Telomerase RNA (TR) conformation determines its function as a template for telomere synthesis and as a scaffold for the assembly of the telomerase nucleoprotein complex. Experimental analyses of TR secondary structure using DMS-Map Seq and SHAPE-Map Seq techniques show its CLOSED conformation as the consensus structure where the template region cannot perform its function. Our data show that the apparent discrepancy between experimental results and predicted TR functional conformation, mostly ignored in published studies, can be explained using data analysis based on single-molecule structure prediction from individual sequencing reads by the recently established DaVinci method. This method results in several clusters of secondary structures reflecting the structural dynamics of TR, possibly related to its multiple functional states. Interestingly, the presumed active (OPEN) conformation of TR corresponds to a minor fraction of TR under in vivo conditions. Therefore, structural polymorphism and dynamic TR transitions between CLOSED and OPEN conformations may be involved in telomerase activity regulation as a switch that functions independently of total TR transcript levels.

• Keywords
• DMS-Map Seq, RNA secondary structure, SHAPE-Map Seq, single molecule analysis, telomerase RNA,
• MeSH
• Nucleic Acid Conformation MeSH
• Bryopsida * MeSH
• RNA * chemistry   genetics   MeSH
• Telomerase * chemistry   genetics   MeSH
• Single Molecule Imaging MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA * MeSH
• Telomerase * MeSH
• telomerase RNA MeSH Browser

Hydrolysis of ATP by Na+/K+-ATPase, a P-Type ATPase, catalyzing active Na+ and K+ transport through cellular membranes leads transiently to a phosphorylation of its catalytical α-subunit. Surprisingly, three-dimensional molecular structure analysis of P-type ATPases reveals that binding of ATP to the N-domain connected by a hinge to the P-domain is much too far away from the Asp369 to allow the transfer of ATP's terminal phosphate to its aspartyl-phosphorylation site. In order to get information for how the transfer of the γ-phosphate group of ATP to the Asp369 is achieved, analogous molecular modeling of the M4-M5 loop of ATPase was performed using the crystal data of Na+/K+-ATPase of different species. Analogous molecular modeling of the cytoplasmic loop between Thr338 and Ile760 of the α2-subunit of Na+/K+-ATPase and the analysis of distances between the ATP binding site and phosphorylation site revealed the existence of two ATP binding sites in the open conformation; the first one close to Phe475 in the N-domain, the other one close to Asp369 in the P-domain. However, binding of Mg2+•ATP to any of these sites in the "open conformation" may not lead to phosphorylation of Asp369. Additional conformations of the cytoplasmic loop were found wobbling between "open conformation" <==> "semi-open conformation <==> "closed conformation" in the absence of 2Mg2+•ATP. The cytoplasmic loop's conformational change to the "semi-open conformation"-characterized by a hydrogen bond between Arg543 and Asp611-triggers by binding of 2Mg2+•ATP to a single ATP site and conversion to the "closed conformation" the phosphorylation of Asp369 in the P-domain, and hence the start of Na+/K+-activated ATP hydrolysis.

• Keywords
• Hinge movement, M4M5 loop, Na+/K+-ATPase phosphorylation, Open and closed conformations,
• Publication type
• Journal Article MeSH

Explicit solvent molecular dynamics simulations (in total almost 800 ns including locally enhanced sampling runs) were applied with different ion conditions and with two force fields (AMBER and CHARMM) to characterize typical geometries adopted by the flanking bases in the RNA kissing-loop complexes. We focus on flanking base positions in multiple x-ray and NMR structures of HIV-1 DIS kissing complexes and kissing complex from the large ribosomal subunit of Haloarcula marismortui. An initial x-ray open conformation of bulged-out bases in HIV-1 DIS complexes, affected by crystal packing, tends to convert to a closed conformation formed by consecutive stretch of four stacked purine bases. This is in agreement with those recent crystals where the packing is essentially avoided. We also observed variants of the closed conformation with three stacked bases, while nonnegligible populations of stacked geometries with bulged-in bases were detected, too. The simulation results reconcile differences in positions of the flanking bases observed in x-ray and NMR studies. Our results suggest that bulged-out geometries are somewhat more preferred, which is in accord with recent experiments showing that they may mediate tertiary contacts in biomolecular assemblies or allow binding of aminoglycoside antibiotics.

