Unwinding by Local Strand Separation Is Critical for the Function of DEAD-Box Proteins as RNA Chaperones

The DEAD-box proteins CYT-19 in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are broadly acting RNA chaperones that function in mitochondria to stimulate group I and group II intron splicing and to activate mRNA translation. Previous studies showed that the S. cerevisiae cytosolic/nuclear DEAD-box protein Ded1p could stimulate group II intron splicing in vitro. Here, we show that Ded1p complements mitochondrial translation and group I and group II intron splicing defects in mss116Δ strains, stimulates the in vitro splicing of group I and group II introns, and functions indistinguishably from CYT-19 to resolve different nonnative secondary and/or tertiary structures in the Tetrahymena thermophila large subunit rRNA-ΔP5abc group I intron. The Escherichia coli DEAD-box protein SrmB also stimulates group I and group II intron splicing in vitro, while the E. coli DEAD-box protein DbpA and the vaccinia virus DExH-box protein NPH-II gave little, if any, group I or group II intron splicing stimulation in vitro or in vivo. The four DEAD-box proteins that stimulate group I and group II intron splicing unwind RNA duplexes by local strand separation and have little or no specificity, as judged by RNA-binding assays and stimulation of their ATPase activity by diverse RNAs. In contrast, DbpA binds group I and group II intron RNAs nonspecifically, but its ATPase activity is activated specifically by a helical segment of E. coli 23S rRNA, and NPH-II unwinds RNAs by directional translocation. The ability of DEAD-box proteins to stimulate group I and group II intron splicing correlates primarily with their RNA-unwinding activity, which, for the protein preparations used here, was greatest for Mss116p, followed by Ded1p, CYT-19, and SrmB. Furthermore, this correlation holds for all group I and group II intron RNAs tested, implying a fundamentally similar mechanism for both types of introns. Our results support the hypothesis that DEAD-box proteins have an inherent ability to function as RNA chaperones by virtue of their distinctive RNA-unwinding mechanism, which enables refolding of localized RNA regions or structures without globally disrupting RNA structure.     

Association of the eukaryotic V1VO ATPase subunits a with d and d with A

Owing to the complex nature of V₁V_O ATPases, identification of neighboring subunits is essential for mechanistic understanding of this enzyme. Here, we describe the links between the V₁ headpiece and the V_O-domain of the yeast V₁V_O ATPase via subunit A and d as well as the V_O subunits a and d using surface plasmon resonance and fluorescence correlation spectroscopy. Binding constants of about 60 and 200 nM have been determined for the a-d and d-A assembly, respectively. The data are discussed in light of subunit a and d forming a peripheral stalk, connecting the catalytic A₃B₃ hexamer with V_O.

Structured summary

MINT-7012054: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by fluorescence correlation spectroscopy (MI:0052)MINT-7012041: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by surface plasmon resonance (MI:0107)MINT-7012028: d (uniprotkb:P32366) binds (MI:0407) to a (uniprotkb:P32563) by surface plasmon resonance (MI:0107)     

Hepatitis C virus NS5A protein modulates template selection by the RNA polymerase in in vitro system

Hepatitis C virus (HCV) NS5A phosphoprotein is a component of virus replicase. Here we demonstrate that in vitro unphosphorylated NS5A protein inhibits HCV RNA-dependent RNA polymerase (RdRp) activity in polyA-oligoU system but has little effect on synthesis of viral RNA. The phosphorylated casein kinase (CK) II NS5A protein causes the opposite effect on RdRp in each of these systems. The phosphorylation of NS5A protein with CKII does not affect its affinity to the HCV RdRp and RNA. The NS5A phosphorylation with CKI does not change the RdRp activity. Herein we report evidence that the NS5A prevents template binding to the RdRp.

Structured summary

MINT-6803697: CKI (uniprotkb:P97633) phosphorylates (MI:0217) NS5A (uniprotkb:P26662) by protein kinase assay (MI:0424)MINT-6803713: CKII (uniprotkb:P67870) phosphorylates (MI:0217) NS5A (uniprotkb:P26662) by protein kinase assay (MI:0424)     

CDF it all: Consensus prediction of intrinsically disordered proteins based on various cumulative distribution functions

Many biologically active proteins are intrinsically disordered. A reasonable understanding of the disorder status of these proteins may be beneficial for better understanding of their structures and functions. The disorder contents of disordered proteins vary dramatically, with two extremes being fully ordered and fully disordered proteins. Often, it is necessary to perform a binary classification and classify a whole protein as ordered or disordered. Here, an improved error estimation technique was applied to develop the cumulative distribution function (CDF) algorithms for several established disorder predictors. A consensus binary predictor, based on the artificial neural networks, NN-CDF, was developed by using output of the individual CDFs. The consensus method outperforms the individual predictors by 4-5% in the averaged accuracy.     

