NUMBERS AND ORGANIZATION OF RNA-POLYMERASES, NASCENT TRANSCRIPTS, ANDTRANSCRIPTION 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 tr anscription sites, and 3) the number of polymerases associated with on e transcription unit. To count engaged polymerases, cells were encapsu lated in agarose, permeabilized, treated with ribonuclease, and the no w-truncated transcripts extended in [P-32]uridine triphosphate; then, the number of growing transcripts was calculated from the total number of nucleotides incorporated and the average increment in length of th e transcripts. Approximately 15,000 transcripts were elongated by poly merase I, and similar to 75,000 were elongated by polymerases II and I II. Transcription sites were detected after the cells were grown in br omouridine for <2.5 min, after which the resulting bromo-RNA was label ed with gold particles; electron microscopy showed that most extranucl eolar transcripts were concentrated in similar to 2400 sites with diam eters of similar to 80 nm. The number of polymerases associated with a transcription unit was counted after templates were spread over a lar ge area; most extranucleolar units were associated with one elongating complex. These results suggest that many templates are attached in a ''cloud'' of loops around a site; each site, or transcription ''factor y,'' would contain similar to 30 active polymerases and associated tra nscripts.