Practice question

Run-off transcription assay evaluates promoter strength and accuracy of initiation in defined in vitro system. A DNA template containing promoter of interest is linearized at defined position downstream of transcription start site using restriction enzyme, creating truncated template. Addition of RNA polymerase holoenzyme with sigma factor and ribonucleoside triphosphates results in transcription initiation at promoter and elongation until enzyme runs off at cut end, producing transcript of predicted length measurable on urea-polyacrylamide gel. Amount of run-off product quantifies transcription rate and enables mapping of promoters recognized by different sigma factors in prokaryotes.