• MeSH
• Models, Chemical * MeSH
• Dimerization MeSH
• HIV-1 chemistry   genetics   MeSH
• Nucleic Acid Conformation MeSH
• Models, Molecular * MeSH
• Base Pairing genetics   MeSH
• Transcription Initiation Site * MeSH
• Computer Simulation MeSH
• RNA, Viral chemistry   MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Viral MeSH

The purpose of this review is to outline our understanding of the nature, mechanism and physiological significance of light-induced reversible reorganizations in closed Type II reaction centre (RC) complexes. In the so-called 'closed' state, purple bacterial RC (bRC) and photosystem II (PSII) RC complexes are incapable of generating additional stable charge separation. Yet, upon continued excitation they display well-discernible changes in their photophysical and photochemical parameters. Substantial stabilization of their charge-separated states has been thoroughly documented-uncovering light-induced reorganizations in closed RCs and revealing their physiological importance in gradually optimizing the operation of the photosynthetic machinery during the dark-to-light transition. A range of subtle light-induced conformational changes has indeed been detected experimentally in different laboratories using different bRC and PSII-containing preparations. In general, the presently available data strongly suggest similar structural dynamics of closed bRC and PSII RC complexes, and similar physical mechanisms, in which dielectric relaxation processes and structural memory effects of proteins are proposed to play important roles.

• Keywords
• Marcus theory, chlorophyll fluorescence, dielectric relaxation, dynamics and structural memory of proteins, photosystem II, purple bacterial reaction centre,
• MeSH
• Photosynthesis * MeSH
• Photosystem II Protein Complex * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Photosystem II Protein Complex * MeSH

The vanilloid transient receptor potential channel TRPV1 is a molecular integrator of noxious stimuli, including capsaicin, heat and protons. Despite clear similarities between the overall architecture of TRPV1 and voltage-dependent potassium (Kv) channels, the extent of conservation in the molecular logic for gating is unknown. In Kv channels, a small contact surface between S1 and the pore-helix is required for channel functioning. To explore the function of S1 in TRPV1, we used tryptophan-scanning mutagenesis and characterized the responses to capsaicin and protons. Wild-type-like currents were generated in 9 out of 17 mutants; three mutants (M445W, A452W, R455W) were non-functional. The conservative mutation R455K in the extracellular extent of S1 slowed down capsaicin-induced activation and prevented normal channel closure. This mutant was neither activated nor potentiated by protons, on the contrary, protons promoted a rapid deactivation of its currents. Similar phenotypes were found in two other gain-of-function mutants and also in the pore-helix mutant T633A, known to uncouple proton activation. We propose that the S1 domain contains a functionally important region that may be specifically involved in TRPV1 channel gating, and thus be important for the energetic coupling between S1-S4 sensor activation and gate opening. Analogous to Kv channels, the S1-pore interface might serve to stabilize conformations associated with TRPV1 channel gating.

• MeSH
• Ion Channel Gating MeSH
• TRPV Cation Channels chemistry   genetics   metabolism   MeSH
• Hydrogen-Ion Concentration MeSH
• Cells, Cultured MeSH
• Kidney cytology   metabolism   MeSH
• Humans MeSH
• Patch-Clamp Techniques MeSH
• Mutation genetics   MeSH
• Protons * MeSH
• Protein Structure, Secondary * MeSH
• Hot Temperature MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• TRPV Cation Channels MeSH
• Protons * MeSH
• TRPV1 protein, human MeSH Browser

Photosystem II (PSII) uses solar energy to oxidize water and delivers electrons for life on Earth. The photochemical reaction center of PSII is known to possess two stationary states. In the open state (PSIIO), the absorption of a single photon triggers electron-transfer steps, which convert PSII into the charge-separated closed state (PSIIC). Here, by using steady-state and time-resolved spectroscopic techniques on Spinacia oleracea and Thermosynechococcus vulcanus preparations, we show that additional illumination gradually transforms PSIIC into a light-adapted charge-separated state (PSIIL). The PSIIC-to-PSIIL transition, observed at all temperatures between 80 and 308 K, is responsible for a large part of the variable chlorophyll-a fluorescence (Fv) and is associated with subtle, dark-reversible reorganizations in the core complexes, protein conformational changes at noncryogenic temperatures, and marked variations in the rates of photochemical and photophysical reactions. The build-up of PSIIL requires a series of light-induced events generating rapidly recombining primary radical pairs, spaced by sufficient waiting times between these events-pointing to the roles of local electric-field transients and dielectric relaxation processes. We show that the maximum fluorescence level, Fm, is associated with PSIIL rather than with PSIIC, and thus the Fv/Fm parameter cannot be equated with the quantum efficiency of PSII photochemistry. Our findings resolve the controversies and explain the peculiar features of chlorophyll-a fluorescence kinetics, a tool to monitor the functional activity and the structural-functional plasticity of PSII in different wild-types and mutant organisms and under stress conditions.

