Supp wk6 - RNA Polymerase Leaves Its Footprint on a...

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RNA Polymerase Leaves Its Footprint on a Promoter Footprinting, a technique derived from principles used in DNA sequencing, identifies the DNA se- quences bound by a particular protein. Researchers isolate a DNA fragment thought to contain sequences recognized by a DNA-binding protein and radiolabel one end of one strand (Fig. 1). They then use chem- ical or enzymatic reagents to introduce random breaks in the DNA fragment (averaging about one per mole- cule). Separation of the labeled cleavage products (bro- ken fragments of various lengths) by high-resolution electrophoresis produces a ladder of radioactive bands. In a separate tube, the cleavage procedure is repeated on copies of the same DNA frag- ment in the presence of the DNA-binding protein. The researchers then subject the two sets of cleavage products to elec- trophoresis and compare them side by side. A gap (“footprint”) in the series of radioactive bands derived from the DNA- protein sample, attributable to protection of the DNA by the bound protein, identi- fies the sequences that the protein binds. The precise location of the protein- binding site can be determined by di- rectly sequencing (see Fig. 8–37) copies of the same DNA fragment and including the sequencing lanes (not shown here) on the same gel with the footprint. Fig- ure 2 shows footprinting results for the binding of RNA polymerase to a DNA fragment containing a promoter. The polymerase covers 60 to 80 bp; protec- tion by the bound enzyme includes the ± 10 and ± 35 regions. Nontemplate strand ±² C ² 1 ± 20 ± 30 ± 40 ± 50 ± 10 Regions bound by RNA polymerase 5 ³ 3 ³ 3 ³ 5 ³ Treat with DNase under conditions in which each strand is cut once (on average).
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Supp wk6 - RNA Polymerase Leaves Its Footprint on a...

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