Practice question

Identification of sigma factor dependent promoters requires direct assessment of initiation specificity rather than steady-state RNA levels. Run-off transcription assay reconstitutes transcription in vitro using purified core RNA polymerase and individual sigma subunits incubated with DNA templates containing putative promoters. Only when functional sigma recognizes -10 and -35 elements does productive initiation occur, generating discrete run-off transcript whose size corresponds to distance between start site and downstream restriction cut. RNA-seq, Northern blot, and RT-PCR measure cellular RNA abundance but cannot discriminate sigma factor specific recognition events in defined biochemical context, making run-off assay suitable.