Numbers and organization of RNA polymerases, nascent transcripts, and transcription units in HeLa nuclei

Using HeLa cells, we have developed methods to determine 1) the number of RNA polymerases that are active at any moment, 2) the number of transcription sites, and 3) the number of polymerases associated with one transcription unit. To count engaged polymerases, cells were encapsulated in agarose, pe...

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Bibliographic Details
Published in:Molecular biology of the cell 1998-06, Vol.9 (6), p.1523-1536
Main Authors: Jackson, D A, Iborra, F J, Manders, E M, Cook, P R
Format: Article
Language:English
Subjects:
Cell Nucleolus
Cell Nucleus
Cytidine Triphosphate - metabolism
DNA-Directed RNA Polymerases - metabolism
Enzyme Activation
HeLa Cells
Humans
RNA Polymerase I - metabolism
RNA Polymerase II - metabolism
Transcription, Genetic
Uridine - analogs & derivatives
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Summary:Using HeLa cells, we have developed methods to determine 1) the number of RNA polymerases that are active at any moment, 2) the number of transcription sites, and 3) the number of polymerases associated with one transcription unit. To count engaged polymerases, cells were encapsulated in agarose, permeabilized, treated with ribonuclease, and the now-truncated transcripts extended in [32P]uridine triphosphate; then, the number of growing transcripts was calculated from the total number of nucleotides incorporated and the average increment in length of the transcripts. Approximately 15, 000 transcripts were elongated by polymerase I, and approximately 75,000 were elongated by polymerases II and III. Transcription sites were detected after the cells were grown in bromouridine for
ISSN:1059-1524
1939-4586
DOI:10.1091/mbc.9.6.1523