COMMUNICATION Identification of a Minimal Binding Element within the T7 RNA Polymerase Promoter

The T7 RNA polymerase promoter has been proposed to contain two domains: the binding region upstream of position ÿ5 is recognized through apparently traditional duplex contacts, while the catalytic domain downstream of position ÿ5 is bound in a melted con®guration. This model is tested by following polymerase binding to a series of syn-thetic oligonucleotides representing truncations of the consensus promo-ter sequence. The increase in the ¯uorescence anisotropy of a rhodamine dye linked to the upstream end of the promoter provides a very sensitive measure of enzyme binding in simple thermodynamic titrations, and allows the determination of both increases and decreases in the dis-sociation constant. The best ®t value of Kd 4.0 nM for the native pro-moter is in good agreement with previous ¯uorescence and steady state measurements. Deletion of the downstream DNA up to position ÿ1 or to position ÿ5 leads to a ®vefold increase in binding, while further sequen-tial single-base deletions upstream result in 20 and 500-fold decreases in binding. These results indicate that the (duplex) region of the promoter upstream of and including position ÿ5 is both necessary and suf®cient for tight binding, and represents the core binding element of the promo-ter. We propose a model in which part of the upstream binding energy is used by T7 RNA polymerase to melt the downstream initiation region of the promoter. We also show that the presence of magnesium is necessary for optimal binding, but not for speci®c enzyme-promoter complex for-mation, and we propose that magnesium is not required for melting of the promoter