Recognition of viral RNA stem–loops by the tandem double-stranded RNA binding domains of PKR

In humans, the double-stranded RNA (dsRNA)-activated protein kinase (PKR) is expressed in late stages of the innate immune response to viral infection by the interferon pathway. PKR consists of tandem dsRNA binding motifs (dsRBMs) connected via a flexible linker to a Ser/Thr kinase domain. Upon interaction with viral dsRNA, PKR is converted into a catalytically active enzyme capable of phosphorylating a number of target proteins that often results in host cell translational repression. A number of high-resolution structural studies involving individual dsRBMs from proteins other than PKR have highlighted the key features required for interaction with perfectly duplexed RNA substrates. However, viral dsRNA molecules are highly structured and often contain deviations from perfect A-form RNA helices. By use of small-angle X-ray scattering (SAXS), we present solution conformations of the tandem dsRBMs of PKR in complex with two imperfectly base-paired viral dsRNA stem–loops; HIV-1 TAR and adenovirus VA_I-AS. Both individual components and complexes were purified by size exclusion chromatography and characterized by dynamic light scattering at multiple concentrations to ensure monodispersity. SAXS ab initio solution conformations of the individual components and RNA–protein complexes were determined and highlight the potential of PKR to interact with both stem and loop regions of the RNA. Excellent agreement between experimental and model-based hydrodynamic parameter determination heightens our confidence in the obtained models. Taken together, these data support and provide a framework for the existing biochemical data regarding the tolerance of imperfectly base-paired viral dsRNA by PKR.     

Spatial variability of nitrous oxide emissions from an Australian irrigated dairy pasture

Understanding spatial variability of emissions of nitrous oxide (N₂O) is essential to understanding of N₂O emissions from soils to the atmosphere and in the design of statistically valid measurement programs to determine plot, farm and regional emission rates. Two afternoon, ‘snap-shot’ experiments were conducted; one in the summer and one in the autumn of 2004, to examine the statistics and soil variables affecting the spatial variability of N₂O emissions at paddock scale. Small, static chambers (mini-chambers) were placed at 100 locations over an 8,100 m² area of irrigated dairy pasture in northern Victoria, Australia. Chamber headspace was sampled for N₂O and soil samples taken below each mini-chamber were analysed for soil nitrate (NO₃ ^-), ammonium (NH₄ ⁺) and other chemical and physical properties known to affect N₂O emissions. The experiments took place immediately after the sequence of grazing, urea application and irrigation. Nitrous oxide emissions and soil variables were analysed using classical statistics to investigate the effect of soil variables on N₂O emissions. Geostatistics were used to investigate spatial patterns of N₂O emissions and soil variables over the measurement area. Nitrous oxide emissions were extremely variable; 45–765 ng N₂O–N m^?2 s^?1 and 20–953 ng N₂O–N m^?2 s^?1 for the two experiments with corresponding averages of 165 and 138 ng N₂O–N m^?2 s^?1. Nitrous oxide emissions showed spatial dependence up to 73 and 51 m for the two experiments. Nitrous oxide emissions showed significant correlation with soil nutrients in decreasing order of NO₃ ^-, NH₄ ⁺ and available-P concentrations. There was no significant correlation of N₂O emissions with measured soil physical properties.