Learn about run-off transcription.
This technique is used when we want to assay accurate transcription in vitro. This technique tells us two things:
- where transcription initiates in the right place, and is therefore accurate.
- How much of this accurate transcription occurred.
We could use S1 mapping or primer extension but they are relatively complicated. This method gives us our answers much more rapidly.
How Run-off Transcription Works
- Start with a DNA fragment containing the gene we want to map, then cut it with a restriction enzyme in the middle of the transcribed region.
- Then we transcribe the truncated gene fragment in vitro with labeled nucleotides so the transcript becomes labeled. Since we have cut the gene in the middle, the polymerase reaches the end of the fragment and simply "runs off".
- Now we can measure the length of the run off transcript. Since we know precisely the location of the restriction site at the 3' end of the truncated gene, the length of the run off transcript tells us that the start of transcription.
S1 mapping and primer extension are well suited to mapping transcripts made in vivo; by contrast, run off transcription relies on transcription in vitro. Therefore it will work only with genes that are accurately transcribed in vitro, and cannot give information about cellular transcript concentrations. However, it is a good method for measuring the rate of in vitro transcription. The more transcript is made, the more intense will be the run off transcription signal.
Run off transcription is most useful as a quantitative method. Only after we have identified the physiological transcription start site by S1 mapping or primer extension do we use run-off transcription in vitro. The test of accurate in vitro transcription is a run-off transcript of the right size- corresponding to the correct start site. Once we have established accurate in vitro transcription this way, we can proceed to manipulate the in vitro system and use the run off assay to quantify the effects of these changes. For example we can mutate the promoter and ask whether this alters the efficiency or the accuracy of transcription. The run-off assay can answer both questions.
See the DNA Molecule in 3-Dimensions