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 P2(1). X-ray diffraction data were collected to 2.44 and 2.75 angstroms resolution, with unit-cell parameters a = 56.8, b = 125.7, c = 66.2 angstroms, beta = 111.9 degrees and a = 119.6, b = 57.2, c = 121.2 angstroms, beta = 99.7 degrees, respectively. The RNase II D209N mutant gave crystals that belonged to space group P6(5), with unit-cell parameters a = b = 86.3, c = 279.2 angstroms, and diffracted to 2.74 angstroms. Diffraction data from the mutant and its SeMet derivative enabled the determination of a partial Se-atom substructure by SIRAS.
McVey
Colin E
CE
Division of Biological Chemistry, ITQB--Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apt. 127, 2781-901 Oeiras, Portugal.
Amblar
Mónica
M
Barbas
Ana
A
Cairrão
Fátima
F
Coelho
Ricardo
R
Romão
Célia
C
Arraiano
Cecília M
CM
Carrondo
Maria A
MA
Frazão
Carlos
C
D003460:Crystallization; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005095:Exoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012645:Selenomethionine; D014961:X-Ray Diffraction
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D012645:Selenomethionine; D005095:Exoribonucleases; C022535:exoribonuclease II