Saccharomyces cerevisiae NineTeen Complex (NTC)-associated Factor Bud31/Ycr063w Assembles on Precatalytic Spliceosomes and Improves First and Second Step Pre-mRNA Splicing Efficiency

Pre-mRNA splicing occurs in spliceosomes whose assembly and activation are critical for splice site selection and catalysis. The highly conserved NineTeen complex protein complex stabilizes various snRNA and protein interactions early in the spliceosome assembly pathway. Among several NineTeen complex-associated proteins is the nonessential protein Bud31/Ycr063w, which is also a component of the Cef1p subcomplex. A role for Bud31 in pre-mRNA splicing is implicated by virtue of its association with splicing factors, but its specific functions and spliceosome interactions are uncharacterized. Here, using in vitro splicing assays with extracts from a strain lacking Bud31, we illustrate its role in efficient progression to the first catalytic step and its requirement for the second catalytic step in reactions at higher temperatures. Immunoprecipitation of functional epitope-tagged Bud31 from in vitro reactions showed that its earliest association is with precatalytic B complex and that the interaction continues in catalytically active complexes with stably bound U2, U5, and U6 small nuclear ribonucleoproteins. In complementary experiments, wherein precatalytic spliceosomes are selected from splicing reactions, we detect the occurrence of Bud31. Cross-linking of proteins to pre-mRNAs with a site-specific 4-thio uridine residue at the -3 position of exon 1 was tested in reactions with WT and bud31 null extracts. The data suggest an altered interaction between a similar to 25-kDa protein and this exonic residue of pre-mRNAs in the arrested bud31 null spliceosomes. These results demonstrate the early spliceosomal association of Bud31 and provide plausible functions for this factor in stabilizing protein interactions with the pre-mRNA.

Pleiotropic Roles of a Ribosomal Protein in Dictyostelium discoideum

The cell cycle phase at starvation influences post-starvation differentiation and morphogenesis in Dictyostelium discoideum. We found that when expressed in Saccharomyces cerevisiae, a D. discoideum cDNA that encodes the ribosomal protein S4 (DdS4) rescues mutations in the cell cycle genes cdc24, cdc42 and bem1. The products of these genes affect morphogenesis in yeast via a coordinated moulding of the cytoskeleton during bud site selection. D. discoideum cells that over-or under-expressed DdS4 did not show detectable changes in protein synthesis but displayed similar developmental aberrations whose intensity was graded with the extent of over-or under-expression. This suggested that DdS4 might influence morphogenesis via a stoichiometric effect - specifically, by taking part in a multimeric complex similar to the one involving Cdc24p, Cdc42p and Bem1p in yeast. In support of the hypothesis, the S. cerevisiae proteins Cdc24p, Cdc42p and Bem1p as well as their D. discoideum cognates could be co-precipitated with antibodies to DdS4. Computational analysis and mutational studies explained these findings: a C-terminal domain of DdS4 is the functional equivalent of an SH3 domain in the yeast scaffold protein Bem1p that is central to constructing the bud site selection complex. Thus in addition to being part of the ribosome, DdS4 has a second function, also as part of a multi-protein complex. We speculate that the existence of the second role can act as a safeguard against perturbations to ribosome function caused by spontaneous variations in DdS4 levels.

Crucial contribution of the multiple copies of the initiator tRNA genes in the fidelity of tRNA(fMet) selection on the ribosomal P-site in Escherichia coli

The accuracy of the initiator tRNA (tRNA(fMet)) selection in the ribosomal P-site is central to the fidelity of protein synthesis. A highly conserved occurrence of three consecutive G-C base pairs in the anticodon stem of tRNA(fMet) contributes to its preferential selection in the P-site. In a genetic screen, using a plasmid borne copy of an inactive tRNA(fMet) mutant wherein the three G-C base pairs were changed, we isolated Escherichia coli strains that allow efficient initiation with the tRNA(fMet) mutant. Here, extensive characterization of two such strains revealed novel mutations in the metZWV promoter severely compromising tRNA(fMet) levels. Low cellular abundance of the chromosomally encoded tRNA(fMet) allows efficient initiation with the tRNA(fMet) mutant and an elongator tRNA(Gln), revealing that a high abundance of the cellular tRNA(fMet) is crucial for the fidelity of initiator tRNA selection on the ribosomal P-site in E. coli. We discuss possible implications of the changes in the cellular tRNA(fMet) abundance in proteome remodeling.