• MeSH
• Chlorophyll analogs & derivatives   chemistry   MeSH
• Diuron pharmacology   MeSH
• Fluorescence MeSH
• Spectrometry, Fluorescence MeSH
• Photosystem II Protein Complex chemistry   drug effects   metabolism   MeSH
• Protein Conformation MeSH
• Spectroscopy, Fourier Transform Infrared MeSH
• Spinacia oleracea chemistry   MeSH
• Light MeSH
• Temperature MeSH
• Thermosynechococcus chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chlorophyll MeSH
• chlorophyll a' MeSH Browser
• Diuron MeSH
• Photosystem II Protein Complex MeSH

Holliday junction (HJ) is a noncanonical four-way DNA structure with a prominent role in DNA repair, recombination, and DNA nanotechnology. By rearranging its four arms, HJ can adopt either closed or open state. With enzymes typically recognizing only a single state, acquiring detailed knowledge of the rearrangement process is an important step toward fully understanding the biological function of HJs. Here, we carried out standard all-atom molecular dynamics (MD) simulations of the spontaneous opening-closing transitions, which revealed complex conformational transitions of HJs with an involvement of previously unconsidered "half-closed" intermediates. Detailed free-energy landscapes of the transitions were obtained by sophisticated enhanced sampling simulations. Because the force field overstabilizes the closed conformation of HJs, we developed a system-specific modification which for the first time allows the observation of spontaneous opening-closing HJ transitions in unbiased MD simulations and opens the possibilities for more accurate HJ computational studies of biological processes and nanomaterials.

• MeSH
• DNA * MeSH
• DNA, Cruciform * MeSH
• Molecular Conformation MeSH
• DNA Repair MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA * MeSH
• DNA, Cruciform * MeSH

Conformational changes of the Na(+)/K(+)-ATPase isolated large cytoplasmic segment connecting transmembrane helices M4 and M5 (C45) induced by the interaction with enzyme ligands (i.e. Mg(2+) and/or ATP) were investigated by means of the intrinsic tryptophan fluorescence measurement and molecular dynamic simulations. Our data revealed that this model system consisting of only two domains retained the ability to adopt open or closed conformation, i.e. behavior, which is expected from the crystal structures of relative Ca(2+)-ATPase from sarco(endo)plasmic reticulum for the corresponding part of the entire enzyme. Our data revealed that the C45 is found in the closed conformation in the absence of any ligand, in the presence of Mg(2+) only, or in the simultaneous presence of Mg(2+) and ATP. Binding of the ATP alone (i.e. in the absence of Mg(2+)) induced open conformation of the C45. The fact that the transmembrane part of the enzyme was absent in our experiments suggested that the observed conformational changes are consequences only of the interaction with ATP or Mg(2+) and may not be related to the transported cations binding/release, as generally believed. Our data are consistent with the model, where ATP binding to the low-affinity site induces conformational change of the cytoplasmic part of the enzyme, traditionally attributed to E2-->E1 transition, and subsequent Mg(2+) binding to the enzyme-ATP complex induces in turn conformational change traditionally attributed to E1-->E2 transition.

• MeSH
• Adenosine Triphosphate metabolism   pharmacology   MeSH
• Biophysical Phenomena MeSH
• Models, Biological MeSH
• DNA Primers genetics   MeSH
• Fluorescence Polarization MeSH
• Spectrometry, Fluorescence MeSH
• Magnesium metabolism   pharmacology   MeSH
• Protein Conformation drug effects   MeSH
• Models, Molecular MeSH
• Mutagenesis, Site-Directed MeSH
• Mice MeSH
• Peptide Fragments chemistry   genetics   metabolism   MeSH
• Recombinant Proteins chemistry   genetics   metabolism   MeSH
• Base Sequence MeSH
• Sodium-Potassium-Exchanging ATPase chemistry   genetics   metabolism   MeSH
• Amino Acid Substitution MeSH
• In Vitro Techniques MeSH
• Thermodynamics MeSH
• Tryptophan chemistry   MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• DNA Primers MeSH
• Magnesium MeSH
• Peptide Fragments MeSH
• Recombinant Proteins MeSH
• Sodium-Potassium-Exchanging ATPase MeSH
• Tryptophan MeSH

