An efficient colorimetric assay for RNA synthesis by viral RNA-dependent RNA polymerases, using thermostable pyrophosphatase.

RNA-dependent RNA polymerase (RdRp) is essential for the replication of RNA genome-containing positive-strand RNA viruses. We developed a simple colorimetric assay to quantify the RNA synthesis activity of RdRp by measuring the pyrophosphates released during nascent RNA synthesis. RNA polymerase reaction was quenched by heating at 70 °C for 5 min, during which thermostable inorganic pyrophosphatase converted the accumulated pyrophosphates into inorganic phosphates. Subsequently, the amount of inorganic phosphate was measured using a color-developing reagent. Using RdRp's from hepatitis C virus and foot-and-mouth disease virus, we demonstrate that this colorimetric assay facilitates the measurement of RNA polymerase activity.

Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of the co-chaperonin XoGroES from Xanthomonas oryzae pv. oryzae.

Bacterial blight (BB), a devastating disease caused by Xanthomonas oryzae pv. oryzae (Xoo), causes serious production losses of rice in Asian countries. Protein misfolding may interfere with the function of proteins in all living cells and must be prevented to avoid cellular disaster. All cells naturally contain molecular chaperones that assist the unfolded proteins in folding into the native structure. One of the well characterized chaperone complexes is GroEL-GroES. GroEL, which consists of two chambers, captures misfolded proteins and refolds them. GroES is a co-chaperonin protein that assists the GroEL protein as a lid that temporarily closes the chamber during the folding process. Xoo4289, the GroES gene from Xoo, was cloned and expressed for X-ray crystallographic study. The purified protein (XoGroES) was crystallized using the hanging-drop vapour-diffusion method and a crystal diffracted to 2.0 Šresolution. The crystal belonged to the hexagonal space group P6(1), with unit-cell parameters a=64.4, c=36.5 Å. The crystal contains a single molecule in the asymmetric unit, with a corresponding VM of 2.05 Å3 Da(-1) and a solvent content of 39.9%.