Environment of TyrZ in photosystem II from Thermosynechococcus elongatus in which PsbA2 is the D1 protein

The main cofactors that determine the photosystem II (PSII) oxygen evolution activity are borne by the D1 and D2 subunits. In the cyanobacterium Thermosynechococcus elongatus, there are three psbA genes coding for D1. Among the 344 residues constituting D1, there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2, and 27 between PsbA2 and PsbA3. Here, we present the first study of PsbA2-PSII. Using EPR and UV-visible time-resolved absorption spectroscopy, we show that: (i) the time-resolved EPR spectrum of Tyr_Z^• in the (S₃Tyr_Z^•)′ is slightly modified; (ii) the split EPR signal arising from Tyr_Z^• in the (S₂Tyr_Z^•)′ state induced by near-infrared illumination at 4.2 K of the S₃Tyr_Z state is significantly modified; and (iii) the slow phases of P₆₈₀^+⋅ reduction by Tyr_Z are slowed down from the hundreds of μs time range to the ms time range, whereas both the S₁Tyr_Z^• → S₂Tyr_Z and the S₃Tyr_Z^• → S₀Tyr_Z + O₂ transition kinetics remained similar to those in PsbA(1/3)-PSII. These results show that the geometry of the Tyr_Z phenol and its environment, likely the Tyr-O···H···Nϵ-His bonding, are modified in PsbA2-PSII when compared with PsbA(1/3)-PSII. They also point to the dynamics of the proton-coupled electron transfer processes associated with the oxidation of Tyr_Z being affected. From sequence comparison, we propose that the C144P and P173M substitutions in PsbA2-PSII versus PsbA(1/3)-PSII, respectively located upstream of the α-helix bearing Tyr_Z and between the two α-helices bearing Tyr_Z and its hydrogen-bonded partner, His-190, are responsible for these changes.     

Investigation of in vivo diferric tyrosyl radical formation in Saccharomyces cerevisiae Rnr2 protein: requirement of Rnr4 and contribution of Grx3/4 AND Dre2 proteins

The β₂ subunit of class Ia ribonucleotide reductase (RNR) contains a diferric tyrosyl radical cofactor (Fe₂^III-Tyr^•) that is essential for nucleotide reduction. The β₂ subunit of Saccharomyces cerevisiae is a heterodimer of Rnr2 (β) and Rnr4 (β′). Although only β is capable of iron binding and Tyr^• formation, cells lacking β′ are either dead or exhibit extremely low Tyr^• levels and RNR activity depending on genetic backgrounds. Here, we present evidence supporting the model that β′ is required for iron loading and Tyr^• formation in β in vivo via a pathway that is likely dependent on the cytosolic monothiol glutaredoxins Grx3/Grx4 and the Fe-S cluster protein Dre2. rnr4 mutants are defective in iron loading into nascent β and are hypersensitive to iron depletion and the Tyr^•-reducing agent hydroxyurea. Transient induction of β′ in a GalRNR4 strain leads to a concomitant increase in iron loading and Tyr^• levels in β. Tyr^• can also be rapidly generated using endogenous iron when permeabilized Δrnr4 spheroplasts are supplemented with recombinant β′ and is inhibited by adding an iron chelator prior to, but not after, β′ supplementation. The growth defects of rnr4 mutants are enhanced by deficiencies in grx3/grx4 and dre2. Moreover, depletion of Dre2 in GalDRE2 cells leads to a decrease in both Tyr^• levels and ββ′ activity. This result, in combination with previous findings that a low level of Grx3/4 impairs RNR function, strongly suggests that Grx3/4 and Dre2 serve in the assembly of the deferric Tyr^• cofactor in RNR.     

