Learn about nuclear run-on transcription.
Nuclear Run-on Transcription
The idea of this assay is to isolate nuclei from cells, then allow them to extend in vitro the transcripts they had already started in vivo. This continuing transcription in isolated nuclei is called run on transcription because the RNA polymerase that has already initiated transcription in vivo simply “runs on”, or continues to elongate the same RNA chains. We usually do the run on reaction in the presence of labeled nucleotides so the products will be labeled. Since the initiation of new RNA chains in isolated nuclei does not generally occur we can be fairly confident that any transcription we observe in the isolated nuclei is simply a continuation of transcription that was already occurring in vivo. Therefore the transcripts obtained in a run-on reaction should tell us which genes are transcribed in vivo. If we are concerned about the possibility of initiation of new RNA chains in vitro, we can add heparin, an anionic polysaccharide that binds any free RNA polymerase and prevents re-initiation.
Once we have produced labeled run-on transcripts, we need a way to identify them. Since few if any of them are complete transcripts, we cannot simply measure their sizes. The easiest way to perform the identification is by dot blotting. We spot samples of known denatured DNAs on a filter and hybridize this dot blot on the labeled run-on RNA. The RNA is identified by the DNA to which it hybridizes. Furthermore, the relative activity of a given gene is proportional to the degree of hybridization to the DNA from that gene. We can also manipulate conditions in the run-on reaction and measure their effects on the products. For example we can include inhibitors of certain RNA polymerases to see if we inhibit transcription of a certain gene. If so, we can identify the RNA polymerase responsible for transcribing that gene.
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