Abstract
Abstract An RNA phosphodiesterase of molecular weight about 100,000 was purified 330-fold from the cell-free culture fluid of a Bacillus species grown on RNA as the sole source of phosphorus. In the presence of Mg2+ or Mn2+, this nuclease catalyzes hydrolysis of high molecular weight RNA and synthetic polyribonucleotides, but not of DNA, dinucleoside monophosphates, 2',3'- or 3',5'-cyclic nucleotides or adenylate oligonucleotides terminating in a 2',3'-cyclic adenylic acid residue. While the initial products are nucleoside 5'-phosphates, nucleosides eventually are formed due to the presence of a 5'-nucleotidase activity which purifies together with the phosphodiesterase. These two activities, which are not separable by acrylamide gel electrophoresis or by sucrose density gradient centrifugation, account for the ability of the organism to grow on RNA as a sole source of phosphorus. Polymer cleavage appears to proceed from the 3'-hydroxyl terminus and to be exonucleolytic. Oligonucleotides characteristic of endonucleolytic degradation are not formed as intermediates during hydrolysis of RNA, and ApCpC and CpCpA are hydrolyzed to ApC plus cytidine and CpC plus adenosine, respectively. Ribose- or deoxyribose-nucleoside 5'-phosphates, but not nucleoside 2'- or 3'-phosphates, are good substrates for the 5'-nucleotidase activity.
Highlights
Oligonucleotides characteristic of endonucleolytic degradation are not formed as intermediates during hydrolysis of RNA, and ApCpC and
We report here the purification, kinetic properties, and substrate specificity of the RNA phosphodiesterase and its associated 5’.nucleotidase activity
Base-specific RNA depolymerases are of great interest with respect to their application to the determination of the primar? structures of RNA’s In the hope of obtaining an RNA depolymerase with a specificity useful for RNA structural studies, we isolated soil bacteria by elective culture on compounds containing phosphodiester bonds as the sole source of phosphorus
Summary
Chemicals-Chemicals from commercial sources includedSephadex G-10 and G-200 (Pharmacia), DEAE-cellulose (Eastman) ; Chelex resin (Bio-Rad Laboratories) ; Tris, ammonium persulfate, acrylamide, and N, N’-methylenebisacrylamide (Canalco); human y-globulin, bovine serum albumin, low and high molecular weight yeast RNA (Mann) ; glycine (NutritionalBiochemicals Corp.); poly(C,Ul.c) (C:U ratio 1.0:1.4) and poly-(G,UI.,) (G:U ratio 1 .O: 1.4) (Miles Laboratories); other polynucleotides, nucleosides, nucleotides, and Aquacide I (Calbiochem) ; cyclic nucleotides, calf thymus DNA, p-nitrophenylphosphate, sodium bis-p-nitrophenylphosphate, nL-a-glycerophosphate, n-glucose B-phosphate, oc-n-glucose l-phosphate, and pancreatic ribonuclease (ribonucleate pyrimidine-nucleotido-Z’-transferase (cyclizing), EC 2.7.7.16) (Sigma).Chromatography and Electrophoresis-Solvents used for descending paper chromatography were: Solvent I, 2-propanol-15N NHIOH-H20, 7: 1: 2 (v/v) [3]; Solvent II, isobutyric acid-15 xNHIOH-H20, 66: 1:33 (v/v) [4] ; Solvent III, saturated aqueous (NH&SO&propanol-1.0M ammonium acetate, 40: 1:9 (v/v)(5); Solvent IV, 95% ethanol-l.0 M ammonium acetate, 7 :3(v/v) [6]. Biochemicals Corp.); poly(C,Ul.c) (C:U ratio 1.0:1.4) and poly-. (G,UI.,) (G:U ratio 1 .O: 1.4) (Miles Laboratories); other polynucleotides, nucleosides, nucleotides, and Aquacide I (Calbiochem) ; cyclic nucleotides, calf thymus DNA, p-nitrophenylphosphate, sodium bis-p-nitrophenylphosphate, nL-a-glycerophosphate, n-glucose B-phosphate, oc-n-glucose l-phosphate, and pancreatic ribonuclease Chromatography and Electrophoresis-Solvents used for descending paper chromatography were: Solvent I, 2-propanol-15. N NHIOH-H20, 7: 1: 2 (v/v) [3]; Solvent II, isobutyric acid-15 x. NHIOH-H20, 66: 1:33 (v/v) [4] ; Solvent III, saturated aqueous Paper electrophoresis in 50 II3M ammonium formate, pH 4.0, or 100 InM sodium borate, pH 9.2, was conducted at a field strength of 30 volts per cm in a Savant FP-3OA flat bed apparatus. Visualization of compounds on chromatograms and electrophoretograms was by ultraviolet quench
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