Nitric oxide suppresses tumor cell migration through N-Myc downstream-regulated gene-1 (NDRG1) expression: role of chelatable iron

N-Myc downstream-regulated gene 1 (NDRG1) is a ubiquitous cellular protein that is up-regulated under a multitude of stress and growth-regulatory conditions. Although the exact cellular functions of this protein have not been elucidated, mutations in this gene or aberrant expression of this protein have been linked to both tumor suppressive and oncogenic phenotypes. Previous reports have demonstrated that NDRG1 is strongly up-regulated by chemical iron chelators and hypoxia, yet its regulation by the free radical nitric oxide (^•NO) has never been demonstrated. Herein, we examine the chemical biology that confers NDRG1 responsiveness at the mRNA and protein levels to ^•NO. We demonstrate that the interaction of ^•NO with the chelatable iron pool (CIP) and the appearance of dinitrosyliron complexes (DNIC) are key determinants. Using HCC 1806 triple negative breast cancer cells, we find that NDRG1 is up-regulated by physiological ^•NO concentrations in a dose- and time-dependant manner. Tumor cell migration was suppressed by NDRG1 expression and we excluded the involvement of HIF-1α, sGC, N-Myc, and c-Myc as upstream regulatory targets of ^•NO. Augmenting the chelatable iron pool abolished ^•NO-mediated NDRG1 expression and the associated phenotypic effects. These data, in summary, reveal a link between ^•NO, chelatable iron, and regulation of NDRG1 expression and signaling in tumor cells.     

The Role of Nitric-oxide Synthase in the Regulation of UVB Light-induced Phosphorylation of the �� Subunit of Eukaryotic Initiation Factor 2

UV light induces phosphorylation of the α subunit of the eukaryotic initiation factor 2 (eIF2α) and inhibits global protein synthesis. Both eIF2 kinases, protein kinase-like endoplasmic reticulum kinase (PERK) and general control of nonderepressible protein kinase 2 (GCN2), have been shown to phosphorylate eIF2α in response to UV irradiation. However, the roles of PERK and GCN2 in UV-induced eIF2α phosphorylation are controversial. The one or more upstream signaling pathways that lead to the activation of PERK or GCN2 remain unknown. In this report we provide data showing that both PERK and GCN2 contribute to UV-induced eIF2α phosphorylation in human keratinocyte (HaCaT) and mouse embryonic fibroblast cells. Reduction of expression of PERK or GCN2 by small interfering RNA decreases phosphorylation of eIF2α after UV irradiation. These data also show that nitric-oxide synthase (NOS)-mediated oxidative stress plays a role in regulation of eIF2α phosphorylation upon UV irradiation. Treating the cells with the broad NOS inhibitor N^G-methyl-l-arginine, the free radical scavenger N-acetyl-l-cysteine, or the NOS substrate l-arginine partially inhibits UV-induced eIF2α phosphorylation. The results presented above led us to propose that NOS mediates UV-induced eIF2α phosphorylation by activation of both PERK and GCN2 via oxidative stress and l-arginine starvation signaling pathways.UV irradiation inhibits translation initiation through activation of kinases that phosphorylate the α-subunit of eukaryotic initiation factor 2 (eIF2α).² Two eIF2α kinases, double strand RNA-dependent protein kinase-like ER kinase (PERK) and general control of amino acid biosynthesis kinase (GCN2), are known to phosphorylate the serine 51 of eIF2α in response to UV irradiation (1–4). However, the one or more upstream pathways that activate eIF2α kinase(s) upon UV irradiation are not known. In this report, we provide evidence that UV-induced nitric-oxide synthase (NOS) activation and nitric oxide (NO^•) production regulate both PERK and GCN2 activation upon UVB irradiation.Expression of inducible nitric-oxide synthase in a mouse macrophage cell line leads to the phosphorylation of eIF2α and inhibition of translation (5). In cultured neuronal and pancreatic cell lines, production of NO^• and peroxynitrite (ONOO⁻) induces endoplasmic reticulum (ER) stress, which activates PERK and results in cell dysfunction and apoptosis (6–9). Cytokine-stimulated inducible nitric-oxide synthase activation in astrocytes depletes l-arginine and activates GCN2, which phosphorylates eIF2α (10). UV irradiation also activates NOS and elevates cellular NO^• (11–13). However, the UV-induced NOS activation and NO^• production have never been shown to be related to the activation of eIF2α kinase(s). Now we demonstrate that UV-induced activation of NOS mediates the activation of both PERK and GCN2, which coordinately regulate the phosphorylation of eIF2α.     

