Expression, purification, crystallization and preliminary diffraction data characterization of Escherichia coli ribonuclease II (RNase II)
RNA degradation is important in the post‐transcriptional control of gene expression. The processing, degradation and quality control of RNA is performed by many different classes of ribonucleases. Ribonuclease II (RNase II) is a 643‐amino‐acid enzyme that degrades single‐stranded RNA from its 3′‐end, releasing ribonucleoside 5′‐monophosphates. RNase II was expressed both as the wild type and as a D209N mutant form. The latter was also produced as an SeMet derivative. The various protein forms were crystallized using the vapour‐diffusion method. Wild‐type RNase II was crystallized in two crystal forms, both of which belonged to space group P21. X‐ray diffraction data were collected to 2.44 and 2.75 Å resolution, with unit‐cell parameters a = 56.8, b = 125.7, c = 66.2 Å, β = 111.9° and a = 119.6, b = 57.2, c = 121.2 Å, β = 99.7°, respectively. The RNase II D209N mutant gave crystals that belonged to space group P65, with unit‐cell parameters a = b = 86.3, c = 279.2 Å, and diffracted to 2.74 Å. Diffraction data from the mutant and its SeMet derivative enabled the determination of a partial Se‐atom substructure by SIRAS.
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