Abstract
We have analyzed the kinetics of assembly and elongation of the mammalian RNA polymerase I complex on endogenous ribosomal genes in the nuclei of living cells with the use of in vivo microscopy. We show that components of the RNA polymerase I machinery are brought to ribosomal genes as distinct subunits and that assembly occurs via metastable intermediates. With the use of computational modeling of imaging data, we have determined the in vivo elongation time of the polymerase, and measurements of recruitment and incorporation frequencies show that incorporation of components into the assembling polymerase is inefficient. Our data provide a kinetic and mechanistic framework for the function of a mammalian RNA polymerase in living cells.
MeSH terms
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Animals
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Catalytic Domain
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Cell Line
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Cell Nucleolus / metabolism
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Cell Nucleus / metabolism*
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Computer Simulation
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DNA, Ribosomal / genetics
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Fluorescence
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Fluorescence Recovery After Photobleaching
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Fluorescent Dyes
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Green Fluorescent Proteins
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Haplorhini
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Humans
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In Situ Hybridization, Fluorescence
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Kinetics
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Least-Squares Analysis
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Luminescent Proteins
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Microscopy
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Pol1 Transcription Initiation Complex Proteins / metabolism
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Probability
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Promoter Regions, Genetic
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Protein Subunits
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RNA Polymerase I / genetics
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RNA Polymerase I / metabolism*
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Recombinant Fusion Proteins / metabolism
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Transcription, Genetic*
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Transfection
Substances
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DNA, Ribosomal
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Fluorescent Dyes
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Luminescent Proteins
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Pol1 Transcription Initiation Complex Proteins
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Protein Subunits
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Recombinant Fusion Proteins
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Green Fluorescent Proteins
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RNA Polymerase I