Function of the SmpB tail in transfer-messenger RNA translation revealed by a nucleus-encoded form

Stalled bacterial ribosomes are freed when they switch to the translation of transfer-messenger RNA (tmRNA). This process requires the tmRNA-binding and ribosome-binding cofactor SmpB, a beta-barrel protein with a protruding C-terminal tail of unresolved structure. Some plastid genomes encode tmRNA, but smpB genes have only been reported from bacteria. Here we identify smpB in the nuclear genomes of both a diatom and a red alga encoding a signal for import into the plastid, where mature SmpB could activate tmRNA. Diatom SmpB was active for tmRNA translation with bacterial components in vivo and in vitro, although less so than Escherichia coli SmpB. The tail-truncated diatom SmpB, the hypothetical product of a misspliced mRNA, was inactive in vivo. Tail-truncated E. coli SmpB was likewise inactive for tmRNA translation but was still able to bind ribosomes, and its affinity for tmRNA was only slightly diminished. This work suggests that SmpB is a universal cofactor of tmRNA. It also reveals a tail-dependent role for SmpB in tmRNA translation that supersedes a simple role of linking tmRNA to the ribosome, which the SmpB body alone could provide.     

The severe acute respiratory syndrome coronavirus Nsp15 protein is an endoribonuclease that prefers manganese as a cofactor

Nonstructural protein 15 (Nsp15) of the severe acute respiratory syndrome coronavirus (SARS-CoV) produced in Escherichia coli has endoribonuclease activity that preferentially cleaved 5' of uridylates of RNAs. Blocking either the 5' or 3' terminus did not affect cleavage. Double- and single-stranded RNAs were both substrates for Nsp15 but with different kinetics for cleavage. Mn(2+) at 2 to 10 mM was needed for optimal endoribonuclease activity, but Mg(2+) and several other divalent metals were capable of supporting only a low level of activity. Concentrations of Mn(2+) needed for endoribonuclease activity induced significant conformation change(s) in the protein, as measured by changes in tryptophan fluorescence. A similar endoribonucleolytic activity was detected for the orthologous protein from another coronavirus, demonstrating that the endoribonuclease activity of Nsp15 may be common to coronaviruses. This work presents an initial biochemical characterization of a novel coronavirus endoribonuclease.     

Chromium stress mitigation by polyamine-brassinosteroid application involves phytohormonal and physiological strategies in Raphanus sativus L

Brassinosteroids (BRs) and polyamines (PAs) are well-established growth regulators playing key roles in stress management among plants. In the present study, we evaluated the effects of epibrassinolide (EBL, an active BR) and spermidine (Spd, an active PA) on the tolerance of radish to oxidative stress induced by Cr (VI) metal. Our investigation aimed to study the impacts of EBL (10(-9) M) and/or Spd (1 mM) on the biochemical and physiological responses of radish (Raphanus sativus L.) under Cr-stress. Applications of EBL and/or Spd were found to improve growth of Cr-stressed seedlings in terms of root length, shoot length and fresh weight. Our data also indicated that applications of EBL and Spd have significant impacts, particularly when applied together, on the endogenous titers of PAs, free and bound forms of IAA and ABA in seedlings treated with Cr-stress. Additionally, co-applications of EBL and Spd modulated more remarkably the titers of antioxidants (glutathione, ascorbic acid, proline, glycine betaine and total phenol) and activities of antioxidant enzymes (guaicol peroxidase, catalase, superoxide dismutase and glutathione reductase) in Cr-stressed plants than their individual applications. Attenuation of Cr-stress by EBL and/or Spd (more efficient with EBL and Spd combination) was also supported by enhanced values of stress indices, such as phytochelatins, photosynthetic pigments and total soluble sugars, and reduction in malondialdehyde and H(2)O(2) levels in Cr-treated seedlings. Diminution of ROS production and enhanced ROS scavenging capacities were also noted for EBL and/or Spd under Cr-stress. However, no significant reduction in Cr uptake was observed for co-application of EBL and Spd when compared to their individual treatments in Cr-stressed seedlings. Taken together, our results demonstrate that co-applications of EBL and Spd are more effective than their independent treatments in lowering the Cr-induced oxidative stress in radish, leading to improved growth of radish seedlings under Cr-stress.     