Kink-turn (K-turn) motifs are asymmetric internal loops found at conserved positions in diverse RNAs, with sharp bends in phosphodiester backbones producing V-shaped structures. Explicit-solvent molecular dynamics simulations were carried out for three K-turns from 23S rRNA, i.e., Kt-38 located at the base of the A-site finger, Kt-42 located at the base of the L7/L12 stalk, and Kt-58 located in domain III, and for the K-turn of human U4 snRNA. The simulations reveal hinge-like K-turn motions on the nanosecond timescale. The first conserved A-minor interaction between the K-turn stems is entirely stable in all simulations. The angle between the helical arms of Kt-38 and Kt-42 is regulated by local variations of the second A-minor (type I) interaction between the stems. Its variability ranges from closed geometries to open ones stabilized by insertion of long-residency waters between adenine and cytosine. The simulated A-minor geometries fully agree with x-ray data. Kt-58 and Kt-U4 exhibit similar elbow-like motions caused by conformational change of the adenosine from the nominally unpaired region. Despite the observed substantial dynamics of K-turns, key tertiary interactions are stable and no sign of unfolding is seen. We suggest that some K-turns are flexible elements mediating large-scale ribosomal motions during the protein synthesis cycle.

• MeSH
• Adenine chemistry   MeSH
• Amino Acid Motifs MeSH
• Biophysics methods   MeSH
• Time Factors MeSH
• Cytosine chemistry   MeSH
• Peptide Elongation Factor G chemistry   MeSH
• Catalysis MeSH
• Nucleic Acid Conformation MeSH
• Protein Conformation MeSH
• Crystallography, X-Ray MeSH
• Macromolecular Substances MeSH
• Molecular Conformation MeSH
• Models, Molecular MeSH
• Molecular Sequence Data MeSH
• Oscillometry MeSH
• Base Pairing * MeSH
• Computer Simulation MeSH
• X-Rays MeSH
• Ribosomes chemistry   MeSH
• RNA, Small Nuclear chemistry   MeSH
• RNA, Ribosomal, 23S chemistry   MeSH
• RNA, Transfer chemistry   MeSH
• RNA chemistry   MeSH
• Protein Structure, Secondary MeSH
• Base Sequence MeSH
• Software MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Names of Substances
• Adenine MeSH
• Cytosine MeSH
• Peptide Elongation Factor G MeSH
• Macromolecular Substances MeSH
• RNA, Small Nuclear MeSH
• RNA, Ribosomal, 23S MeSH
• RNA, Transfer MeSH
• RNA MeSH
• U4 small nuclear RNA MeSH Browser

Retroviruses can create endogenous forms on infiltration into the germline cells of their hosts. These forms are then vertically transmitted and can be considered as genetic fossils of ancient viruses. All retrovirus genera, with the exception of deltaretroviruses, have had their representation identified in the host genome as a virus fossil record. Here we describe an endogenous Deltaretrovirus, identified in the germline of long-fingered bats (Miniopteridae). A single, heavily deleted copy of this retrovirus has been found in the genome of miniopterid species, but not in the genomes of the phylogenetically closest bat families, Vespertilionidae and Cistugonidae. Therefore, the endogenization occurred in a time interval between 20 and 45 million years ago. This discovery closes the last major gap in the retroviral fossil record and provides important insights into the history of deltaretroviruses in mammals.

• Keywords
• Chiroptera, Deltaretroviruses, endogenous retroviruses,
• MeSH
• Chiroptera classification   genetics   MeSH
• Deltaretrovirus genetics   MeSH
• Endogenous Retroviruses genetics   MeSH
• Phylogeny MeSH
• Genome * MeSH
• Genomics methods   MeSH
• Nucleic Acid Conformation MeSH
• Consensus Sequence MeSH
• Evolution, Molecular MeSH
• Open Reading Frames MeSH
• Base Sequence MeSH
• Genes, Viral MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH