A fluorescent derivative of ribosomal protein S18 which permits direct observation of messenger RNA binding.
70 S Escherichia coli ribosomes were reacted with the fluorescent dye N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid for 10 min under mild conditions. The resulting ribosomes were fully active. 30 S subunits isolated from these particles were also fully active. They contain approximately 0.7 eq of fluorescent dye. Nearly all of it is attached to protein S18. Competitive reaction with N-ethylmaleimide implies that the fluorescent dye is located at cysteine 10 of the protein. The labeled 30 S particles will recombine with 50 S subunits to form stable 70 S particles. Thus the procedures we have developed allow the large scale preparation of an active fluorescent conjugate of the 70 S ribosome. The fluorescence of the 70 S particles is sensitive to the binding of mRNA, showing both quenching and a shift in emission spectra. Thus it affords a simple way to quantitate mRNA binding directly. In pilot studies without tRNA, the binding constant of the initiation triplet codon adenylyl-(3' leads to 5')-uridylyl-(3' leads to 5')-guanosine to 70 S ribosome was found to be an order of magnitude larger than that of polyuridylic acid.
D004926:Escherichia coli / Q000378:metabolism*; D005456:Fluorescent Dyes; D007460:Iodoacetamide / Q000031:analogs & derivatives; D007700:Kinetics; D009282:Naphthalenesulfonates; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D012333:RNA, Messenger; D012269:Ribosomal Proteins; D007460:Iodoacetamide