Affinity labeling of the ribosomal decoding site with an AUG-substrate analog.
The trinucleotide AUG was condensed at the 5'-end with N-bromoacetyl-p-aminophenylphosphate. This bromoactylated AUG analog reacted irreversibly with the mRNA binding site of Escherichia coli 70S ribosomes. After reaction of 70S ribosomes with the AUG analog, labeled 30S subunits could be isolated that were programmed for initiation-factor-dependent binding of fMet-tRNAfMet. This shows that this AUG-affinity label reacted in the decoding site for fMet-tRNAfMet. By combination of sodium dodecyl sulfate-, Sarkosyl-, and ureapolyacrylamide gel electrophoresis the AUG-affinity label was found to be irreversibly bound to ribosomal proteins S4, the ram gene product, and S18.
D001665:Binding Sites; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D009239:N-Formylmethionine / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism*; D010448:Peptide Initiation Factors; D012329:RNA, Bacterial / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D011955:Receptors, Drug; D012270:Ribosomes / Q000378:metabolism*; D013329:Structure-Activity Relationship
D009841:Oligonucleotides; D010448:Peptide Initiation Factors; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011955:Receptors, Drug; D009239:N-Formylmethionine; D012343:RNA, Transfer