Antibacterial peptide microcin J25 inhibits transcription by binding within and obstructing the RNA polymerase secondary channel.

The antibacterial peptide microcin J25 (MccJ25) inhibits transcription by bacterial RNA polymerase (RNAP). Biochemical results indicate that inhibition of transcription occurs at the level of NTP uptake or NTP binding by RNAP. Genetic results indicate that inhibition of transcription requires an extensive determinant, comprising more than 50 amino acid residues, within the RNAP secondary channel (also known as the "NTP-uptake channel" or "pore"). Biophysical results indicate that inhibition of transcription involves binding of MccJ25 within the RNAP secondary channel. Molecular modeling indicates that binding of MccJ25 within the RNAP secondary channel obstructs the RNAP secondary channel. We conclude that MccJ25 inhibits transcription by binding within and obstructing the RNAP secondary channel--acting essentially as a "cork in a bottle." Obstruction of the RNAP secondary channel represents an attractive target for drug discovery.

[1]  F. Neidhart.  Escherichia coli and Salmonella. , 1996 .

[2]  R. Ebright,et al.  Transcription Activation at Class II CAP-Dependent Promoters: Two Interactions between CAP and RNA Polymerase , 1996, Cell.

[3]  S. Darst,et al.  A Structural Model of Transcription Elongation , 2000 .

[4]  Ulf Göransson,et al.  Microcin J25 has a threaded sidechain-to-backbone ring structure and not a head-to-tail cyclized backbone. , 2003, Journal of the American Chemical Society.

[5]  J. Gralla,et al.  Supercoiling response of the lac ps promoter in vitro. , 1985, Journal of molecular biology.

[6]  Hiroshi Handa,et al.  NTP-driven Translocation by Human RNA Polymerase II* , 2003, The Journal of Biological Chemistry.

[7]  O. Laptenko,et al.  Transcript cleavage factors GreA and GreB act as transient catalytic components of RNA polymerase , 2003, The EMBO journal.

[8]  S. Blacklow,et al.  A reliable method for random mutagenesis: the generation of mutant libraries using spiked oligodeoxyribonucleotide primers. , 1989, Gene.

[9]  A. Sali,et al.  Domain Organization of Escherichia coli Transcript Cleavage Factors GreA and GreB* , 1997, The Journal of Biological Chemistry.

[10]  Paul R. Selvin,et al.  The renaissance of fluorescence resonance energy transfer , 2000, Nature Structural Biology.

[11]  Escherichia coli rpoC397 encodes a temperature-sensitive C-terminal frameshift in the beta' subunit of RNA polymerase that blocks growth of bacteriophage P2 , 1996, Journal of bacteriology.

[12]  G. Montelione,et al.  Structure of antibacterial peptide microcin J25: a 21-residue lariat protoknot. , 2003, Journal of the American Chemical Society.

[13]  C. Chan,et al.  Discontinuous movements of DNA and RNA in RNA polymerase accompany formation of a paused transcription complex , 1995, Cell.

[14]  R. Ebright,et al.  Site-specific protein-DNA photocrosslinking. Analysis of bacterial transcription initiation complexes. , 2001, Methods in molecular biology.

[15]  Michelle D. Wang,et al.  Molecular mechanism of transcription inhibition by peptide antibiotic Microcin J25. , 2004, Molecular cell.

[16]  M. Kozlov,et al.  Donation of catalytic residues to RNA polymerase active center by transcription factor Gre , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[17]  K. Severinov,et al.  Crystal Structure of Thermus aquaticus Core RNA Polymerase at 3.3 Å Resolution , 1999, Cell.

[18]  X P Zhang,et al.  Random mutagenesis of gene-sized DNA molecules by use of PCR with Taq DNA polymerase. , 1991, Nucleic acids research.

[19]  Y. Cheng,et al.  Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. , 1973, Biochemical pharmacology.

[20]  P. Cramer,et al.  Architecture of the RNA Polymerase II-TFIIS Complex and Implications for mRNA Cleavage , 2003, Cell.

