370,804 research outputs found
G-quadruplex formation of FXYD1 pre-mRNA indicates the possiblity of regulating expression of its protein product
G-quadruplexes are higher-order nucleic acid structures formed of square-planar arrangements of four guanine bases held together by Hoogsteen-type hydrogen bonds. Stacks of guanine tetrads are stabilised by intercalating potassium ions. FXYD1 encodes for phospholemman, a regulatory subunit of the cardiac Na+/K+-ATPase. Computational sequence analysis of FXYD1 pre-mRNA predicted the formation of stable intramolecular G-quadruplexes in human and orthologue sequences. Multiple sequence alignment indicated that G-rich sequences are conserved in evolution suggesting a potential role of G-quadruplexes in FXYD1 gene expression. The existence of a non-functional alternative splicing product indicated that the G-quadruplex formation may control alternative splicing. Quadruplex formation of human and bovine oligonucleotides was confirmed in vitro by native polyacrylamide gel electrophoresis and intrinsic fluorescence emission spectroscopy. Taking together the evolutionary conservation of G-quadruplex forming sequences with the confirmation of G-quadruplex formation in vitro by two FXYD1 homologues the results point to a potential role of these structures in regulating the expression of FXYD1 and thus regulate indirectly the activity of the cardiac Na+/K+ -ATPase.Peer reviewe
Glutamine Phosphoribosylpyrophosphate Amidotransferase-independent Phosphoribosyl Amine Synthesis from Ribose 5-Phosphate and Glutamine or Asparagine
Phosphoribosylamine (PRA) is the first intermediate in the common pathway to purines and thiamine and is generated in bacteria by glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase (EC 2.4.2.14) from PRPP and glutamine. Genetic data have indicated that multiple, non-PRPP amidotransferase mechanisms exist to generate PRA sufficient for thiamine but not purine synthesis. Here we describe the purification and identification of an activity (present in both Escherichia coli and Salmonella enterica) that synthesizes PRA from ribose 5-phosphate and glutamine/asparagine. A purification resulting in greater than a 625-fold increase in specific activity identified 8 candidate proteins. Of the candidates, overexpression of AphA (EC 3.1.3.2), a periplasmic class B nonspecific acid phosphatase, significantly increased activity in partially purified extracts. Native purification of AphA to >95% homogeneity determined that the periplasmic L-asparaginase II, AnsB (EC 3.5.1.1), co-purified with AphA and was also necessary for PRA formation. The potential physiological relevance of AphA and AnsB in contributing to thiamine biosynthesis in vivo is discussed
Dynamics of Electron Transport in Cytochrome P450 Systems Studied at Sub-Zero Temperature
Experimentation in fluid mixed solvents (1 : 1 v/v phosphate
buffer ethylene glycol) at sub-zero temperatures has permitted us
to record the two univalent reductions of the bacterial cytochrome
P450 by the natural electron donor putidaredoxin, without recycling
or alternative pathway reactions. Dynamic evidence shows the formation of putidaredoxincytochrome complexes prior to electron
transfer. The complex formation is rate limiting in the first reduction
and in our experimental conditions. The kinetics of binding
between the two oxidized proteins has also been recorded in the
same medium under various conditions of concentration, temperature
and ionic strength. At very low ionic strength, the rate is
limited by electrostatic repulsion between the two negatively charge
proteins; above I = 0.03 this effect appears negligible and the affinity
seems to be governed by hydrophobic interaction free energy
Elongation Factor TFIIS Prevents Transcription Stress and R-Loop Accumulation to Maintain Genome Stability
Although correlations between RNA polymerase II (RNAPII) transcription stress, R-loops, and genome instability have been established, the mechanisms underlying these connections remain poorly understood. Here, we used a mutant version of the transcription elongation factor TFIIS (TFIISmut), aiming to specifically induce increased levels of RNAPII pausing, arrest, and/or backtracking in human cells. Indeed, TFIISmut expression results in slower elongation rates, relative depletion of polymerases from the end of genes, and increased levels of stopped RNAPII; it affects mRNA splicing and termination as well. Remarkably, TFIISmut expression also dramatically increases R-loops, which may form at the anterior end of backtracked RNAPII and trigger genome instability, including DNA strand breaks. These results shed light on the relationship between transcription stress and R-loops and suggest that different classes of R-loops may exist, potentially with distinct consequences for genome stability.Cancer Research UK FC001166UK Medical Research Council FC001166Wellcome Trust FC001166European Research Council 693327, ERC2014 AdG669898Ministerio de Economía y Competitividad BFU2013-42918-P, BFU2016-75058-
Toward an Alternative Intrinsic Probe for Spectroscopic Characterization of a Protein
The intrinsic fluorescent amino acid tryptophan is the unanimous choice for the spectroscopic investigation
of proteins. However, several complicacies in the interpretation of tryptophan fluorescence in a protein are
inevitable and an alternative intrinsic protein probe is a longstanding demand. In this contribution, we report
an electron-transfer reaction in a human transporter protein (HSA) cavity which causes the tryptophan residue
(Trp214) to undergo chemical modification to form one of its metabolites kynurenine (Kyn214). Structural
integrity upon modification of the native protein is confirmed by dynamic light scattering (DLS) as well as
