Exahustive degradation of nucleotide triphosphates
Ontology highlight
ABSTRACT: The degradation kinetics of nucleotide triphosphates (ATP, GTP, UTP and CTP) were evaluated under boiling ethanol extraction conditions (95°C) during 0 to 300 minutes.
Project description:The effect of the Verduyn culture medium on the degradation profiles of nucleotide triphosphates extracted under typical boiling ethanol conditions was evaluated.
Project description:The influence of particular groups of compounds/metabolites, mainly comprising major central carbon metabolites including amino acids, organic acids, sugar phosphates, coenzymes, etc, on the degradation profiles of nucleotide triphosphates extracted under typical boiling ethanol conditions was evaluated.
Project description:The effect of a complex cellular matrix extracted from yeast (S. cerevisiae, strain YSBN6 (MATa; genotype: FY3 ho::HphMX4 derived from the S288C parental strain)) on the degradation profiles of nucleotide triphosphates extracted under typical boiling ethanol conditions was evaluated.
Project description:RNA synthesis and decay rates determine the steady-state levels of cellular RNAs. Metabolic tagging of newly transcribed RNA by 4-thiouridine (4sU) can reveal the relative contributions of RNA synthesis and decay rates. The kinetics of RNA processing, however, so far remained unresolved. Here, we show that ultra-short 4sU-tagging not only provides snap-shot pictures of eukaryotic gene expression but, when combined with progressive 4sU-tagging and RNA-seq, reveals global RNA processing kinetics at nucleotide resolution. Using this method, we identified classes of rapidly and slowly spliced/degraded introns. Interestingly, each class of splicing kinetics was characterized by a distinct association with intron length, gene length and splice site strength. For a large group of introns, we also observed long lasting retention in the primary transcript, but efficient secondary splicing or degradation at later time points. Finally, we show that processing of most, but not all small nucleolar (sno)RNA-containing introns is remarkably inefficient with the majority of introns being spliced and degraded rather than processed into mature snoRNAs. In summary, our study yields unparalleled insights into the kinetics of RNA processing and provides the tools to study molecular mechanisms of RNA processing and their contribution to the regulation of gene expression. 4sU-tagging was performed in human DG75 B-cells by adding 500 uM 4sU to cell culture medium for 5, 10, 15, 20 or 60 min. Following isolation of total cellular RNA, this was separated into nascent and untagged, pre-existing RNA. Nascent RNA as well as total and untagged RNA from 60 min 4sU-tagging were subjected to SOLiD sequencing (SOLiD II) obtaining 35 nt reads.
Project description:AAA+ ATPases constitute a large family of proteins that are involved in a plethora of cellular processes including DNA disassembly, protein degradation and protein complex disassembly. They typically form a hexametric ring-shaped structure with six subunits in a (pseudo) six-fold symmetry. In a subset of AAA+ ATPases that facilitate protein unfolding and degradation, six subunits cooperate to translocate protein substrates through a central pore in the ring. The number and type of nucleotides in an AAA+ ATPase hexamer is inherently linked to the mechanism that underlies cooperation among subunits and couples ATP hydrolysis with substrate translocation. We conducted a native mass spectrometry study of a monodispersed form of PAN, an archaeal proteasome AAA+ ATPase, to determine the number of nucleotides bound to each hexamer of the wild-type protein. We utilized ADP and its analogues (TNP-ADP and mant-ADP), and a non-hydrolyzable ATP analogue (AMP-PNP) to study nucleotide site occupancy within the PAN hexamer in ADP- and ATP-binding states, respectively. Throughout all experiments we used a Walker A mutant (PANK217A) that is impaired in nucleotide binding as an internal standard to mitigate the effects of residual solvation on mass measurement accuracy and to serve as a reference protein to control for non-specific nucleotide binding. This approach led to the unambiguous finding that a wild-type PAN hexamer carried, from expression host six tightly bound ADP molecules that could be exchanged for ADP and ATP analogues. While the Walker A mutant did not bind ADP analogues, it did bind AMP-PNP, albeit at multiple stoichiometries. We observed variable levels of hexamer dissociation and an appearance of multimeric species with the over-charged molecular ion distributions across repeated experiments. We posit that these phenomena originated during ESI process at the final stages of ESI droplet evolution.
Project description:RNA synthesis and decay rates determine the steady-state levels of cellular RNAs. Metabolic tagging of newly transcribed RNA by 4-thiouridine (4sU) can reveal the relative contributions of RNA synthesis and decay rates. The kinetics of RNA processing, however, so far remained unresolved. Here, we show that ultra-short 4sU-tagging not only provides snap-shot pictures of eukaryotic gene expression but, when combined with progressive 4sU-tagging and RNA-seq, reveals global RNA processing kinetics at nucleotide resolution. Using this method, we identified classes of rapidly and slowly spliced/degraded introns. Interestingly, each class of splicing kinetics was characterized by a distinct association with intron length, gene length and splice site strength. For a large group of introns, we also observed long lasting retention in the primary transcript, but efficient secondary splicing or degradation at later time points. Finally, we show that processing of most, but not all small nucleolar (sno)RNA-containing introns is remarkably inefficient with the majority of introns being spliced and degraded rather than processed into mature snoRNAs. In summary, our study yields unparalleled insights into the kinetics of RNA processing and provides the tools to study molecular mechanisms of RNA processing and their contribution to the regulation of gene expression.
Project description:Outbred D.melanogaster populations subjected to >300 generations of natural selection on either control, or 12% ethanol, or variable food (2 replicates each) and exposed, as first instar larvae, to either water control or 12% ethanol.
Project description:We sequenced microRNA from heads of two D. melanogaster populations: an ethanol treated group (30 minutes after sedation with ethanol saturated vapor), and an age-matched untreated control group; and calculated differential microRNA expression between the two groups.