Identification of the 30-S ribosomal proteins at the decoding site by affinity labelling with a reactive oligonucleotide.
Proteins S18 and S1 were identified by immunological and electrophoretic techniques as constituents of the 30-S ribosomal decoding site by the use of G-U-U-i[3H]acn5U (where i[3H]acn5U=5-iodo[3H]acetylaminouridine) as a chemical affinity label. S12 was modified to a minor extent and mainly when assays were carried out in the absence of tRNA. 1. Evidence for the specificity of the covalent binding of the oligonucleotide probe is given by the following experiments: G-U-U-iacn5U bound twice as much Val-tRNA as compared to G-U-U-U. The covalent binding was strongly inhibited by prebound poly(U)-Phe-tRNA. 'Programmed' ribosomes, i.e. ribosomes which contained only covalent bound oligonucleotide, stimulated the binding of Val-tRNA. 2. Val-tRNA bound to G-U-U-iacn5U labelled ribosomes could not be transferred to puromycin. This might indicate that binding of the oligonucleotide occurred at the aminoacyl site. 3. 80-90% of the radioactivity was found in the 30-S subunit and only the protein moiety of the ribosome was modified. 4. Unambiguous identification of the labelled proteins was achieved by immunological techniques and revealed proteins S1 and S18 as constituents of the 30-S ribosomal decoding site.
D000345:Affinity Labels; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism; D005779:Immunodiffusion; D007700:Kinetics; D009841:Oligonucleotides; D009843:Oligoribonucleotides; D010649:Phenylalanine; D011485:Protein Binding; D012343:RNA, Transfer / Q000378:metabolism; D012269:Ribosomal Proteins / Q000276:immunology / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D014633:Valine
D000345:Affinity Labels; D009841:Oligonucleotides; D009843:Oligoribonucleotides; D012269:Ribosomal Proteins; D010649:Phenylalanine; D012343:RNA, Transfer; D014633:Valine