near and far circular dichroism (CD) spectroscopy. Femtosecond-resolved fluorescence transients of the modified
protein describe the dynamics of solvent molecules in the protein cavity in both the native and denatured
states. In order to establish general use of the probe, we have studied the dipolar interaction of Kyn214 with
a surface-bound ligand (crystal violet, CV) of the protein. By using the sensitivity of FRET, we have determined
the distance between Kyn214 (donor) and CV (acceptor). Our study is an attempt to explore an alternative
intrinsic fluorescence probe for the spectroscopic investigation of a protein. In order to establish the efficacy
of the modification technique we have converted the tryptophan residues of other proteins (bovine serum
albumin, chymotrypsin and subtilisin Carlsberg) to kynurenine and confirmed their structural integrity. We
have also shown that catalytic activity of the enzymes remains intact upon the modification
The Bentonite Barrier - Swelling Properties, Redox Chemistry and Mineral Evolution
Bentonite is planned for use as a buffer material in high-level radioactive waste repositories, where safety assessment is performed for very long periods (100-1000 ka). This thesis focuses on the swelling of smectites in liquid water, and analysis of bentonite from field experiments at Äspö Hard Rock Laboratory, Sweden. Four field experiments were analyzed (Alternative Buffer Material experiment, ABM1, ABM2; Temperature Buffer Test, TBT; and Prototype) with focus on Fe- redox chemistry and formation of trioctahedral smectite. The techniques used were mainly synchrotron X-ray diffraction and X-ray absorption spectroscopy. In ABM1 and Prototype the Fe(II)/Fe-total ratio had increased. In TBT no significant increase in Fe(II) was found; instead the corrosion products were dominated by Fe(III). Formation of trioctahedral clays was found in the iron-bentonite experiments (ABM1, ABM2, TBT), but not in Prototype where the heater instead was of copper. In swelling experiments, Ca-Wyoming montmorillonite was shown to expand and partly form a four-water-layer hydrate at lower temperatures in water. This was studied in more detail, and the influence of divalent interlayer cation, temperature, layer charge, salt and irradiation was investigated. Among the investigated smectites, decreased temperature increased the crystalline swelling until ice was formed. Lower smectite layer charge increased the crystalline swelling. Increasing the Gibbs hydration energy of the divalent interlayer cation increased the crystalline swelling. Introduction of salt in the water partly dehydrated the montmorillonite at 20°C, but minimized the dehydration of montmorillonite upon ice formation at low temperatures (-50, -100°C), especially with CaCl2. It was found that in a gradient of ethylene glycol and water a 21 Å basal distance was formed in the montmorillonite, which was higher than in the pure liquids
The actinobacterial transcription factor RbpA binds to the principal sigma subunit of RNA polymerase
RbpA is a small non-DNA-binding transcription factor that associates with RNA polymerase holoenzyme and stimulates transcription in actinobacteria, including Streptomyces coelicolor and Mycobacterium tuberculosis. RbpA seems to show specificity for the vegetative form of RNA polymerase as opposed to alternative forms of the enzyme. Here, we explain the basis of this specificity by showing that RbpA binds directly to the principal σ subunit in these organisms, but not to more diverged alternative σ factors. Nuclear magnetic resonance spectroscopy revealed that, although differing in their requirement for structural zinc, the RbpA orthologues from S. coelicolor and M. tuberculosis share a common structural core domain, with extensive, apparently disordered, N- and C-terminal regions. The RbpA-σ interaction is mediated by the C-terminal region of RbpA and σ domain 2, and S. coelicolor RbpA mutants that are defective in binding σ are unable to stimulate transcription in vitro and are inactive in vivo. Given that RbpA is essential in M. tuberculosis and critical for growth in S. coelicolor, these data support a model in which RbpA plays a key role in the σ cycle in actinobacteria
Inactivation of mammalian Ero 1α is catalysed by specific protein disulfide isomerases
Disulfide formation within the endoplasmic reticulum is a complex process requiring a disulfide exchange protein such as protein disulfide isomerase and a mechanism to form disulfides de novo. In mammalian cells, the major pathway for de novo disulfide formation involves the enzyme Ero1α which couples oxidation of thiols to the reduction of molecular oxygen to form hydrogen peroxide. Ero1α activity is tightly regulated by a mechanism that requires the formation of regulatory disulfides. These regulatory disulfides are reduced to activate and reform to inactive the enzyme. To investigate the mechanism of inactivation we analysed regulatory disulfide formation in the presence of various oxidants under controlled oxygen concentration. Neither molecular oxygen, nor hydrogen peroxide was able to oxidise Ero1α efficiently to form the correct regulatory disulfides. However, specific members of the PDI family such as PDI or ERp46 were able to catalyse this process. Further studies showed that both active sites of PDI contribute to the formation of regulatory disulfides in Ero1α and that the PDI substrate binding domain is crucial to allow electron transfer between the two enzymes. These results demonstrate a simple feedback mechanism of regulation of mammalian Ero1α involving its primary substrate
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