Novel insights through the integration of structural and functional genomics data with protein networks

In recent years, major advances in genomics, proteomics, macromolecular structure determination, and the computational resources capable of processing and disseminating the large volumes of data generated by each have played major roles in advancing a more systems-oriented appreciation of biological organization. One product of systems biology has been the delineation of graph models for describing genome-wide protein-protein interaction networks. The network organization and topology which emerges in such models may be used to address fundamental questions in an array of cellular processes, as well as biological features intrinsic to the constituent proteins (or "nodes") themselves. However, graph models alone constitute an abstraction which neglects the underlying biological and physical reality that the network's nodes and edges are highly heterogeneous entities. Here, we explore some of the advantages of introducing a protein structural dimension to such models, as the marriage of conventional network representations with macromolecular structural data helps to place static node and edge constructs in a biologically more meaningful context. We emphasize that 3D protein structures constitute a valuable conceptual and predictive framework by discussing examples of the insights provided, such as enabling in silico predictions of protein-protein interactions, providing rational and compelling classification schemes for network elements, as well as revealing interesting intrinsic differences between distinct node types, such as disorder and evolutionary features, which may then be rationalized in light of their respective functions within networks.     

Butterfly communities along an elevational gradient in the Tons valley,Western Himalayas: Implications of rapid assessment for insect conservation

As time and money is limited, explicit, cost-effective, quick, and appropriate methods are needed to assist conservation planners and managers for making quick decisions. Butterflies promise to be a good model for rapid assessment and habitat monitoring studies because they are widespread, conspicuous, and easily recognizable and they are effective indicators of forest health. We conducted a rapid assessment of butterflies at five disturbance gradient sites that varied in elevation from 900 m a.s.l. to 3500 m a.s.l. for 20 days during March–April 2010 and recorded 79 butterfly species and 1504 individuals in the Tons valley in Western Himalayas. We were able to sample approximately 77% (123 species) of the estimated species richness on continuing the sampling until July 2010. Species richness at the study site is estimated to be 159 (95% CI: 145–210) species. Diversity was highest in heterogeneous habitats and decreased towards homogeneous habitats. Unique species were highly restricted to lowest disturbed sites. Using Pearson's correlation analysis, the strongest vegetative predictors of butterfly richness were plant species richness, canopy cover, and herb and shrub density. Butterfly species richness and abundance were highly correlated with altitude, temperature, relative humidity, fire signs, and livestock abundance. We also found positive cross-taxon correlation among butterflies, moths, and beetles across sites, indicating that butterflies can be used as surrogate or indicator taxa for insect conservation. Short sampling periods providing comprehensive estimates of species richness were reliable for identifying habitats and sites with the most conservation value in the Tons valley landscape.     

Small terminase couples viral DNA binding to genome-packaging ATPase activity

Packaging of viral genomes into empty procapsids is powered by a large DNA-packaging motor. In most viruses, this machine is composed of a large (L) and a small (S) terminase subunit complexed with a dodecamer of portal protein. Here we describe the 1.75?? crystal structure of the bacteriophage P22 S-terminase in a nonameric conformation. The structure presents a central channel ～23?? in diameter, sufficiently large to accommodate hydrated B-DNA. The last 23 residues of S-terminase are essential for binding to DNA and assembly to L-terminase. Upon binding to its own DNA, S-terminase functions as a specific activator of L-terminase ATPase activity. The DNA-dependent stimulation of ATPase activity thus rationalizes the exclusive specificity of genome-packaging motors for viral DNA in the crowd of host DNA, ensuring fidelity of packaging and avoiding wasteful ATP hydrolysis. This posits?a model for DNA-dependent activation of genome-packaging motors of general interest in virology.     

Structural and functional analyses of the severe acute respiratory syndrome coronavirus endoribonuclease Nsp15

The severe acute respiratory syndrome (SARS) coronavirus encodes several RNA-processing enzymes that are unusual for RNA viruses, including Nsp15 (nonstructural protein 15), a hexameric endoribonuclease that preferentially cleaves 3' of uridines. We solved the structure of a catalytically inactive mutant version of Nsp15, which was crystallized as a hexamer. The structure contains unreported flexibility in the active site of each subunit. Substitutions in the active site residues serine 293 and proline 343 allowed Nsp15 to cleave at cytidylate, whereas mutation of leucine 345 rendered Nsp15 able to cleave at purines as well as pyrimidines. Mutations that targeted the residues involved in subunit interactions generally resulted in the formation of catalytically inactive monomers. The RNA-binding residues were mapped by a method linking reversible cross-linking, RNA affinity purification, and peptide fingerprinting. Alanine substitution of several residues in the RNA-contacting portion of Nsp15 did not affect hexamer formation but decreased the affinity of RNA binding and reduced endonuclease activity. This suggests a model for Nsp15 hexamer interaction with RNA.