Cation-dependent folding of 30 cap-independent translation elements facilitates interaction of a 17-nucleotide conserved sequence with eIF4G

The 3\u27-untranslated regions of many plant viral RNAs contain cap-independent translation elements (CITEs) that drive translation initiation at the 5\u27-end of the mRNA. The barley yellow dwarf virus-like CITE (BTE) stimulates translation by binding the eIF4G subunit of translation initiation factor eIF4F with high affinity. To understand this interaction, we characterized the dynamic structural properties of the BTE, mapped the eIF4G-binding sites on the BTE and identified a region of eIF4G that is crucial for BTE binding. BTE folding involves cooperative uptake of magnesium ions and is driven primarily by charge neutralization. Footprinting experiments revealed that functional eIF4G fragments protect the highly conserved stem-loop I and a downstream bulge. The BTE forms a functional structure in the absence of protein, and the loop that base pairs the 5\u27-untranslated region (5\u27-UTR) remains solventaccessible at high eIF4G concentrations. The region in eIF4G between the eIF4E-binding site and the MIF4G region is required for BTE binding and translation. The data support the model in which the eIF4F complex binds directly to the BTE which base pairs simultaneously to the 5\u27-UTR, allowing eIF4F to recruit the 40S ribosomal subunit to the 5\u27-end

Complexity, Collective Effects and Modelling of Ecosystems: formation, function and stability

We discuss the relevance of studying ecology within the framework of Complexity Science from a statistical mechanics approach. Ecology is concerned with understanding how systems level properties emerge out of the multitude of interactions amongst large numbers of components, leading to ecosystems that possess the prototypical characteristics of complex systems. We argue that statistical mechanics is at present the best methodology available to obtain a quantitative description of complex systems, and that ecology is in urgent need of ``integrative'' approaches that are quantitative and non-stationary. We describe examples where combining statistical mechanics and ecology has led to improved ecological modelling and, at the same time, broadened the scope of statistical mechanics.Comment: 11 pages and 1 figur

Substitution of the premembrane and envelope protein genes of Modoc virus with the homologous sequences of West Nile virus generates a chimeric virus that replicates in vertebrate but not mosquito cells

Background: Most known flaviviruses, including West Nile virus (WNV), are maintained in natural transmission cycles between hematophagous arthropods and vertebrate hosts. Other flaviviruses such as Modoc virus (MODV) and Culex flavivirus (CxFV) have host ranges restricted to vertebrates and insects, respectively. The genetic elements that modulate the differential host ranges and transmission cycles of these viruses have not been identified. Methods: Fusion polymerase chain reaction (PCR) was used to replace the capsid (C), premembrane (prM) and envelope (E) genes and the prM-E genes of a full-length MODV infectious cDNA clone with the corresponding regions of WNV and CxFV. Fusion products were directly transfected into baby hamster kidney-derived cells that stably express T7 RNA polymerase. At 4 days post-transfection, aliquots of each supernatant were inoculated onto vertebrate (BHK-21 and Vero) and mosquito (C6/36) cells which were then assayed for evidence of viral infection by reverse transcription-PCR, Western blot and plaque assay. Results: Chimeric virus was recovered in cells transfected with the fusion product containing the prM-E genes of WNV. The virus could infect vertebrate but not mosquito cells. The in vitro replication kinetics and yields of the chimeric virus were similar to MODV but the chimeric virus produced larger plaques. Chimeric virus was not recovered in cells transfected with any of the other fusion products. Conclusions: Our data indicate that genetic elements outside of the prM-E gene region of MODV condition its vertebrate-specific phenotype

Determination of the Secondary Structure of an RNA fragment in Solution: Selective 2`- Hydroxyl Acylation Analyzed by Primer Extension Assay (SHAPE)

This protocol describes the methodology for the determination of the secondary structure of an RNA fragment in solution using Selective 2´-Hydroxyl Acylation analyzed by Primer Extension, abbreviation SHAPE. It consists in the very fast chemical modification of flexible and therefore possibly single-stranded nucleotides in a sequence-independent manner using benzoyl cyanide (BzCN), forming 2´-O-adducts. The modifications in the RNA are then analyzed by primer extension. Reverse transcriptase is blocked by the 2´-O-adducts formed. The advantage of the method is, first, that not each RNA molecule studied but the primer used in the extension reaction is labelled and, second, that the resulting cDNA analyzed in sequencing gels is much more stable than the modified RN

The Impact of Consumerism on Health Care Change: Alternatives for the Future?

The quest for consumer participation in the management of health care delivery may have experienced its first signs of success, but the implications of that success are as yet unclear. The establishment of consumer majorities on the newly developed health systems agency (HSA) boards was seen as an important milestone in the development of the consumer movement in America over the last ten years. The initial wave of optimism over the Great Society programs that in part gave birth to the consumer movement has long since vanished, but some of the organizational results of those attempts at innovation have become routinely established, as the requirements for consumer participation specified in wave after wave of health related amendments clearly indicates. But what are the results of this participation, and what can we reasonably expect in the future

Debt maturity, risk, and asymmetric information

We test the implications of Flannery’s (1986) and Diamond’s (1991) models concerning the effects of risk and asymmetric information in determining debt maturity, and we examine the overall importance of informational asymmetries in debt maturity choices. We employ data from more than 6,000 commercial loans from 53 large U.S. banks. Our results for low-risk firms are consistent with the predictions of both theoretical models, but our findings for high-risk firms conflict with the predictions of Diamond’s model and with much of the empirical literature. Our findings also suggest a strong quantitative role for asymmetric information in explaining debt maturity.

Why do borrowers pledge collateral? new empirical evidence on the role of asymmetric information

An important theoretical literature motivates collateral as a mechanism that mitigates adverse selection, credit rationing, and other inefficiencies that arise when borrowers hold ex ante private information. There is no clear empirical evidence regarding the central implication of this literature—that a reduction in asymmetric information reduces the incidence of collateral. We exploit exogenous variation in lender information related to the adoption of an information technology that reduces ex ante private information, and compare collateral outcomes before and after adoption. Our results are consistent with this central implication of the private-information models and support the empirical importance of this theory.

Control of translation during the unfolded protein response in maize seedlings: Life without PERKs

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) defines a condition called ER stress that induces the unfolded protein response (UPR). The UPR in mammalian cells attenuates protein synthesis initiation, which prevents the piling up of misfolded proteins in the ER. Mammalian cells rely on Protein Kinase RNA‐Like Endoplasmic Reticulum Kinase (PERK) phosphorylation of eIF2α to arrest protein synthesis, however, plants do not have a PERK homolog, so the question is whether plants control translation in response to ER stress. We compared changes in RNA levels in the transcriptome to the RNA levels protected by ribosomes and found a decline in translation efficiency, including many UPR genes, in response to ER stress. The decline in translation efficiency is due to the fact that many mRNAs are not loaded onto polyribosomes (polysomes) in proportion to their increase in total RNA, instead some of the transcripts accumulate in stress granules (SGs). The RNAs that populate SGs are not derived from the disassembly of polysomes because protein synthesis remains steady during stress. Thus, the surge in transcription of UPR genes in response to ER stress is accompanied by the formation of SGs, and the sequestration of mRNAs in SGs may serve to temporarily relieve the translation load during ER stress