Purification and crystallization of Escherichia coli oligoribonuclease.

Oligoribonuclease (Orn) is an essential 3'-to-5' hydrolytic exoribonuclease which degrades short oligoribonucleotides to 5' mononucleotides. Escherichia coli Orn has been crystallized under several different conditions using ammonium sulfate, sodium citrate and sodium acetate as precipitants. Both native and selenomethionine-labeled oligoribonuclease (SeMet-Orn) can be crystallized at room temperature in 1.4-1.55 M sodium citrate. The SeMet-Orn crystals diffract to 2.2 A resolution and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 70.43, b = 72.87, c = 147.76 A, and two dimers in the asymmetric unit. When grown in the presence of manganese, a second crystal form (Mn-SeMet-Orn) was obtained containing a single dimer per asymmetric unit (P2(1)2(1)2(1); a = 63.74, b = 74.31, c = 74.19 A). Finally, a hexagonal crystal form was obtained using sodium acetate as a precipitant (a = 91.5, b = 91.5, c = 111.1 A). This crystal (Zn-ApUp-Orn) belongs to the P6(5) space group and has three oligoribonuclease molecules per asymmetric unit.

[1]  M. Deutscher,et al.  Oligoribonuclease is distinct from the other known exoribonucleases of Escherichia coli , 1995, Journal of bacteriology.

[2]  K. Isono,et al.  The physical map of the whole E. coli chromosome: Application of a new strategy for rapid analysis and sorting of a large genomic library , 1987, Cell.

[3]  D. Kennell.  Processing Endoribonucleases and mRNA Degradation in Bacteria , 2002, Journal of bacteriology.

[4]  Susan E. Brown,et al.  Structural basis for proofreading during replication of the Escherichia coli chromosome. , 2002, Structure.

[5]  R F Standaert,et al.  Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin. , 1993, Journal of molecular biology.

[6]  Sung-Hou Kim,et al.  Sparse matrix sampling: a screening method for crystallization of proteins , 1991 .

[7]  B. Matthews.  Solvent content of protein crystals. , 1968, Journal of molecular biology.

[8]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[9]  G. Mackie,et al.  Degradation of mRNA in Escherichia coli: an old problem with some new twists. , 1999, Progress in nucleic acid research and molecular biology.

[10]  A. J. Carpousis,et al.  mRNA degradation. A tale of poly(A) and multiprotein machines. , 1999, Trends in genetics : TIG.

[11]  A. Thomson,et al.  mRNA Stability and the Control of Gene Expression: Implications for Human Disease , 2002, Neurochemical Research.

[12]  M. Deutscher,et al.  Oligoribonuclease Is Encoded by a Highly Conserved Gene in the 3′-5′ Exonuclease Superfamily , 1998, Journal of bacteriology.

[13]  T. Steitz.  DNA Polymerases: Structural Diversity and Common Mechanisms* , 1999, The Journal of Biological Chemistry.

[14]  M. Deutscher,et al.  Exoribonucleases and their multiple roles in RNA metabolism. , 2001, Progress in nucleic acid research and molecular biology.

[15]  S. Doublié.  [29] Preparation of selenomethionyl proteins for phase determination. , 1997, Methods in enzymology.

[16]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[17]  K. Niyogi,et al.  A novel oligoribonuclease of Escherichia coli. II. Mechanism of action. , 1975, The Journal of biological chemistry.

[18]  M. Deutscher,et al.  Exoribonuclease superfamilies: structural analysis and phylogenetic distribution. , 2001, Nucleic acids research.

[19]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[20]  M. Deutscher.  Promiscuous exoribonucleases of Escherichia coli , 1993, Journal of Bacteriology.

[21]  J. P. Erzberger,et al.  The Human Homolog of Escherichia coli Orn Degrades Small Single-stranded RNA and DNA Oligomers* , 2000, The Journal of Biological Chemistry.