Stabilization of unstable CGC+ triplex DNA by single-walled carbon nanotubes under physiological conditions

Triplex formation is a promising strategy for realizing artificially controlling of gene expression, reversible assembly of nanomaterials and DNA nanomachine and single-walled nanotubes (SWNTs) have been widely used as gene and drug delivery vector or as ‘building blocks’ in nano-/microelectronic devices. CGC⁺ triplex is not as stable as TAT triplex. The poor stability of CGC⁺ triplex limits its use in vitro and in vivo. There is no ligand that has been reported to selectively stabilize CGC⁺ triplets rather than TAT. Here, we report that SWNTs can cause d(CT)•d(AG) duplex disproportionation into triplex d(C⁺T)•d(AG)•d(CT) and single-strand d(AG) under physiological conditions. SWNTs can reduce the stringency of conditions for CGC⁺ triplex formation studied by UV–vis, CD, DNA melting, light scattering and atomic force microscopy. Further studies indicate that electrostatic interaction is crucial for d(CT)•d(AG) repartition into triplex d(C⁺T)•d(AG)•d(CT). Our findings may facilitate utilization of SWNTs–DNA complex in artificially controlling of gene expression, nanomaterials assembly and biosensing.     

The three faces of riboviral spontaneous mutation: spectrum, mode of genome replication, and mutation rate

Riboviruses (RNA viruses without DNA replication intermediates) are the most abundant pathogens infecting animals and plants. Only a few riboviral infections can be controlled with antiviral drugs, mainly because of the rapid appearance of resistance mutations. Little reliable information is available concerning i) kinds and relative frequencies of mutations (the mutational spectrum), ii) mode of genome replication and mutation accumulation, and iii) rates of spontaneous mutation. To illuminate these issues, we developed a model in vivo system based on phage Qß infecting its natural host, Escherichia coli. The Qß RT gene encoding the Read-Through protein was used as a mutation reporter. To reduce uncertainties in mutation frequencies due to selection, the experimental Qß populations were established after a single cycle of infection and selection against RT ⁻ mutants during phage growth was ameliorated by plasmid-based RT complementation in trans. The dynamics of Qß genome replication were confirmed to reflect the linear process of iterative copying (the stamping-machine mode). A total of 32 RT mutants were detected among 7,517 Qß isolates. Sequencing analysis of 45 RT mutations revealed a spectrum dominated by 39 transitions, plus 4 transversions and 2 indels. A clear template•primer mismatch bias was observed: A•C>C•A>U•G>G•U> transversion mismatches. The average mutation rate per base replication was ≈9.1×10⁻⁶ for base substitutions and ≈2.3×10⁻⁷ for indels. The estimated mutation rate per genome replication, μ_g, was ≈0.04 (or, per phage generation, ≈0.08), although secondary RT mutations arose during the growth of some RT mutants at a rate about 7-fold higher, signaling the possible impact of transitory bouts of hypermutation. These results are contrasted with those previously reported for other riboviruses to depict the current state of the art in riboviral mutagenesis.     

Jellyfish support high energy intake of leatherback sea turtles (Dermochelys coriacea): video evidence from animal-borne cameras

The endangered leatherback turtle is a large, highly migratory marine predator that inexplicably relies upon a diet of low-energy gelatinous zooplankton. The location of these prey may be predictable at large oceanographic scales, given that leatherback turtles perform long distance migrations (1000s of km) from nesting beaches to high latitude foraging grounds. However, little is known about the profitability of this migration and foraging strategy. We used GPS location data and video from animal-borne cameras to examine how prey characteristics (i.e., prey size, prey type, prey encounter rate) correlate with the daytime foraging behavior of leatherbacks (n = 19) in shelf waters off Cape Breton Island, NS, Canada, during August and September. Video was recorded continuously, averaged 1:53 h per turtle (range 0:08–3:38 h), and documented a total of 601 prey captures. Lion''s mane jellyfish (Cyanea capillata) was the dominant prey (83–100%), but moon jellyfish (Aurelia aurita) were also consumed. Turtles approached and attacked most jellyfish within the camera''s field of view and appeared to consume prey completely. There was no significant relationship between encounter rate and dive duration (p = 0.74, linear mixed-effects models). Handling time increased with prey size regardless of prey species (p = 0.0001). Estimates of energy intake averaged 66,018 kJ•d⁻¹ but were as high as 167,797 kJ•d⁻¹ corresponding to turtles consuming an average of 330 kg wet mass•d⁻¹ (up to 840 kg•d⁻¹) or approximately 261 (up to 664) jellyfish•d^-1. Assuming our turtles averaged 455 kg body mass, they consumed an average of 73% of their body mass•d⁻¹ equating to an average energy intake of 3–7 times their daily metabolic requirements, depending on estimates used. This study provides evidence that feeding tactics used by leatherbacks in Atlantic Canadian waters are highly profitable and our results are consistent with estimates of mass gain prior to southward migration.     

