Conserved nucleotides in an RNA essential for hepatitis B virus replication show distinct mobility patterns

The number of regulatory RNAs with identified non-canonical structures is increasing, and structural transitions often play a role in their biological function. This stimulates interest in internal motions of RNA, which can underlie structural transitions. Heteronuclear NMR relaxation measurements, which are commonly used to study internal motion, only report on local motions of few sites within the molecule. Here we have studied a 27-nt segment of the human hepatitis B virus (HBV) pregenomic RNA, which is essential for viral replication. We combined heteronuclear relaxation with the new off-resonance ROESY technique, which reports on internal motions of H,H contacts. Using off-resonance ROESY, we could for the first time detect motion of through-space H,H contacts, such as in intra-residue base-ribose contacts or inter-nucleotide contacts, both essential for NMR structure determination. Motions in non-canonical structure elements were found primarily on the sub-nanosecond timescale. Different patterns of mobility were observed among several mobile nucleotides. The most mobile nucleotides are highly conserved among different HBV strains, suggesting that their mobility patterns may be necessary for the RNA’s biological function

Cytosine ribose flexibility in DNA: a combined NMR 13C spin relaxation and molecular dynamics simulation study

Using 13C spin relaxation NMR in combination with molecular dynamic (MD) simulations, we characterized internal motions within double-stranded DNA on the pico- to nano-second time scale. We found that the C–H vectors in all cytosine ribose moieties within the Dickerson–Drew dodecamer (5′-CGCGAATTCGCG-3′) are subject to high amplitude motions, while the other nucleotides are essentially rigid. MD simulations showed that repuckering is a likely motional model for the cytosine ribose moiety. Repuckering occurs with a time constant of around 100 ps. Knowledge of DNA dynamics will contribute to our understanding of the recognition specificity of DNA-binding proteins such as cytosine methyltransferase

Anaplerotic flux into the Calvin–Benson cycle: hydrogen isotope evidence for in vivo occurrence in C 3 metabolism

As the central carbon uptake pathway in photosynthetic cells, the Calvin–Benson cycle is among the most important biochemical cycles for life on Earth. A carbon flux of anaplerotic origin (i.e. through the chloroplast-localized oxidative branch of the pentose phosphate pathway) into the Calvin–Benson cycle was proposed recently. Here, we measured intramolecular deuterium abundances in leaf starch of Helianthus annuus grown at varying ambient CO2 concentrations, Ca. Additionally, we modelled deuterium fractionations expected for the anaplerotic pathway and compared modelled with measured fractionations. We report deuterium fractionation signals at H1 and H2 of starch glucose. Below a Ca change point, these signals increase with decreasing Ca consistent with modelled fractionations by anaplerotic flux. Under standard conditions (Ca = 450 ppm corresponding to intercellular CO2 concentrations, Ci, of 328 ppm), we estimate negligible anaplerotic flux. At Ca = 180 ppm (Ci = 140 ppm), more than 10% of the glucose-6-phosphate entering the starch biosynthesis pathway is diverted into the anaplerotic pathway. In conclusion, we report evidence consistent with anaplerotic carbon flux into the Calvin–Benson cycle in vivo. We propose the flux may help to: maintain high levels of ribulose 1,5-bisphosphate under source-limited growth conditions to facilitate photorespiratory nitrogen assimilation required to build-up source strength; and counteract oxidative stress

A model of photosynthetic CO2 assimilation in C3 leaves accounting for respiration and energy recycling by the plastidial oxidative pentose phosphate pathway

Recently, we reported estimates of anaplerotic carbon flux through the oxidative pentose phosphate pathway (OPPP) in chloroplasts into the Calvin–Benson cycle. These estimates were based on intramolecular hydrogen isotope analysis of sunflower leaf starch. However, the isotope method is believed to underestimate the actual flux at low atmospheric CO2 concentration (Ca). Since the OPPP releases CO2 and reduces NADP+, it can be expected to affect leaf gas exchange under both rubisco- and RuBP-regeneration-limited conditions. Therefore, we expanded Farquhar-von Caemmerer–Berry models to account for OPPP metabolism. Based on model parameterisation with values from the literature, we estimated OPPP-related effects on leaf carbon and energy metabolism in the sunflowers analysed previously. We found that flux through the plastidial OPPP increases both above and below Ca ≈ 450 ppm (the condition the plants were acclimated to). This is qualitatively consistent with our previous isotope-based estimates, yet gas-exchange-based estimates are larger at low Ca. We discuss our results in relation to regulatory properties of the plastidial and cytosolic OPPP, the proposed variability of CO2 mesophyll conductance, and the contribution of day respiration to the A/Ci curve drop at high Ca. Furthermore, we critically examine the models and parameterisation and derive recommendations for follow-up studies

Carbon flux around leaf-cytosolic glyceraldehyde-3-phosphate dehydrogenase introduces a 13C signal in plant glucose

Within the plant and earth sciences, stable isotope analysis is a versatile tool conveying information (inter alia) about plant physiological and paleoclimate variability across scales. Here, we identify a 13C signal (i.e., systematic 13C/ 12C variation) at tree-ring glucose C-4 and report an experimentally testable theory on its origin. We propose the signal is introduced by glyceraldehyde-3-phosphate dehydrogenases in the cytosol of leaves. It conveys two kinds of (potentially convoluted) information: (i) commitment of glyceraldehyde 3-phosphate to 3-phosphoglycerate vs. fructose 1,6-bisphosphate metabolism, and (ii) the contribution of non-phosphorylating vs. phosphorylating glyceraldehyde-3-phosphate dehydrogenase to catalysing the glyceraldehyde 3-phosphate to 3-phosphoglycerate forward reaction of glycolysis. Theory is supported by 13C fractionation modelling. Modelling results provide first evidence in support of the cytosolic oxidation-reduction (COR) cycle, a carbon-neutral mechanism supplying NADPH at the expense of ATP and NADH which may help to maintain leaf-cytosolic redox balances. In line with expectations related to COR cycling, we found a positive correlation between air vapour pressure deficit and 13C discrimination at glucose C-4. Overall, 13C-4 signal analysis may enable an improved understanding of leaf carbon and energy metabolism.ISSN:1460-2431ISSN:0022-095

Carbon flux around leaf-cytosolic glyceraldehyde-3-phosphate dehydrogenase introduces a 13C signal in plant glucose

Within the plant and earth sciences, stable isotope analysis is a versatile tool conveying information (inter alia) about plant physiological and paleoclimate variability across scales. Here, we identify a 13C signal (i.e., systematic 13C/12C variation) at tree-ring glucose C-4 and report an experimentally testable theory on its origin. We propose the signal is introduced by glyceraldehyde-3-phosphate dehydrogenases in the cytosol of leaves. It conveys two kinds of (potentially convoluted) information: (i) commitment of glyceraldehyde 3-phosphate to 3-phosphoglycerate vs. fructose 1,6-bisphosphate metabolism, and (ii) the contribution of non-phosphorylating vs. phosphorylating glyceraldehyde-3-phosphate dehydrogenase to catalysing the glyceraldehyde 3-phosphate to 3-phosphoglycerate forward reaction of glycolysis. Theory is supported by 13C fractionation modelling. Modelling results provide first evidence in support of the cytosolic oxidation-reduction (COR) cycle, a carbon-neutral mechanism supplying NADPH at the expense of ATP and NADH which may help to maintain leaf-cytosolic redox balances. In line with expectations related to COR cycling, we found a positive correlation between air vapour pressure deficit and 13C discrimination at glucose C-4. Overall, 13C-4 signal analysis may enable an improved understanding of leaf carbon and energy metabolism