%0 Figure
%A Polacek, Norbert
%A Swaney, Steven
%A Shinabarger, Dean
%A Mankin, Alexander S.
%D 2002
%T SPARKA Novel Method To Monitor Ribosomal Peptidyl Transferase Activity†
%U https://acs.figshare.com/articles/figure/SPARK_A_Novel_Method_To_Monitor_Ribosomal_Peptidyl_Transferase_Activity_sup_sup_/3586035
%R 10.1021/bi026040s.s007
%2 https://acs.figshare.com/ndownloader/files/5671959
%K chemical catalysis
%K peptide bond formation
%K covalent attachment
%K tritiated radioligand
%K 2602 mutations
%K reaction product
%K antibiotic action
%K 23 S rRNA
%K method
%K peptidyl transferase activity
%K biotin moiety
%K Transpeptidation results
%K catalyze peptide bond formation
%K Escherichia coli
%K tritiated compound
%K Similar results
%K ribosome
%K mechanism
%K peptidyl transferase center
%K subunit
%K peptidyl transferase forms
%K peptidyl transfer reaction
%K nucleotide
%K peptide bond
%K monitoring peptidyl transferase activity
%X 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.
%I ACS Publications