title: SPARK--a novel method to monitor ribosomal peptidyl transferase activity.
abstract: The key enzymatic activity of the ribosome is catalysis of peptide bond formation. This reaction is a target for many clinically important antibiotics. However, the molecular mechanisms of the peptidyl transfer reaction, the catalytic contribution of the ribosome, and the mechanisms of antibiotic action are still poorly understood. Here we describe a novel, simple, convenient, and sensitive method for monitoring peptidyl transferase activity (SPARK). In this method, the ribosomal peptidyl transferase forms a peptide bond between two ligands, one of which is tritiated whereas the other is biotin-tagged. Transpeptidation results in covalent attachment of the biotin moiety to a tritiated compound. The amount of the reaction product is then directly quantified using the scintillation proximity assay technology: binding of the tritiated radioligand to the commercially available streptavidin-coated beads causes excitation of the bead-embedded scintillant, resulting in detection of radioactivity. The reaction is readily inhibited by known antibiotics, inhibitors of peptide bond formation. The method we developed is amenable to simple automation which makes it useful for screening for new antibiotics. The method is useful for different types of ribosomal research. Using this method, we investigated the effect of mutations at a universally conserved nucleotide of the active site of 23S rRNA, A2602 (Escherichia coli numbering), on the peptidyl transferase activity of the ribosome. The activities of the in vitro reconstituted mutant subunits, though somewhat reduced, were comparable with those of the subunits assembled with the wild-type 23S rRNA, indicating that A2602 mutations do not abolish the ability of the ribosome to catalyze peptide bond formation. Similar results were obtained with double mutants carrying mutations at A2602 and another universally conserved nucleotide in the peptidyl transferase center, A2451. The obtained results agree with our previous conclusion that the ribosome accelerates peptide bond formation primarily through entropic rather than chemical catalysis.
authors.affiliation: Center for Pharmaceutical Biotechnology, m/c 870, University of Illinois, 900 South Ashland Avenue, Chicago, IL 60607, USA.
authors.forename: Norbert
authors.initials: N
authors.lastname: Polacek
authors.forename: Steven
authors.initials: S
authors.lastname: Swaney
authors.forename: Dean
authors.initials: D
authors.lastname: Shinabarger
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Mankin
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001710:Biotin / Q000737:chemistry; D004789:Enzyme Activation / Q000187:drug effects; D007118:Immunoassay / Q000379:methods*; D007700:Kinetics; D010458:Peptidyl Transferases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D021122:Protein Subunits; D011691:Puromycin / Q000737:chemistry; D012335:RNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D012345:RNA, Transfer, Amino Acid-Specific / Q000737:chemistry; D012269:Ribosomal Proteins / Q000096:biosynthesis / Q000737:chemistry*; D012270:Ribosomes / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D012588:Scintillation Counting / Q000379:methods; D012680:Sensitivity and Specificity; D013824:Thermus / Q000201:enzymology / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D021122:Protein Subunits; D012335:RNA, Ribosomal; D012345:RNA, Transfer, Amino Acid-Specific; D012269:Ribosomal Proteins; D011691:Puromycin; D001710:Biotin; D010458:Peptidyl Transferases