Topology of the RNA Polymerase Active Center Probed by Chimeric Rifampicin-Nucleotide Compounds

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 91; no. 25; pp. 12036 - 12040
• Main Authors: Mustaev, Arkady, Zaychikov, Evgeny, Severinov, Konstatin, Kashlev, Mikhail, Polyakov, Andrey, Nikiforov, Vadim, Goldfarb, Alex
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 06.12.1994; National Acad Sciences; National Academy of Sciences
• Subjects: Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - chemical synthesis; Adenosine Triphosphate - chemistry; Adenosine Triphosphate - metabolism; Alkylation; Bacteria; Binding Sites; Biochemistry; Cross-Linking Reagents; Crosslinking; DNA; DNA-Directed RNA Polymerases - chemistry; DNA-Directed RNA Polymerases - metabolism; Escherichia coli; Escherichia coli - enzymology; Gels; Molecules; Nucleotides; Promoter Regions, Genetic; Reagents; Ribonucleic acid; Rifampin - analogs & derivatives; Rifampin - chemical synthesis; Rifampin - chemistry; Rifampin - metabolism; RNA; Sequencing; Substrate Specificity; Templates, Genetic; Transcription, Genetic
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.91.25.12036

Spatial organization of the binding sites for the priming substrate, the template DNA, and the transcription inhibitor rifampicin (Rif) in Escherichia coli RNA polymerase (EC 2.7.7.6) was probed with chimeric compounds in which Rif is covalently attached to a ribonucleotide. The compounds bind to RNA polymerase in bifunctional manner and serve as substrates 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 the two ligands as ruler, we determined the distance between the sites for Rif and the priming nucleotide to be ≈15 Å. A reactive side group placed in the linker next to Rif crosslinks to the template strand of DNA at the -2 or -3 position of the promoter. Thus, bound Rif is juxtaposed to DNA immediately upstream of the start site, suggesting that Rif plugs the channel leading RNA out of the active center.