Production of RNA by a polymerase protein encapsulated within phospholipid vesicles |
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Authors: | Ajoy C Chakrabarti Ronald R Breaker Gerald F Joyce David W Deamer |
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Institution: | (1) Department of Chemistry and Biochemistry, University of California, 95064 Santa Cruz, Santa Cruz, CA, USA;(2) Departments of Chemistry and Molecular Biology, The Scripps Research Institute, 10666 N. Torrey Pines Road, 92037 CA, La Jolla, USA |
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Abstract: | Catalyzed polymerization reactions represent a primary anabolic activity of all cells. It can be assumed that early cells carried out such reactions, in which macromolecular catalysts were encapsulated within some type of boundary membrane. In the experiments described here, we show that a template-independent RNA polymerase (polynucleotide phosphorylase) can be encapsulated in dimyristoyl phosphatidylcholine vesicles without substrate. When the substrate adenosine diphosphate (ADP) was provided externally, long-chain RNA polymers were synthesized within the vesicles. Substrate flux was maximized by maintaining the vesicles at the phase transition temperature of the component lipid. A protease was introduced externally as an additional control. Free enzyme was inactivated under identical conditions. RNA products were visualized in situ by ethidium bromide fluorescence. The products were harvested from the liposomes, radiolabeled, and analyzed by polyacrylamide gel electrophoresis. Encapsulated catalysts represent a model for primitive cellular systems in which an RNA polymerase was entrapped within a protected microenvironment.Abbreviations ADP
adenosine diphosphate
- DMPC
dimyristoyl phosphatidylcholine
- EDTA
ethylenediaminetetraacetic acid
- LUV
large unilamellar vesicle
- MLV
multilamellar vesicle
- PAGE
polyacrylamide gel electrophoresis
- PNPase or PNP
polynucleotide phosphorylase
- SUV
small unilamellar vesicle
Correspondence to.: A.C. Chakrabarti |
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Keywords: | RNA Liposome Biogenesis Origin of life Polynucleotide phosphorylase Polymerase Permeability |
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