Crosslinking of phenylalanyl-tRNA to the ribosomal A site via a photoaffinity probe attached to the 4-thiouridine residue is exclusively to ribosomal protein S19.
Phe-tRNA of Escherichia coli, specifically derivatized at the S4U8 position with the 9 A long p-azidophenacyl photoaffinity probe, can be crosslinked to 30 S ribosomal protein when the tRNA is placed at the ribosomal A site. This protein has now been identified by immunological methods. The protein-[3H]Phe-tRNA covalent complex, obtained by extraction with 6 M-urea, was reacted separately with each of the 21 purified antisera to 30 S ribosomal proteins. The double antibody technique was used. Anti-S19 was the only antiserum able to precipitate the radioactivity, and 66 to 81% of the added radioactivity could be precipitated. The same result was obtained with three different ribosome preparations, at low as well as high crosslinking yield, with dipeptidyl-tRNA in the A site as well as aminoacyl-tRNA, and when binding and crosslinking were performed at 20 mM-Mg2+ instead of at 5 mM. Therefore, when aminoacyl-tRNA or peptidyl-tRNA is in the ribosomal A site, position 8, which is always uridine or 4-thiouridine, must be within 9 A of protein S19.
D000345:Affinity Labels / Q000494:pharmacology; D000906:Antibodies / Q000276:immunology; D001386:Azides / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D002499:Centrifugation, Density Gradient; D004926:Escherichia coli; D010444:Peptide Elongation Factor Tu; D010445:Peptide Elongation Factors / Q000494:pharmacology; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000276:immunology / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D013891:Thiouridine / Q000378:metabolism*
D000345:Affinity Labels; D000906:Antibodies; D001386:Azides; D010445:Peptide Elongation Factors; D012346:RNA, Transfer, Amino Acyl; D012269:Ribosomal Proteins; C040343:ribosomal protein S19; D013891:Thiouridine; D010444:Peptide Elongation Factor Tu; C011171:4-azidophenacyl bromide