Open AccessTopology of the RNA polymerase active center probed by chimeric rifampicin-nucleotide compounds.
Author(s) -
Arkady Mustaev,
Evgeny Zaychikov,
K. Severinov,
Mikhail Kashlev,
A. Polyakov,
Vadim Nikiforov,
Alex Goldfarb
Publication year - 1994
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.91.25.12036
Subject(s) - polymerase , rna polymerase , t7 rna polymerase , microbiology and biotechnology , rna dependent rna polymerase , nucleotide , transcription (linguistics) , dna , linker , rna , chemistry , ribonucleotide , biology , escherichia coli , biochemistry , bacteriophage , gene , linguistics , philosophy , computer science , operating system
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 approximately 15 A. 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.