Regulatory volume decrease in cardiomyocytes is modulated by calcium influx and reactive oxygen species

We investigated the role of Ca²⁺ in generating reactive oxygen species (ROS) induced by hyposmotic stress (Hypo) and its relationship to regulatory volume decrease (RVD) in cardiomyocytes. Hypo-induced increases in cytoplasmic and mitochondrial Ca²⁺. Nifedipine (Nife) inhibited both Hypo-induced Ca²⁺ and ROS increases. Overexpression of catalase (CAT) induced RVD and a decrease in Hypo-induced blebs. Nife prevented CAT-dependent RVD activation. These results show a dual role of Hypo-induced Ca²⁺ influx in the control of cardiomyocyte viability. Hypo-induced an intracellular Ca²⁺ increase which activated RVD and inhibited necrotic blebbing thus favoring cell survival, while simultaneously increasing ROS generation, which in turn inhibited RVD and induced necrosis.     

Genetic tests of rapid parallel speciation of flightless birds from an extant volant ancestor

Prior to the extinction wave that followed the human colonization of Oceania, flightless rails (Aves: Rallidae) were among the largest radiations of island birds, and perhaps the most species-rich example of convergent evolution in vertebrates. Insular flightless species are thought to have evolved from extant, volant species that colonized from continental sources and rapidly followed parallel adaptive pathways to flightlessness. The present study provides the first test of this model of speciation using genetic data sampled throughout the range of a putative ancestral species. Mitochondrial control region sequences from 71 individuals of the Gallirallus philippensis species complex reveal essentially no geographic structure within archipelagos and only weak structure among archipelagos, with no major genetic breaks except for birds sampled in the Philippines. Demographic tests of coalescent models support a recent rapid expansion into Oceania (including Australia) out of the Philippines approximately 20 000 years ago. The estimated coalescence of G. philippensis mitochondrial alleles approximately 33 000 years ago closely corresponds to the expansion of humans into the archipelagoes of Near Oceania, suggesting that humans may have facilitated its colonization by exterminating flightless competitors and clearing lowland forests. Phylogenetic analyses that included all G. philippensis haplotypes and samples from 11 single-island endemic flightless species of Gallirallus indicate that G. philippensis is polyphyletic, but is not the ancestor of most of its flightless congeners, as previously thought. Nuclear gene sequences (β-actin inron 3) suggest that G. philippensis polyphyly is at least partly due to hybridization. The flightless condition evolves in rails before reproductive isolation is complete.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 601–616.     

The reaction force and the transition region of a reaction

The reaction force F(R) and the position-dependent reaction force constant κF(R) are defined by F(R)=-∂V(R)/∂R and κ(R)=∂²V(R)/∂R², where V(R) is the potential energy of a reacting system along a coordinate R. The minima and maxima of F(R) provide a natural division of the process into several regions. Those in which F(R) is increasing are where the most dramatic changes in electronic properties take place, and where the system goes from activated reactants (at the force minimum) to activated products (at the force maximum). κ(R) is negative throughout such a region. We summarize evidence supporting the idea that a reaction should be viewed as going through a transition region rather than through a single point transition state. A similar conclusion has come out of transition state spectroscopy. We describe this region as a chemically-active, or electronically-intensive, stage of the reaction, while the ones that precede and follow it are structurally-intensive. Finally, we briefly address the time dependence of the reaction force and the reaction force constant.     

雀麦上雀麦腥黑粉菌Tilletia bromi的分子检测

雀麦是重要的牧草,近年来进口量激增。寄生于雀麦上的腥黑粉菌共有4种,即小麦矮腥黑穗菌Tilletia controversa(TCK)、雀麦腥黑粉菌T.bromi、T.bolayi和小麦网腥黑穗菌T.caries(TCT),根据冬孢子形态难以直接区分这些种类。本文在形态、自发荧光和萌发生理三方面的比较研究基础上,依据beta-微管蛋白tub2基因序列设计一套引物,转化为特异SCAR分子标记,建立了雀麦上T.bromi的菌丝基因组DNA的特异PCR检测方法和冬孢子的套式特异PCR检测方法,为病害提供了快速、可靠的检测方法。