Mechanism of adsorption effects on electrode kinetics: Part I: Selection rules in the case of low coverage by molecular surfactants

It is shown that the effect of adsorption of inert molecules on electrode reaction rates is completely accounted for, by introducing into the rate equation, adsorption-induced changes in both the effective electrode area as well as in the electrostatic potential at the reaction site with an additional term for the noncoulombic interaction between the reactant and the adsorbate. The electrostatic potential at the reaction site due to the adsorbed layer is calculated using a model of discretely-distributed molecules in parallel orientation when adsorbed on the electrode with an allowance for thermal agitation. The resulting expression, which is valid for the limiting case of low coverages, is used to predict the types of molecular surfactants that are most likely to be useful for acceleration and inhibition of electrode reactions.

Characterizing the pocketome of Mycobacterium tuberculosis and application in rationalizing polypharmacological target selection

Polypharmacology is beginning to emerge as an important concept in the field of drug discovery. However, there are no established approaches to either select appropriate target sets or design polypharmacological drugs. Here, we propose a structural-proteomics approach that utilizes the structural information of the binding sites at a genome-scale obtained through in-house algorithms to characterize the pocketome, yielding a list of ligands that can participate in various biochemical events in the mycobacterial cell. The pocket-type space is seen to be much larger than the sequence or fold-space, suggesting that variations at the site-level contribute significantly to functional repertoire of the organism. All-pair comparisons of binding sites within Mycobacterium tuberculosis (Mtb), pocket-similarity network construction and clustering result in identification of binding-site sets, each containing a group of similar binding sites, theoretically having a potential to interact with a common set of compounds. A polypharmacology index is formulated to rank targets by incorporating a measure of druggability and similarity to other pockets within the proteome. This study presents a rational approach to identify targets with polypharmacological potential along with possible drugs for repurposing, while simultaneously, obtaining clues on lead compounds for use in new drug-discovery pipelines.

Site-Selective Addition of Maleimide to Indole at the C-2 Position: Ru(II)-Catalyzed C-H Activation

Synthesis of 3-(indol-2-yl)succinimide derivatives is presented using a directing group strategy. Selective functionalization of C-2 in the presence of highly reactive C-3 in indole derivatives has been achieved. A conjugate addition product instead of Heck-type product has been brought about by careful selection of the alkene partner (maleimides and maleate esters) such that a beta-hydride elimination is avoided.

Structural studies on a non-toxic homologue of type II RIPs from bitter gourd: Molecular basis of non-toxicity, conformational selection and glycan structure

The structures of nine independent crystals of bitter gourd seed lectin (BGSL), a non-toxic homologue of type II RIPs, and its sugar complexes have been determined. The four-chain, two-fold symmetric, protein is made up of two identical two-chain modules, each consisting of a catalytic chain and a lectin chain, connected by a disulphide bridge. The lectin chain is made up of two domains. Each domain carries a carbohydrate binding site in type II RIPs of known structure. BGSL has a sugar binding site only on one domain, thus impairing its interaction at the cell surface. The adenine binding site in the catalytic chain is defective. Thus, defects in sugar binding as well as adenine binding appear to contribute to the non-toxicity of the lectin. The plasticity of the molecule is mainly caused by the presence of two possible well defined conformations of a surface loop in the lectin chain. One of them is chosen in the sugar complexes, in a case of conformational selection, as the chosen conformation facilitates an additional interaction with the sugar, involving an arginyl residue in the loop. The N-glycosylation of the lectin involves a plant-specific glycan while that in toxic type II RIPs of known structure involves a glycan which is animal as well as plant specific.

Electronic Control of Facial Selection in Additions to Sterically Unbiased Ketones and Olefins

By definition, the two faces of a pi bond are equivalent.1 However, they are rendered nonequivalent in most molecules because of the absence of a plane of symmetry encompassing the double bond and the adjacent substituents. As a result, additions to trigonal centers from the two faces need not be equally facile. Exploiting this stereodifferentiation in a controlled manner represents one of the core problems in organic synthesis. Evidently, the factors which determine such diastereoselection need to be delineated in as much detail as possible.

