Open AccessIdentification of the 30‐S Ribosomal Proteins at the Decoding Site by Affinity Labelling with a Reactive Oligonucleotide
Author(s) -
LÜHRMANN Reinhard,
GASSEN Hans Günter,
STÖFFLER Georg
Publication year - 1976
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1976.tb10419.x
Subject(s) - ribosome , oligonucleotide , puromycin , ribosomal rna , ribosomal protein , transfer rna , biochemistry , covalent bond , a site , binding site , eukaryotic ribosome , eukaryotic large ribosomal subunit , protein subunit , chemistry , biology , microbiology and biotechnology , protein biosynthesis , rna , dna , gene , organic chemistry
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[ 3 H]acn 5 U (where i[ 3 H]acn 5 U =5‐iodo[ 3 H]acetylaminouridine) as a chemical affinity label. S12 was modified to a minor extent and mainly wheel assays were carried out in the absence of tRNA1 Evidence for the specificity of the covalent binding of the oligonucleotide probe is given by the following experiments: G‐U‐U‐iacn 5 U 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‐iacn 5 U 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.