title: Molecular mechanism of transcription inhibition by peptide antibiotic Microcin J25.
abstract: 21 amino acid peptide Microcin J25 (MccJ25) inhibits transcription by bacterial RNA polymerase (RNAP). MccJ25-resistance mutations cluster in the RNAP secondary channel through which incoming NTP substrates are thought to reach the catalytic center and the 3' end of the nascent RNA is likely to thread in backtracked transcription complexes. The secondary channel also accepts transcript cleavage factors GreA and GreB. Here, we demonstrate that MccJ25 inhibits GreA/GreB-dependent transcript cleavage, impedes formation of backtracked complexes, and can be crosslinked to the 3'-end of the nascent RNA in elongation complexes. These results place the MccJ25 binding site within the secondary channel. Moreover, single-molecule assays reveal that MccJ25 binding to a transcribing RNAP temporarily stops transcript elongation but has no effect on the elongation velocity between pauses. Kinetic analysis of single-molecule data allows us to put forward a model of transcription inhibition by MccJ25 that envisions the complete occlusion of the secondary channel by bound inhibitor.
authors.affiliation: Department of Molecular Biology and Biochemistry, Laboratory of Atomic and Solid State Physics, Cornell University, Ithaca, New York 14853, USA. ka42@cornell.edu
authors.forename: Karen
authors.initials: K
authors.lastname: Adelman
authors.forename: Julia
authors.initials: J
authors.lastname: Yuzenkova
authors.forename: Arthur
authors.initials: A
authors.lastname: La Porta
authors.forename: Nikolay
authors.initials: N
authors.lastname: Zenkin
authors.forename: Jookyung
authors.initials: J
authors.lastname: Lee
authors.forename: John T
authors.initials: JT
authors.lastname: Lis
authors.forename: Sergei
authors.initials: S
authors.lastname: Borukhov
authors.forename: Michelle D
authors.initials: MD
authors.lastname: Wang
authors.forename: Konstantin
authors.initials: K
authors.lastname: Severinov
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001430:Bacteriocins / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology*; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D010455:Peptides / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects*; D035602:Transcriptional Elongation Factors / Q000502:physiology
chemical_list: D000900:Anti-Bacterial Agents; D001430:Bacteriocins; D010455:Peptides; D035602:Transcriptional Elongation Factors; C020411:microcin; D012321:DNA-Directed RNA Polymerases