Purification and characterization of a chloroplast DNA-dependent soluble RNA polymerase from pea.
Author
Boyer, Angelique Sun.Issue Date
1994Committee Chair
Hallick, Richard B.
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The University of Arizona.Rights
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.Abstract
A chloroplast DNA-dependent RNA polymerase has been purified 350,000-fold from pea. The purification procedure includes the following steps: 100,000 x g centrifugation, DE-52 ion exchange chromatography, ammonium sulfate precipitation, Sephacryl S-300 gel filtration and two sequential Mono-Q columns. The purified protein preparation was resolved into a predominant protein complex at 669 kDa by non-denaturing gel electrophoresis. Based on gel filtration chromatography the native molecular weight of the purified enzyme is approximately 620,000. Using SDS-PAGE the purified enzyme is separated into ten polypeptides of 120, 110, 95, 84, 81, 75, 54, 51, 42, and 35 kDa. The enzyme is completely dependent on an exogenous DNA template for activity. The 110 kDa polypeptide binds nucleotide triphosphates. The 42 kDa polypeptide cross-reacts with antiserum raised to the plastid encoded α-subunit protein. The enzymatic properties of the soluble RNA polymerase from pea chloroplasts were determined. The apparent energy of activation for the incorporation of UMP into RNA is 34 kcal/mole. The apparent Km value is 0.1 mM for GTP and the Vmax is 200 pmol/min/mg. The optimal conditions for maximum enzyme activity were 2 mM KCl, 15 mM MgCl₂ (or MnCl₂), 40°C and pH 7.9. The soluble enzyme activity is inhibited by (NH₄)₂SO₄, tagetitoxin and heparin. The bacterial RNA polymerase inhibitor rifampicin inhibits chloroplast RNA polymerase enzyme activity completely at high concentration (1 mg/ml). Slot blot hybridization, S1 nuclease protection, and in vitro transcription assays were employed to demonstrate that the purified RNA polymerase selectively initiates transcription from higher plant psbA and rbcL promoters and the Euglena tRNAᴾʰᵉ promoter. Transcription occurs from supercoiled DNA templates but not from linear DNA templates. The soluble RNA polymerase does not transcribe from the higher plant 16S rRNA or tRNA promoter, and does not recognize the threonine attenuator (thra).Type
textDissertation-Reproduction (electronic)
Degree Name
Ph.D.Degree Level
doctoralDegree Program
BiochemistryGraduate College