Accurate placement of substrate RNA by Gar1 in H/ACA RNA-guided pseudouridylation

H/ACA RNA-guided ribonucleoprotein particle (RNP), the most complicated RNA pseudouridylase so far known, uses H/ACA guide RNA for substrate capture and four proteins (Cbf5, Nop10, L7Ae and Gar1) for pseudouridylation. Although it was shown that Gar1 not only facilitates the product release, but also enhances the catalytic activity, the chemical role that Gar1 plays in this complicated machinery is largely unknown. Kinetics measurement on Pyrococcus furiosus RNPs at different temperatures making use of fluorescence anisotropy showed that Gar1 reduces the catalytic barrier through affecting the activation entropy instead of enthalpy. Site-directed mutagenesis combined with molecular dynamics simulations demonstrated that V149 in the thumb loop of Cbf5 is critical in placing the target uridine to the right position toward catalytic D85 of Cbf5. The enzyme elegantly aligns the position of uridine in the catalytic site with the help of Gar1. In addition, conversion of uridine to pseudouridine results in a rigid syn configuration of the target nucleotide in the active site and causes Gar1 to pull out the thumb. Both factors guarantee the efficient release of the product.     

Chiral Recognition of Tyrosine Enantiomers Based on Decreased Resonance Scattering Signals With Silver Nanoparticles as Optical Sensor

A novel chiral sensing platform, employing silver nanoparticles capped with N‐acetyl‐L‐cysteine (NALC‐Ag NPs), was utilized for the discrimination of L‐tyrosine and D‐tyrosine. This nanosensor, which could be used as an optical sensing unit and chiral probe, was characterized by transmission electron microscopy (TEM) and resonance Rayleigh scattering (RRS) spectroscopy. After the proposed sensing platform interacted with L‐tyrosine and D‐tyrosine, a decreased resonance scattering signal was only obtained from L‐tyrosine. This phenomenon offered a useful assay for the selectivity and determination of L‐tyrosine with the RRS method. The linear range and detection limit of L‐tyrosine were 0.2838–20.0 µg⋅mL^‐1 and 0.0860 µg⋅mL^‐1, respectively. In addition, experimental factors such as acidity, interaction time, and the concentration of enantiomers were investigated with regard to the effect on enantioselective interaction. Chirality 27:194–198, 2015. © 2014 Wiley Periodicals, Inc.