Ribosomal Pausing at a Frameshifter RNA Pseudoknot Is Sensitive to Reading Phase but Shows Little Correlation with Frameshift Efficiency

Here we investigated ribosomal pausing at sites of programmed -1 ribosomal frameshifting, using translational elongation and ribosome heelprint assays. The site of pausing at the frameshift signal of infectious bronchitis virus (IBV) was determined and was consistent with an RNA pseudoknot-induced pause that placed the ribosomal P- and A-sites over the slippery sequence. Similarly, pausing at the simian retrovirus 1 gag/pol signal, which contains a different kind of frameshifter pseudoknot, also placed the ribosome over the slippery sequence, supporting a role for pausing in frameshifting. However, a simple correlation between pausing and frameshifting was lacking. Firstly, a stem-loop structure closely related to the IBV pseudoknot, although unable to stimulate efficient frameshifting, paused ribosomes to a similar extent and at the same place on the mRNA as a parental pseudoknot. Secondly, an identical pausing pattern was induced by two pseudoknots differing only by a single loop 2 nucleotide yet with different functionalities in frameshifting. The final observation arose from an assessment of the impact of reading phase on pausing. Given that ribosomes advance in triplet fashion, we tested whether the reading frame in which ribosomes encounter an RNA structure (the reading phase) would influence pausing. We found that the reading phase did influence pausing but unexpectedly, the mRNA with the pseudoknot in the phase which gave the least pausing was found to promote frameshifting more efficiently than the other variants. Overall, these experiments support the view that pausing alone is insufficient to mediate frameshifting and additional events are required. The phase dependence of pausing may be indicative of an activity in the ribosome that requires an optimal contact with mRNA secondary structures for efficient unwinding.     

Investigating molecular mechanisms of 2A-stimulated ribosomal pausing and frameshifting in Theilovirus

The 2A protein of Theiler''s murine encephalomyelitis virus (TMEV) acts as a switch to stimulate programmed –1 ribosomal frameshifting (PRF) during infection. Here, we present the X-ray crystal structure of TMEV 2A and define how it recognises the stimulatory RNA element. We demonstrate a critical role for bases upstream of the originally predicted stem–loop, providing evidence for a pseudoknot-like conformation and suggesting that the recognition of this pseudoknot by beta-shell proteins is a conserved feature in cardioviruses. Through examination of PRF in TMEV-infected cells by ribosome profiling, we identify a series of ribosomal pauses around the site of PRF induced by the 2A-pseudoknot complex. Careful normalisation of ribosomal profiling data with a 2A knockout virus facilitated the identification, through disome analysis, of ribosome stacking at the TMEV frameshifting signal. These experiments provide unparalleled detail of the molecular mechanisms underpinning Theilovirus protein-stimulated frameshifting.     

Antioxidant protection against iron toxicity: Plasma changes during cardiopulmonary bypass in neonates, infants, and children

Cardiopulmonary bypass surgery is associated with the release of low molecular mass iron, which increases the saturation of plasma transferrin to over 50% in all adult patients treated. In a significant minority, however plasma transferrin becomes 100% iron saturated and non-transferrin bound iron can be detected in the plasma. An iron-saturated transferrin is also a common physiological finding in normal term and pre-term infants at a time when their plasma antioxidants, which protect against iron toxicity and radical scavening, are profoundly different from those seen in adults. This study was conducted to assess the extent to which antioxidants, which protect against iron toxicity, are altered in neonates, infants, and children undergoing cardiopulmonary bypass surgery.     

Post-European changes to the fluvial geomorphology of Bega catchment, Australia: implications for river ecology

1. Within a few decades of European disturbance in the mid-nineteenth century, river character and behaviour were transformed in Bega catchment on the south coast of New South Wales, Australia. Ecological impacts of geomorphic changes to river structure and function throughout the catchment are assessed. 2. At the time of European settlement, many water courses in Bega catchment were discontinuous, with extensive swamps along middle and upper courses. Following a series of direct and indirect human impacts, channels became continuous in the middle and upper parts of the catchment, as extensive valley fills at the base of the escarpment were incised. Along the lowland plain, the channel widened by over 300%, fundamentally altering the relationship between the channel and its adjacent floodplain. 3. Geomorphic changes to river structure have modified habitat availability throughout Bega catchment. The impacts have been least pronounced in headwater streams, but have been dramatic along virtually all river courses beyond the base of the escarpment. 4. Changes in river structure have been directly related to altered riparian vegetation cover, and vice versa. As a consequence of changes to river structure, bed substrate calibre (and supply volume/rate) has been modified along most streams. 5. A series of indirect, secondary impacts have modified habitat viability along river courses. Lateral, longitudinal and vertical linkages within the river system have been altered, affecting the transfer of water, sediment, organic matter, nutrients and other biotic interactions. 6. These direct and indirect consequences of geomorphic changes in river structure suggest that ecologists need to adopt a longer-term, catchment-framed view of human disturbance to river ecosystems. 7. Effective, sustainable ecological rehabilitation of river courses is dependent on an understanding of geomorphic processes and determination of appropriate river structure at differing positions in catchments.     

Ion transport in heart mitochondria. XIII. The effect of ethylenediaminetetraacetate on monovalent ion uptake

1. Ethylenediaminetetraacetate (EDTA) markedly activates the accumulation of Na⁺ and Li⁺ and the swelling which accompanies the ion uptake by isolated heart mitochondria. This activation is reflected in the removal of limited amounts of endogenous Mg²⁺ and extensive loss of K⁺. The removal of these cations requires the presence of Na⁺, a source of energy, and a permeant anion as well as EDTA. The effects of EDTA on the activation of Na⁺ uptake and cation removal are duplicated by chelators with a high affinity for Mg²⁺, but not by ethyleneglycol-bis-(β-aminoethylether)-N, N′-tetraacetic acid. Mg²⁺ at concentrations 5 to 6 times less than EDTA prevents both activation of Na⁺ uptake and cation removal.

2. EDTA does not appear to be bound by heart mitochondria. At neutral pH the chelator penetrates into the mitochondrial water volume to the same extent as sucrose and mannitol. At pH 8.1 where the removal of mitochondrial Mg²⁺ by EDTA is more effective, EDTA penetrates virtually the entire water volume. This penetration requires the presence of a source of energy, a transported cation such as Na⁺, and a permeant anion. It appears possible that the oscillations in ion uptake and swelling observed in the presence of EDTA at pH 8.1 may be related to the presence of the chelator in the interior compartment under these conditions.     

Sequence specificity in the interaction of Bluetongue virus non-structural protein 2 (NS2) with viral RNA

The non-structural protein NS2 of Bluetongue virus (BTV) is synthesized abundantly in virus-infected cells and has been suggested to be involved in virus replication. The protein, with a high content of charged residues, possesses a strong affinity for single-stranded RNA species but, to date, all studies have failed to identify any specificity in the NS2-RNA interaction. In this report, we have examined, through RNA binding assays using highly purified NS2, the specificity of interaction with different single-stranded RNA (ssRNA) species in the presence of appropriate competitors. The data obtained show that NS2 indeed has a preference for BTV ssRNA over nonspecific RNA species and that NS2 recognizes a specific region within the BTV10 segment S10. The secondary structure of this region was determined and found to be a hairpin-loop with substructures within the loop. Modification-inhibition experiments highlighted two regions within this structure that were protected from ribonuclease cleavage in the presence of NS2. Overall, these data imply that a function of NS2 may be to recruit virus messenger RNAs (that also act as templates for synthesis of genomic RNAs) selectively from other RNA species within the infected cytosol of the cell during virus replication.