[21]  R. Farías,et al.  Microcin 25, a novel antimicrobial peptide produced by Escherichia coli , 1992, Journal of bacteriology.

[22]  Arkady Mustaev,et al.  Structural Mechanism for Rifampicin Inhibition of Bacterial RNA Polymerase , 2001, Cell.

[23]  S. Yokoyama,et al.  Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 Å resolution , 2002, Nature.

[24]  R. Farías,et al.  Escherichia coli RNA Polymerase Is the Target of the Cyclopeptide Antibiotic Microcin J25 , 2001, Journal of bacteriology.

[25]  R. Ebright,et al.  Translocation of σ70 with RNA Polymerase during Transcription Fluorescence Resonance Energy Transfer Assay for Movement Relative to DNA , 2001, Cell.

[26]  L. Yarbrough,et al.  Spectroscopic techniques for study of phosphodiester bond formation by Escherichia coli RNA polymerase. , 1979, The Journal of biological chemistry.

[27]  J. Solbiati,et al.  Sequence Analysis of the Four Plasmid Genes Required To Produce the Circular Peptide Antibiotic Microcin J25 , 1999, Journal of bacteriology.

[28]  Clement S. Chu,et al.  A New Class of Bacterial RNA Polymerase Inhibitor Affects Nucleotide Addition , 2003, Science.

[29]  K. Severinov,et al.  Mutations in and Monoclonal Antibody Binding to Evolutionary Hypervariable Region of Escherichia coli RNA Polymerase β′ Subunit Inhibit Transcript Cleavage and Transcript Elongation* , 1998, The Journal of Biological Chemistry.

[30]  Jennifer L. Knight,et al.  Structural Organization of Bacterial RNA Polymerase Holoenzyme and the RNA Polymerase-Promoter Open Complex , 2002, Cell.

[31]  V. Markovtsov,et al.  Swing-gate model of nucleotide entry into the RNA polymerase active center. , 2002, Molecular cell.

[32]  M. Smith,et al.  A simple and efficient procedure for generating random point mutations and for codon replacements using mixed oligodeoxynucleotides. , 1988, DNA.

[33]  William J. Rice,et al.  Structure and Function of the Transcription Elongation Factor GreB Bound to Bacterial RNA Polymerase , 2003, Cell.

[34]  K. Severinov,et al.  Tethering of the Large Subunits of Escherichia coli RNA Polymerase* , 1997, The Journal of Biological Chemistry.

[35]  R. Ebright,et al.  Transcription activation at Class II CAP‐dependent promoters , 1993, Molecular microbiology.

[36]  S. Nechaev,et al.  Mutations of Bacterial RNA Polymerase Leading to Resistance to Microcin J25* , 2002, The Journal of Biological Chemistry.

[37]  P. Cramer,et al.  Structural Basis of Transcription : RNA Polymerase II at 2 . 8 Å Resolution , 2022 .

[38]  P. Cramer,et al.  Structural Basis of Transcription: An RNA Polymerase II Elongation Complex at 3.3 Å Resolution , 2001, Science.

[39]  D. Bushnell,et al.  Structural Basis of Transcription Nucleotide Selection by Rotation in the RNA Polymerase II Active Center , 2004, Cell.

[40]  B. Chait,et al.  Structure of microcin J25, a peptide inhibitor of bacterial RNA polymerase, is a lassoed tail. , 2003, Journal of the American Chemical Society.

[41]  P. Cramer,et al.  Structural Basis of Transcription: RNA Polymerase II at 2.8 Ångstrom Resolution , 2001, Science.

[42]  Patrick Cramer,et al.  Structural basis of transcription: α-Amanitin–RNA polymerase II cocrystal at 2.8 Å resolution , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[43]  S. Rebuffat,et al.  The cyclic structure of microcin J25, a 21-residue peptide antibiotic from Escherichia coli. , 2001, European journal of biochemistry.