Topology of the RNA polymerase active center probed by chimeric rifampicin-nucleotide compounds

Arkady Mustaev, Evgeny Zaychikov, Konstantin Severinov, Mikhail Kashlev, Andrey Polyakov, Vadim Nikiforov, Alex Goldfarb

Research output: Contribution to journalArticlepeer-review

48 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)12036-12040
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume91
Issue number25
DOIs
Publication statusPublished - 6 Dec 1994
Externally publishedYes

Keywords

  • protein-DNA interaction
  • ternary complex
  • transcription initiation

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