Intraphagosomal peroxynitrite as a macrophage-derived cytotoxin against internalized Trypanosoma cruzi: consequences for oxidative killing and role of microbial peroxiredoxins in infectivity

Macrophage-derived radicals generated by the NADPH oxidase complex and inducible nitric-oxide synthase (iNOS) participate in cytotoxic mechanisms against microorganisms. Nitric oxide (^•NO) plays a central role in the control of acute infection by Trypanosoma cruzi, the causative agent of Chagas disease, and we have proposed that much of its action relies on macrophage-derived peroxynitrite (ONOO⁻ + ONOOH) formation, a strong oxidant arising from the reaction of ^•NO with superoxide radical (O₂^˙̄). Herein, we have shown that internalization of T. cruzi trypomastigotes by macrophages triggers the assembly of the NADPH oxidase complex to yield O₂^˙̄ during a 60–90-min period. This does not interfere with IFN-γ-dependent iNOS induction and a sustained ^•NO production (∼24 h). The major mechanism for infection control via reactive species formation occurred when ^•NO and O₂^˙̄ were produced simultaneously, generating intraphagosomal peroxynitrite levels compatible with microbial killing. Moreover, biochemical and ultrastructural analysis confirmed cellular oxidative damage and morphological disruption in internalized parasites. Overexpression of cytosolic tryparedoxin peroxidase in T. cruzi neutralized macrophage-derived peroxynitrite-dependent cytotoxicity to parasites and favored the infection in an animal model. Collectively, the data provide, for the first time, direct support for the action of peroxynitrite as an intraphagosomal cytotoxin against pathogens and the premise that microbial peroxiredoxins facilitate infectivity via decomposition of macrophage-derived peroxynitrite.     

Fidelity of tRNA 5'-maturation: a possible basis for the functional dependence of archaeal and eukaryal RNase P on multiple protein cofactors

RNase P, which catalyzes tRNA 5′-maturation, typically comprises a catalytic RNase P RNA (RPR) and a varying number of RNase P proteins (RPPs): 1 in bacteria, at least 4 in archaea and 9 in eukarya. The four archaeal RPPs have eukaryotic homologs and function as heterodimers (POP5•RPP30 and RPP21•RPP29). By studying the archaeal Methanocaldococcus jannaschii RPR''s cis cleavage of precursor tRNA^Gln (pre-tRNA^Gln), which lacks certain consensus structures/sequences needed for substrate recognition, we demonstrate that RPP21•RPP29 and POP5•RPP30 can rescue the RPR''s mis-cleavage tendency independently by 4-fold and together by 25-fold, suggesting that they operate by distinct mechanisms. This synergistic and preferential shift toward correct cleavage results from the ability of archaeal RPPs to selectively increase the RPR''s apparent rate of correct cleavage by 11 140-fold, compared to only 480-fold for mis-cleavage. Moreover, POP5•RPP30, like the bacterial RPP, helps normalize the RPR''s rates of cleavage of non-consensus and consensus pre-tRNAs. We also show that archaeal and eukaryal RNase P, compared to their bacterial relatives, exhibit higher fidelity of 5′-maturation of pre-tRNA^Gln and some of its mutant derivatives. Our results suggest that protein-rich RNase P variants might have evolved to support flexibility in substrate recognition while catalyzing efficient, high-fidelity 5′-processing.     

Reductions in maize root-tip elongation by salt and osmotic stress do not correlate with apoplastic O2*- levels

Background and Aims

Experimental evidence in the literature suggests that O₂^•− produced in the elongation zone of roots and leaves by plasma membrane NADPH oxidase activity is required for growth. This study explores whether growth changes along the root tip induced by hyperosmotic treatments in Zea mays are associated with the distribution of apoplastic O₂^•−.