The nature of the charge-ordered state in Y0.5Ca0.5MnO3 with a very small average radius of the A-site cations

A detailed investigation of Y0.5Ca0.5MnO3 with a very small radius of the A-site cations ([r(A)] approximate to 1.13 Angstrom reveals the occurrence of a charge-ordering transition in the paramagnetic state, at a relatively high temperature of 260 K. The orthorhombic lattice distortion, as measured by the dimensionless index D, is large (similar to 1.75%) over the entire 300-100 K range, but the antiferromagnetic interactions become prominent only at low temperatures (< 160 K). The charge-ordering gap in Y0.5Ca0.5MnO3, measured by low-temperature vacuum tunnelling spectroscopy, is large (similar to 0.5 eV) and the charge-ordered state is unaffected by the application of a magnetic field of 6 T. The study indicates that the nature of charge-ordering in Y0.5Ca0.5MnO3 which is dominated by the cooperative Jahn-Teller effect and the associated lattice distortion is distinctly different from analogous manganates with larger [r(A)].

The role of lysine-41 in RNase A catalysis - A quantum chemical study on the active site ligand complex

Several mechanisms have been proposed to explain the action of enzymes at the atomic level. Among them, the recent proposals involving short hydrogen bonds as a step in catalysis by Gerlt and Gassman [1] and proton transfer through low barrier hydrogen bonds (LBHBs) [2, 3] have attracted attention. There are several limitations to experimentally testing such hypotheses, Recent developments in computational methods facilitate the study of active site-ligand complexes to high levels of accuracy, Our previous studies, which involved the docking of the dinucleotide substrate UpA to the active site of RNase A [4, 5], enabled us to obtain a realistic model of the ligand-bound active site of RNase A. From these studies, based on empirical potential functions, we were able to obtain the molecular dynamics averaged coordinates of RNase A, bound to the ligand UpA. A quantum mechanical study is required to investigate the catalytic process which involves the cleavage and formation of covalent bonds. In the present study, we have investigated the strengths of some of the hydrogen bonds between the active site residues of RNase A and UpA at the ab initio quantum chemical level using the molecular dynamics averaged coordinates as the starting point. The 49 atom system and other model systems were optimized at the 3-21G level and the energies of the optimized systems were obtained at the 6-31G* level. The results clearly indicate the strengthening of hydrogen bonds between neutral residues due to the presence of charged species at appropriate positions. Such a strengthening manifests itself in the form of short hydrogen bonds and a low barrier for proton transfer. In the present study, the proton transfer between the 2'-OH of ribose (from the substrate) and the imidazole group from the H12 of RNase A is influenced by K41, which plays a crucial role in strengthening the neutral hydrogen bond, reducing the barrier for proton transfer.

Perception of ultraviolet light by crab spiders and its role in selection of hunting sites

The perception of ultraviolet (UV) light by spiders has so far been only demonstrated in salticids. Crab spiders (Thomisidae) hunt mostly on flowers and need to find appropriate hunting sites. Previous studies have shown that some crab spiders that reflect UV light use UV contrast to enhance prey capture. The high UV contrast can be obtained either by modulation of body colouration or active selection of appropriate backgrounds for foraging. We show that crab spiders (Thomisus sp.)hunting on Spathiphyllum plants use chromatic contrast, especially UV contrast, to make themselves attractive to hymenopteran prey. Apart from that, they are able to achieve high UV contrast by active selection of non-UV reflecting surfaces when given a choice of UV-reflecting and non-UV reflecting surfaces in the absence of odour cues. Honeybees (Apis cerana) approached Spathiphyllum plants bearing crab spiders on which the spiders were high UV-contrast targets with greater frequency than those plants on which the UV-contrast of the spiders was low. Thus, crab spiders can perceive UV and may use it to choose appropriate backgrounds to enhance prey capture, by exploiting the attraction of prey such as honeybees to UV.

