Probing the interaction of T7 RNA polymerase with promoter

Transcription is the fundamental process by which RNA is synthesized by RNA polymerases on double-stranded DNA templates. One structurally simple RNA polymerase is encoded by bacteriophage T7. T7 RNA polymerase is an excellen t candidate for studying structural aspects of transcription, because unlik e the eucaryotic and bacterial RNA polymerases, it is a single subunit enzy me and does not require additional factors to carry out the entire process of transcription from start to finish. An important advantage of studying t ranscription using this enzyme is that the high-resolution crystal structur e of T7 RNA polymerase has been solved. However, a cocrystal structure of t he polymerase complexed with promoter has not yet been published. Here, we have used cross-linking techniques to understand the interaction of promote r with T7 RNA polymerase. We constructed promoters that were substituted wi th the photocross-linkable nucleotide 5-iodo uracil at every dT in the prom oter from -17 to -1. This substitution replaces the 5-methyl in dT with an iodine atom. The substituted promoters were photo-cross-linked to T7 RNAP, and the efficiency of cross-linking was quantitated at every position. In t he melting domain, the strongest contacts occurred at -3 and at -1 on the t emplate strand while very weak cross-linking was seen at -2 and at -4 on th e nontemplate strand. In the binding domain, the strongest contacts were se en at -16, -15, and -13 and at -10 on the template strand while at -17 and -14 on the nontemplate strand very weak cross-linking was observed. Cross-l inking was poor in the intervening region between the binding and the melti ng domains. These results suggested that, in the T7 RNA polymerase-promoter complex, the polymerase molecule mainly contacts the template bases in the TATA box while the upstream contacts are used as an anchor for DNA binding . For a systematic study designed to probe the nature of base-specific inte ractions in the polymerase-promoter complex, we used neutral salts from the Hofmeister series. In general, the order of perturbation was sulfate, citr ate > acetate for anions and ammonium > magnesium > potassium for cations. Using acrylamide, a neutral hydrophobic agent to probe for nonionic contact s, we observed that at -2, -4, and -17 the contacts had a hydrophobic compo nent, while at many other positions there was no significant effect, sugges ting that the contacts in the promoter-polymerase complexes were predominan tly ionic but at certain positions nonionic interactions also existed. To l ocalize a specific interaction in the melting domain, we proteolyzed the cr oss-linked T7 RNAP and analyzed the fragments using gel electrophoresis, ma ss spectrometry, and amino acid composition. High-resolution mapping indica ted that amino acid residues 614-627 may be in the vicinity of the melting domain. Specifically, Y623 may contact -3 on the template strand.