Purification and characterization of chloroplast transfer RNA processing enzymes.
AuthorHibbert, Catherine Sanders.
Committee ChairHallick, Richard B.
MetadataShow full item record
PublisherThe University of Arizona.
RightsCopyright © 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.
AbstractMonomeric and dimeric chloroplast tRNA precursors from Euglena gracilis DNA were synthesized using an in vitro transcription system. The lengths of the 3'- and 5'- ends of these precursors were varied to identify processing intermediates, and to study the effect of the structure of the tRNA precursors on the processing reactions. All of these pre-tRNAs, independent of their structure, are processed to mature tRNAs in both spinach and pea chloroplast soluble extracts. 5'- and 3'- endonucleases are involved in the cleavage of 5'- and 3'- ends of the pre-tRNAs. These two reactions are not ordered in vitro. Exonuclease and CCA-adding activities can be detected in the soluble extract. RNase P catalyzes the 5'-endonucleolytic cleavage of pre-tRNA transcripts. An RNase P was purified > 68,000-fold to near homogeneity from isolated chloroplasts of Pisum sativum. Purification was achieved by chromatography on DEAE-cellulose, heparin-agarose and FPLC Mono S columns. The purified enzyme migrates as a 27,000 dalton polypeptide during SDS-PAGE. The molecular weight of the native form of the enzyme is estimated to be 22,000 based on gel filtration chromatography. The enzyme does not appear to contain an essential RNA component. The highly purified enzyme has an absolute requirement for divalent cations (5 mM optimal Mg²⁺), and is stimulated by K⁺ (150 mM optimal). The enzyme activity is inhibited by heparin (50% at 18 ug/ml), and by yeast tRNA (80% at 1 mg/ml), but is slightly stimulated by poly A (0.1-2 mg/ml). Ribonuclease P2 catalyzes the cleavage of large multimeric tRNA precursors to smaller immature tRNA precursors in E. coli. A distinct pea chloroplast 5'-tRNA endonuclease resembling the RNase P2 activity was identified. The novel RNase P2-like activity was separated from the previously described RNase P-like enzyme by ammonium sulfate precipitation and phenyl sepharose, FPLC Mono Q, and heparin agarose chromatography. The >65-fold partially pure RNase P2-like activity cleaves multimeric precursor-tRNAs differently than RNase P. The dimeric pre-tRNA^(Phe-Cys} is processed but not the resulting 5'-extended intermediates. The novel activity is resistant to micrococcal nuclease treatment. A tRNA 3'-endonuclease activity from pea and spinach chloroplasts was characterized based on chromatographic properties. Molecular mass was estimated to be 55,000 by rate zonal glycerol gradient centrifugation.
Degree ProgramMolecular and Cellular Biology