Synthesis and characterisation of heterometallic molecular triangles using ambidentate linker: self-selection of a single linkage isomer

The coordination driven self-assembly of discrete molecular triangles from a non-symmetric ambidentate linker 5-pyrimidinecarboxylate (5-pmc) and Pd(II)/Pt(II) based 90◦ acceptors is presented. Despite the possibility of formation of a mixture of isomeric macrocycles (linkage isomers) due to different connectivity of the ambidentate linker, formation of a single and symmetrical linkage somer in both the cases is an interesting observation. Moreover, the reported macrocycles represent the first example of discrete metallamacrocycles of bridging 5-pmc. While solution composition in both the cases was characterised by multinuclear NMR study and electrospray ionization mass spectrometry (ESI-MS), the identity of the assemblies in the solid state was established by X-ray single crystals structure analysis. Variable temperature NMR study clearly ruled out the formation of any other macrocycles by [4 + 4] or [2 + 2] self-assembly of the reacting components.

Fast Multiple Access Selection through variable power transmissions

Many wireless applications demand a fast mechanism to detect the packet from a node with the highest priority ("best node") only, while packets from nodes with lower priority are irrelevant. In this paper, we introduce an extremely fast contention-based multiple access algorithm that selects the best node and requires only local information of the priorities of the nodes. The algorithm, which we call Variable Power Multiple Access Selection (VP-MAS), uses the local channel state information from the accessing nodes to the receiver, and maps the priorities onto the receive power. It is based on a key result that shows that mapping onto a set of discrete receive power levels is optimal, when the power levels are chosen to exploit packet capture that inherently occurs in a wireless physical layer. The VP-MAS algorithm adjusts the expected number of users that contend in each step and their respective transmission powers, depending on whether previous transmission attempts resulted in capture, idle channel, or collision. We also show how reliable information regarding the total received power at the receiver can be used to improve the algorithm by enhancing the feedback mechanism. The algorithm detects the packet from the best node in 1.5 to 2.1 slots, which is considerably lower than the 2.43 slot average achieved by the best algorithm known to date.

Impact of rRNA methylations on ribosome recycling and fidelity of initiation in Escherichia coli

Ribosomal RNA (rRNA) contains a number of modified nucleosides in functionally important regions including the intersubunit bridge regions. As the activity of ribosome recycling factor (RRF) in separating the large and the small subunits of the ribosome involves disruption of intersubunit bridges, we investigated the impact of rRNA methylations on ribosome recycling. We show that deficiency of rRNA methylations, especially at positions 1518 and 1519 of 16S rRNA near the interface with the 50S subunit and in the vicinity of the IF3 binding site, adversely affects the efficiency of RRF-mediated ribosome recycling. In addition, we show that a compromise in the RRF activity affords increased initiation with a mutant tRNA(fMet) wherein the three consecutive G-C base pairs ((29)GGG(31):39CCC41), a highly conserved feature of the initiator tRNAs, were mutated to those found in the elongator tRNA(Met) ((29)UCA(31):(39)psi GA(41)). This observation has allowed us to uncover a new role of RRF as a factor that contributes to fidelity of initiator tRNA selection on the ribosome. We discuss these and earlier findings to propose that RRF plays a crucial role during all the steps of protein synthesis.

On the moulding of population viscosity by natural selection

In this paper, we explore the conjoint evolution of dispersal and social behaviour. The model investigated is of a population distributed over a number of sites each with a carrying capacity of two adults and an episode of dispersal in the juvenile stage. The fertilities are governed by whether an individual and its neighbour are selfish or co-operative. It is shown that the best dispersal strategy for the co-operative genotype always involves lower levels of dispersal; and further that ecological conditions favouring low levels of dispersal increase the selective advantage of a co-operative genotype. Given this positive feedback, we suggest that in any taxon viscosity and co-operativity will tend to be correlated and bimodally distributed. Hence we predict the existence of two kinds of animal societies; viscous and co-operative (e.g. quasi-social wasps such as Mischocyttarus), and non-viscous and selfish (e.g. communal sphecid wasps such as Cerceris), and relatively few social groups with intermediate levels of co-operativity and viscosity. We also suggest that when one of the two sexes disperses, it will be the sex with lower potential for co-operative behaviour.