52,374 expressed sequence tags from pre- and peri-implantation embryos, embryonic day (E) 12.5 female gonad/mesonephros, and newborn ovary. From these cDNA collections, a microarray representing 15,264 unique genes (78% novel and 22% known) was assembled. In initial applications, the divergence of placental and embryonic gene expression profiles was assessed. At stage E12.5 of development, based on triplicate experiments, 720 genes (6.5%) displayed statistically significant differences in expression between placenta and embryo. Among 289 more highly expressed in placenta, 61 placenta-specific genes encoded, for example, a novel prolactin-like protein. The number of genes highly expressed (and frequently specific) for placenta has thereby been increased 5-fold over the total previously reported, illustrating the potential of the microarrays for tissue-specific gene discovery and analysis of mammalian developmental programs.
Methyl farnesoate (MF), an analogue of the insect juvenile hormone III, is believed to play important roles in the regulation of the growth and reproductive development in crustaceans. Farnesoic acid ...O-methyltransferase (FAMeT) is the key enzyme in the juvenile hormone biosynthetic pathway, involved in the conversion of farnesoic acid (FA) to MF in the final step of MF synthesis. In this study, a FAMeT cDNA (named EcFAMeT) was cloned from the hemocytes of ridgetail white prawn Exopalaemon carinicauda by rapid amplification of cDNA ends (RACE) methods. The fulllength cDNA of EcFAMeT was 1,620 bp, including contains a 5'-untranslated region (UTR) of 75 bp, 3'-UTR of 714 bp with a poly (A) tail, an open reading frame (ORF) of 831 bp, encoding a 276-amino-acid polypeptide with the predicted molecular weight of 31.57 kDa and estimated isoelectric point of 4.67. BLAST analysis revealed that amino acids of EcFAMeT shared high identity (75-90 %) with that of other crustaceans. Two conserved signatures domains of Methyltransf-FA superfamily were also identified in EcFAMeT. Real time quantitative RT-PCR analysis indicated that EcFAMeT could be detected in all the tested tissues and strongly expressed in hepatopancreas and ovary of E. carinicauda. After Vibrio anguillarum and WSSV challenge, EcFAMeT transcripts both in hemocytes and hepatopancreas increased significantly in the first 3 h, respectively. The results indicated that EcFAMeT might be associated with the immune defenses to V. anguillarum and WSSV in E. carinicauda.
Type I polyketide synthases (PKSs) are multifunctional enzymes that are organized into modules, each of which minimally contains a β-ketoacyl synthase, an acyltransferase (AT), and an acyl carrier ...protein. Here we report that the leinamycin (LNM) biosynthetic gene cluster from Streptomyces atroolivaceus S-140 consists of two PKS genes, lnmI and lnmJ, that encode six PKS modules, none of which contain the cognate AT domain. The only AT activity identified within the lnm gene cluster is a discrete AT protein encoded by lnmG. Inactivation of lnmG, lnmI, or lnmJ in vivo abolished LNM biosynthesis. Biochemical characterization of LnmG in vitro showed that it efficiently and specifically loaded malonyl CoA to all six PKS modules. These findings unveiled a previously unknown PKS architecture that is characterized by a discrete, iteratively acting AT protein that loads the extender units in trans to "AT-less" multifunctional type I PKS proteins for polyketide biosynthesis. This PKS structure provides opportunities for PKS engineering as exemplified by overexpressing lnmG to improve LNM production.
This article examines the development of different forms of spectator violence in terms of the socio-temporal structure of situational dynamics at Gaelic football matches in Ireland. The nature of ...violent encounters has shifted from a collective form based on local solidarity and a reciprocal code of honour, through a transitional collective form based on deferred emotional satisfaction and group pride, towards increasing individualization of spectator violence. This occurs due to the shifting objects of emotional involvement. As the functional specialization of the various roles in the game is partially accepted by spectators, the referee becomes the target of anger. Violence becomes more individualized as 'mutually expected self-restraint' proceeds within the context of relative state pacification beyond the field of play and the formation of a less volatile habitus. We use Elias's figurational perspective on violence over the micro-interactional approach of Randall Collins, but support Collins' emphasis on state legitimacy.
The Class Bdelloidea of the Phylum Rotifera is the largest metazoan taxon in which males, hermaphrodites, and meiosis are unknown. We conducted a molecular genetic test of this indication that ...bdelloid rotifers may have evolved without sexual reproduction or genetic exchange. The test is based on the expectation that after millions of years without these processes, genomes will no longer contain pairs of closely similar haplotypes and instead will contain highly divergent descendants of formerly allelic nucleotide sequences. We find that genomes of individual bdelloid rotifers, representing four different species, appear to lack pairs of closely similar sequences and contain representatives of two ancient lineages that began to diverge before the bdelloid radiation many millions of years ago when sexual reproduction and genetic exchange may have ceased.
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