Interactions between σ70-RNA polymerase and promoter DNA has been examined by a technique called footprinting (FIGuRE 13. 11). A particular piece of double-stranded DNA, labeled in one strand at its 5'-termmus with 32p (or in its 3'-terminus with a labeled nucleotide), is mixed with RNA polymerase holoenzyme (or some other protein of interest that binds to DNA). Then DNase is added, but so briefly that, on average, each DNA molecule receives no more than one single-strand break. (This brief exposure to DNase is in marked contrast to the long exposure used in the DNase protection method.) Nicking occurs at all positions except those protected by σ70-RNA polymerase. The DNA is then isolated and denatured. The radioactively labeled DNA consists of a set of molecules with sizes that are determined by the nick positions in relation to the labeled 5'-end. If the DNA contains n base pairs and RNA polymerase is not added, n sizes of DNA fragments will be present. However, if RNA polymerase binds to x base pairs and thereby prevents access of the DNA to the nuclease, only n-x different sizes of DNA fragments will be represented. These fragments are separated by gel electrophoresis.
Two DNA samples are compared, one without RNA polymerase (to obtain the positions of the n bands), and one with RNA polymerase (to determine the positions of the missing bands). The results of a DNase footprinting experiment in which a modified lactose promoter designated lac UV5 was examined. The lac UV5 promoter has the same sequence as the normal lactose promoter except that two bases in the -10 box have been changed so that the lac UV5 -10 box now has a sequence that matches the consensus sequence, which makes it a stronger promoter.
RNA polymerase holoenzyme need not make direct contact with a base to protect it from DNase because the large DNase molecule cannot pass through tiny gaps in the holoenzyme · promoter complex. Therefore, some bases within the promoter that are protected from DNase cleavage do not make direct contact with the holoenzyme. Small chemical reagents such as dimethyl sulfate can distinguish bases that make direct contact with the holoenzyme from those that do not. Dimethyl sulfate methylates the ring nitrogens N7 of G (major groove) and N3 of A (minor groove) that are not protected by RNA polymerase holoenzyme. Piperidine in formic acid cleaves both methylated guanine and adenine nucleotides, whereas piperidine alone selectively cleaves the methylated guanine nucleotides. DNA fragments produced by the chemical cleavage are separated by gel electrophoresis. Fragment sizes indicate methylation positions in reIation to the labeled 5'-end. The bases protected by RNA polymerase holoenzyme tend to be clustered around the -10 and -35 boxes.