Methods

Stress treatments were imposed using 150 mm NaCl or 300 mm sorbitol. Root elongation rates and the spatial distribution of growth rates in the root tip were measured. Apoplastic O₂^•− was determined using nitro blue tetrazolium, and H₂O₂ was determined using 2′, 7′-dichlorofluorescin.

Key Results

In non-stressed plants, the distribution of accelerating growth and highest O₂^•− levels coincided along the root tip. Salt and osmotic stress of the same intensity had similar inhibitory effects on root elongation, but O₂^•− levels increased in sorbitol-treated roots and decreased in NaCl-treated roots.

Conclusions

The lack of association between apoplastic O₂^•− levels and root growth inhibition under hyper-osmotic stress leads us to hypothesize that under those conditions the role of apoplastic O₂^•− may be to participate in signalling processes, that convey information on the nature of the substrate that the growing root is exploring.Key words: Root tip growth, Zea mays, salt stress, reactive oxygen species, ROS     

Quadruplex formation by both G-rich and C-rich DNA strands of the C9orf72 (GGGGCC)8?(GGCCCC)8 repeat: effect of CpG methylation

Unusual DNA/RNA structures of the C9orf72 repeat may participate in repeat expansions or pathogenesis of amyotrophic lateral sclerosis and frontotemporal dementia. Expanded repeats are CpG methylated with unknown consequences. Typically, quadruplex structures form by G-rich but not complementary C-rich strands. Using CD, UV and electrophoresis, we characterized the structures formed by (GGGGCC)8 and (GGCCCC)8 strands with and without 5-methylcytosine (5mCpG) or 5-hydroxymethylcytosine (5hmCpG) methylation. All strands formed heterogenous mixtures of structures, with features of quadruplexes (at pH 7.5, in K⁺, Na⁺ or Li⁺), but no feature typical of i-motifs. C-rich strands formed quadruplexes, likely stabilized by G•C•G•C-tetrads and C•C•C•C-tetrads. Unlike G•G•G•G-tetrads, some G•C•G•C-tetrad conformations do not require the N7-Guanine position, hence C9orf72 quadruplexes still formed when N7-deazaGuanine replace all Guanines. 5mCpG and 5hmCpG increased and decreased the thermal stability of these structures. hnRNPK, through band-shift analysis, bound C-rich but not G-rich strands, with a binding preference of unmethylated > 5hmCpG > 5mCpG, where methylated DNA-protein complexes were retained in the wells, distinct from unmethylated complexes. Our findings suggest that for C-rich sequences interspersed with G-residues, one must consider quadruplex formation and that methylation of quadruplexes may affect epigenetic processes.     

Arabidopsis annexin1 mediates the radical-activated plasma membrane Ca²+- and K+-permeable conductance in root cells

Plant cell growth and stress signaling require Ca²⁺ influx through plasma membrane transport proteins that are regulated by reactive oxygen species. In root cell growth, adaptation to salinity stress, and stomatal closure, such proteins operate downstream of the plasma membrane NADPH oxidases that produce extracellular superoxide anion, a reactive oxygen species that is readily converted to extracellular hydrogen peroxide and hydroxyl radicals, OH^•. In root cells, extracellular OH^• activates a plasma membrane Ca²⁺-permeable conductance that permits Ca²⁺ influx. In Arabidopsis thaliana, distribution of this conductance resembles that of annexin1 (ANN1). Annexins are membrane binding proteins that can form Ca²⁺-permeable conductances in vitro. Here, the Arabidopsis loss-of-function mutant for annexin1 (Atann1) was found to lack the root hair and epidermal OH^•-activated Ca²⁺- and K⁺-permeable conductance. This manifests in both impaired root cell growth and ability to elevate root cell cytosolic free Ca²⁺ in response to OH^•. An OH^•-activated Ca²⁺ conductance is reconstituted by recombinant ANN1 in planar lipid bilayers. ANN1 therefore presents as a novel Ca²⁺-permeable transporter providing a molecular link between reactive oxygen species and cytosolic Ca²⁺ in plants.     

