Molecular mechanism of drug-dependent ribosome stalling.
Inducible expression of the erm erythromycin resistance genes relies on drug-dependent ribosome stalling. The molecular mechanisms underlying stalling are unknown. We used a cell-free translation system to elucidate the contribution of the nascent peptide, the drug, and the ribosome toward formation of the stalled complex during translation of the ermC leader cistron. Toe-printing mapping, selective amino acid labeling, and mutational analyses revealed the peptidyl transferase center (PTC) as the focal point of the stalling mechanism. In the ribosome exit tunnel, the C-terminal sequence of the nascent peptide, critical for stalling, is in the immediate vicinity of the universally conserved A2062 of 23S rRNA. Mutations of this nucleotide eliminate stalling. Because A2062 is located in the tunnel, it may trigger a conformational change in the PTC, responding to the presence of a specific nascent peptide. The cladinose-containing macrolide antibiotic in the tunnel positions the nascent peptide for interaction with the tunnel sensory elements.
Vazquez-Laslop
Nora
N
Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, 900 South Ashland Avenue, m/c 870, Chicago, IL 60607, USA. nvazquez@uic.edu
Thum
Celine
C
Mankin
Alexander S
AS
D020121:5' Untranslated Regions / Q000378:metabolism; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001483:Base Sequence; D002474:Cell-Free System; D018983:DNA Footprinting; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004917:Erythromycin / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010458:Peptidyl Transferases / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis / Q000235:genetics; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D020121:5' Untranslated Regions; D000900:Anti-Bacterial Agents; D012346:RNA, Transfer, Amino Acyl; C019037:tRNA, peptidyl-; D004917:Erythromycin; D008780:Methyltransferases; C035181:rRNA (adenosine-O-2'-)methyltransferase; D010458:Peptidyl Transferases