RNA polymerase: the mechanism of initiation

A novel mechanism of rotational translocation for transcription by DNA dependent RNA polymerase has been proposed by Dennis and Sylvester. Inherent in this model is an active surface that contains two ribose binding loci and two catalytic loci positions such that a two-fold rotational symmetry is described. Recent evidence has shown the existence of two topographically distinct, alternately and successively employed elongations sites. An augmentation of this model has speculated the existence of an asymmetric unique starting point (initiation site) in order to establish the 3' → 5' polarity of phosphodiester-bond formation. Subsequent elongation events employ only the two symmetric active sites. This study tests the hypothesis that a unique initiation site exists by directly derivatizing the enzyme with the chemically synthesized photoaffinity probes 9(3'-deoxy-3'-azido-β-D-xylofuranosyl)adenine-5'-mono and triphosphate. These compounds were first kinetically analyzed and found to be competitive inhibitors of the initiation binding site i. The 32P analogues of these compounds photoderivatized a unique initiation binding domain located on the σ subunit of RNA polymerase which is topographically distinct from the sites employed during elongation