Expression, purification, crystallization and preliminary diffraction data characterization of Escherichia coli ribonuclease II (RNase II)
| Autor: | Colin E. McVey, Fátima Cairrão, Célia V. Romão, Cecília M. Arraiano, Maria Arménia Carrondo, Ana Barbas, Carlos Frazão, Ricardo Coelho, Mónica Amblar |
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| Rok vydání: | 2006 |
| Předmět: |
RNase P
Mutant Biophysics medicine.disease_cause Biochemistry X-Ray Diffraction Structural Biology Gene expression Genetics medicine Escherichia coli Ribonuclease III Selenomethionine biology Chemistry Escherichia coli Proteins Wild type RNA Condensed Matter Physics Ribonucleoside Recombinant Proteins Mutagenesis Crystallization Communications Exoribonucleases biology.protein Crystallization |
| Zdroj: | Acta crystallographica. Section F, Structural biology and crystallization communications. 62(Pt 7) |
| ISSN: | 1744-3091 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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