TOPOLOGY OF THE RNA-POLYMERASE ACTIVE-CENTER PROBED BY CHIMERIC RIFAMPICIN-NUCLEOTIDE COMPOUNDS

Spatial organization of the binding sites for the priming substrate, t he template DNA, and the transcription inhibitor rifampicin (Rif) in E scherichia coli RNA polymerase (EC 2.7.7.6) was probed with chimeric c ompounds in which Rif is covalently attached to a ribonucleotide. The compounds bind to RNA polymerase in bifunctional manner and serve as s ubstrates for RNA chain extension, yielding chains up to 8 nucleotides in length, with Rif linked to their 5' termini. These products act as potent inhibitors of normal transcription. Using the linker between t he two ligands as ruler, we determined the distance between the sites for Rif and the priming nucleotide to be approximate to 15 Angstrom. A reactive side group placed in the linker next to Rif crosslinks to th e template strand of DNA at the -2 or -3 position of the promoter. Thu s, bound Rif is juxtaposed to DNA immediately upstream of the start si te, suggesting that Rif plugs the channel leading RNA out of the activ e center.