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Antimicrobials that Bind to the 50S Ribosomal Subunit Chloramphenicol, Lincomycin, Clindamycin (bacteriostatic) • Mode of action - These antimicrobials bind to the 50S ribosome and inhibit peptidyl transferase activity. • Spectrum of activity - Chloramphenicol - Broad range; Lincomycin and clindamycin - Restricted range • Resistance - Common • Adverse effects - Chloramphenicol is toxic (bone marrow suppression) but is used in the treatment of bacterial meningitis. 1 Macrolides (bacteriostatic) erythromycin, clarithromycin, azithromycin, spiramycin • Mode of action - The macrolides inhibit translocation. • Spectrum of activity - Gram-positive bacteria, Mycoplasma, Legionella • Resistance - Common Antimicrobials that Interfere with Elongation Factors Selectivity due to differences in prokaryotic and eukaryotic elongation factors 2 Fusidic acid (bacteriostatic) • Mode of action - Fusidic acid binds to elongation factor G (EF-G) and inhibits release of EF-G from the EF-G/GDP complex. • Spectrum of activity - Gram-positive cocci 3 4 5 6 7 8 ENHANCEMENT FOOTPRINTING ANTIBIOTIC BINDIN SITES ON 16S rRNA IN 30S SUBUNIT NUCLEOTIDES IN 16S RNA THAT ARE PROTECTED FROM CHEMICAL MODIFICATION WHEN VARIOUS ANTIBIOTICS ARE BOUND TO THE 30S RIBOSOMAL SUBUNIT WEAVER: FIG. 19.27 1 CORRELATION OF (1) SITES OF CHEMICAL PROTECTION BY CHLORAMPHENICOL WITH (2) SITES OF MUTATION TO CHLORAMPHENICOL RESISTANCE IN DOMAIN V OF THE 23S RNA 2 WEAVER: FIG. 19.28 9 Antibiotics that inhibit protein synthesis by binding to ribosomes. Chloramphenicol inhibits peptidyl transferase (PT) activity! Inhibits PT on 80S cytoplasmic ribosomes Fig. 18.11 Antibiotics that inhibit PT bind to a loop in Domain V of 23S rRNA Antibiotic footprints (circled bases) PT loop PT loop PT loop Antibiotic resistance mutations (circled bases) PT loop – peptidyl transferase loop 10 11 Tetracycline Mode of Action Vancomycin Binds to D-Ala-D-Ala terminus; inhibits transpeptidation Penicillin Inhibits transpeptidation enzymes. Activates lyctic enzymes of cell wall Carbenicillin Inhibits transpeptidation enzymes. Activates lyctic enzymes of cell wall Rifampin Blocks RNA synthesis: binds to, inhibits DNAdependent RNA polymerase Ciprofloxacin Inhibits bacterial DNA gyrase; interferes with DNAinvolved activities like DNA replication Chloramphenicol Interferes with protein synthesis by binding to the bacterial ribosome Streptomycin Causes misreading of mRNA: binds to 30S ribosomal subunit Sulfonamides Interferes with synthesis of folic acid by competition with p-aminobenzoic acid Polymyxin B Disrupts structure and permeability of plasma mambrane by binding with it from: http://project.bio.iastate.edu/Courses/MIPM302/302new/9_1chemother.html Chloramphenicol Antibiotic Modes of Action Drug 12 Tunnel for nascent peptide Role of 23S rRNA (in red) Nissen et al. (2000) Science,289, 920-930 Tunnel of nascent peptide Interactions with chaperonines or SRP? Nissen et al. (2000) Science,289, 920-930 13 Tunnel for nascent peptide Diameter18 à 25 Å: no helical formation Nissen et al. (2000) Science,289, 920-930 Tunnel of nascent peptide Nissen et al. (2000) Science,289, 920-930 14 Tunnel for nascent peptide Nissen et al. (2000) Science,289, 920-930 Journal of Molecular Biology Volume 330, Issue 5 , 25 July 2003, Pages 1061-1075 Structures of Five Antibiotics Bound at the Peptidyl Transferase Center of the Large Ribosomal Subunit Jeffrey L. Hansen1, Peter B. Moore1, 2 and Thomas A. Steitz, , 1, 2, 3 15 Anisomycin, chloramphenicol, sparsomycin, blasticidin S, and virginiamycin M bind to sites that overlap those of either peptidyl-tRNA or aminoacyltRNA, consistent with their functioning as competitive inhibitors of peptide bond formation. Two hydrophobic crevices, one at the peptidyl transferase center and the other at the entrance to the peptide exit tunnel play roles in binding these antibiotics. Midway between these crevices, nucleotide A2103 of H. marismortui (2062 Escherichia coli) varies in its conformation and thereby contacts antibiotics bound at either crevice. The aromatic ring of anisomycin binds to the active-site hydrophobic crevice, as does the aromatic ring of puromycin, while the aromatic ring of chloramphenicol binds to the exit tunnel hydrophobic crevice. Sparsomycin contacts primarily a P-site bound substrate, but also extends into the active-site hydrophobic crevice. Virginiamycin M occupies portions of both the A and P-site, and induces a conformational change in the ribosome. Blasticidin S base-pairs with the P-loop and thereby mimics C74 and C75 of a P-site bound tRNA. 16 17 18 19