Thermodynamic examination of the pyrophosphate sensor helix in the thiamine pyrophosphate riboswitch

Riboswitches are functional mRNA that control gene expression. Thiamine pyrophosphate (TPP) binds to thi-box riboswitch RNA and allosterically inhibits genes that code for proteins involved in the biosynthesis and transport of thiamine. Thiamine binding to the pyrimidine sensor helix and pyrophosphate binding to the pyrophosphate sensor helix cause changes in RNA conformation that regulate gene expression. Here we examine the thermodynamic properties of the internal loop of the pyrophosphate binding domain by comparing the wild-type construct (RNA WT) with six modified 2 × 2 bulged RNA and one 2 × 2 bulged DNA. The wild-type construct retains five conserved bases of the pyrophosphate sensor domain, two of which are in the 2 × 2 bulge (C65 and G66). The RNA WT construct was among the most stable (ΔG°₃₇ = −7.7 kcal/mol) in 1 M KCl at pH 7.5. Breaking the A•G mismatch of the bulge decreases the stability of the construct ∼0.5–1 kcal/mol, but does not affect magnesium binding to the RNA WT. Guanine at position 48 is important for RNA–Mg²⁺ interactions of the TPP-binding riboswitch at pH 7.5. In the presence of 9.5 mM magnesium at pH 5.5, the bulged RNA constructs gained an average of 1.1 kcal/mol relative to 1 M salt. Formation of a single A+•C mismatch base pair contributes about 0.5 kcal/mol at pH 5.5, whereas two tandem A+•C mismatch base pairs together contribute about 2 kcal/mol.     

Lewis acid catalysis of phosphoryl transfer from a copper(II)-NTP complex in a kinase ribozyme

The chemical strategies used by ribozymes to enhance reaction rates are revealed in part from their metal ion and pH requirements. We find that kinase ribozyme K28(1-77)C, in contrast with previously characterized kinase ribozymes, requires Cu²⁺ for optimal catalysis of thiophosphoryl transfer from GTPγS. Phosphoryl transfer from GTP is greatly reduced in the absence of Cu²⁺, indicating a specific catalytic role independent of any potential interactions with the GTPγS thiophosphoryl group. In-line probing and ATPγS competition both argue against direct Cu²⁺ binding by RNA; rather, these data establish that Cu²⁺ enters the active site within a Cu²⁺•GTPγS or Cu²⁺•GTP chelation complex, and that Cu²⁺•nucleobase interactions further enforce Cu²⁺ selectivity and position the metal ion for Lewis acid catalysis. Replacing Mg²⁺ with [Co(NH₃)₆]³⁺ significantly reduced product yield, but not k_obs, indicating that the role of inner-sphere Mg²⁺ coordination is structural rather than catalytic. Replacing Mg²⁺ with alkaline earths of increasing ionic radii (Ca²⁺, Sr²⁺ and Ba²⁺) gave lower yields and approximately linear rates of product accumulation. Finally, we observe that reaction rates increased with pH in log-linear fashion with an apparent pKa = 8.0 ± 0.1, indicating deprotonation in the rate-limiting step.     

Solution structure and DNA-binding properties of the phosphoesterase domain of DNA ligase D

The phosphoesterase (PE) domain of the bacterial DNA repair enzyme LigD possesses distinctive manganese-dependent 3′-phosphomonoesterase and 3′-phosphodiesterase activities. PE exemplifies a new family of DNA end-healing enzymes found in all phylogenetic domains. Here, we determined the structure of the PE domain of Pseudomonas aeruginosa LigD (PaePE) using solution NMR methodology. PaePE has a disordered N-terminus and a well-folded core that differs in instructive ways from the crystal structure of a PaePE•Mn²⁺• sulfate complex, especially at the active site that is found to be conformationally dynamic. Chemical shift perturbations in the presence of primer-template duplexes with 3′-deoxynucleotide, 3′-deoxynucleotide 3′-phosphate, or 3′ ribonucleotide termini reveal the surface used by PaePE to bind substrate DNA and suggest a more efficient engagement in the presence of a 3′-ribonucleotide. Spectral perturbations measured in the presence of weakly catalytic (Cd²⁺) and inhibitory (Zn²⁺) metals provide evidence for significant conformational changes at and near the active site, compared to the relatively modest changes elicited by Mn²⁺.     

