Probing the functional importance of the hexameric ring structure of RNase PH.
RNase PH is a phosphate-dependent exoribonuclease that catalyzes the removal of nucleotides at the 3' end of the tRNA precursor, leading to the release of nucleoside diphosphate, and generates the CCA end during the maturation process. The 1.9-A crystal structures of the apo and the phosphate-bound forms of RNase PH from Pseudomonas aeruginosa reveal a monomeric RNase PH with an alpha/beta-fold tightly associated into a hexameric ring structure in the form of a trimer of dimers. A five ion pair network, Glu-63-Arg-74-Asp-116-Arg-77-Asp-118 and an ion-pair Glu-26-Arg-69 that are positioned symmetrically in the trimerization interface play critical roles in the formation of a hexameric ring. Single or double mutations of Arg-69, Arg-74, or Arg-77 in these ion pairs leads to the dissociation of the RNase PH hexamer into dimers without perturbing the overall monomeric structure. The dissociated RNase PH dimer completely lost its binding affinity and catalytic activity against a precursor tRNA. Our structural and mutational analyses of RNase PH demonstrate that the hexameric ring formation is a critical feature for the function of members of the RNase PH family.
Choi
Jung Min
JM
National Creative Research Initiative Center for Structural Biology and Department of Life Science, Pohang University of Science and Technology, Hyo-ja dong, San31, Pohang, KyungBook 790-784, South Korea.
Park
Eun Young
EY
Kim
Jun Hyun
JH
Chang
Sung Key
SK
Cho
Yunje
Y
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000201:enzymology; D018360:Crystallography, X-Ray; D005095:Exoribonucleases / Q000737:chemistry*; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013816:Thermodynamics
D046911:Macromolecular Substances; D011994:Recombinant Proteins; C056502:ribonuclease PH; D005095:Exoribonucleases