Cleavage of model substrates by archaeal RNase P: role of protein cofactors in cleavage-site selection

RNase P is a catalytic ribonucleoprotein primarily involved in tRNA biogenesis. Archaeal RNase P comprises a catalytic RNase P RNA (RPR) and at least four protein cofactors (RPPs), which function as two binary complexes (POP5•RPP30 and RPP21• RPP29). Exploiting the ability to assemble a functional Pyrococcus furiosus (Pfu) RNase P in vitro, we examined the role of RPPs in influencing substrate recognition by the RPR. We first demonstrate that Pfu RPR, like its bacterial and eukaryal counterparts, cleaves model hairpin loop substrates albeit at rates 90- to 200-fold lower when compared with cleavage by bacterial RPR, highlighting the functionally comparable catalytic cores in bacterial and archaeal RPRs. By investigating cleavage-site selection exhibited by Pfu RPR (±RPPs) with various model substrates missing consensus-recognition elements, we determined substrate features whose recognition is facilitated by either POP5•RPP30 or RPP21•RPP29 (directly or indirectly via the RPR). Our results also revealed that Pfu RPR + RPP21•RPP29 displays substrate-recognition properties coinciding with those of the bacterial RPR-alone reaction rather than the Pfu RPR, and that this behaviour is attributable to structural differences in the substrate-specificity domains of bacterial and archaeal RPRs. Moreover, our data reveal a hierarchy in recognition elements that dictates cleavage-site selection by archaeal RNase P.     

ON THE NATURE OF FORCES OPERATING IN BLOOD CLOTTING : I. THE PARTICIPATION OF ELECTROSTATIC ATTRACTION

The results of the study of the inhibiting effect of neutral salts upon the clotting tendency of fibrinogen by thrombin may be summarised as follows: Salts like NaCl and KCl inhibit only weakly. Salts of the same cation (K^•) with monovalent anions of different ionic radius are the more active the larger the anion (Cl'',Br'',I''). Salts of the same cation with anions of different valency are the more active the higher the charge of the anion (1–1 <1–2 <1–3 <1–4). Salts with the same anion with cations of different valency show stronger inhibition in the case of cations of higher charge (K^•,Na^• < Mg^••, Ca^••, Sr^••, Ba^••). Salts with the same anion and cations of the same charge, but of different radius, are the more active the larger the cation (but with an inversion between Mg^•• and Ca^•• in the series of the alkali earths, which is not infrequent in biocolloids). These results show that the clotting of fibrinogen with thrombin is, at least partly, caused by a coacervation process, due to electrostatic attraction between positive and negative groups. Its nature and localisation will be dealt with in the next paper of this series.     

Pre-power-stroke cross-bridges contribute to force transients during imposed shortening in isolated muscle fibers

When skeletal muscles are activated and mechanically shortened, the force that is produced by the muscle fibers decreases in two phases, marked by two changes in slope (P₁ and P₂) that happen at specific lengths (L₁ and L₂). We tested the hypothesis that these force transients are determined by the amount of myosin cross-bridges attached to actin and by changes in cross-bridge strain due to a changing fraction of cross-bridges in the pre-power-stroke state. Three separate experiments were performed, using skinned muscle fibers that were isolated and subsequently (i) activated at different Ca²⁺ concentrations (pCa²⁺ 4.5, 5.0, 5.5, 6.0) (n = 13), (ii) activated in the presence of blebbistatin (n = 16), and (iii) activated in the presence of blebbistatin at varying velocities (n = 5). In all experiments, a ramp shortening was imposed (amplitude 10%L_o, velocity 1 L_o•sarcomere length (SL)•s⁻¹), from an initial SL of 2.5 µm (except by the third group, in which velocities ranged from 0.125 to 2.0 L_o•s⁻¹). The values of P₁, P₂, L₁, and L₂ did not change with Ca²⁺ concentrations. Blebbistatin decreased P₁, and it did not alter P₂, L₁, and L₂. We developed a mathematical cross-bridge model comprising a load-dependent power-stroke transition and a pre-power-stroke cross-bridge state. The P₁ and P₂ critical points as well as the critical lengths L₁ and L₂ were explained qualitatively by the model, and the effects of blebbistatin inhibition on P₁ were also predicted. Furthermore, the results of the model suggest that the mechanism by which blebbistatin inhibits force is by interfering with the closing of the myosin upper binding cleft, biasing cross-bridges into a pre-power-stroke state.