Photoaffinity labeling of the ribosomal A site with S-(p-azidophenacyl)valyl-tRNA.
S-(p-Azidophenacyl)valyl-tRNA, an analog of valyl-tRNA which has a photoaffinity label attached to its 4-thiouridine residue, was bound to the ribosomal A site at 10 mM Mg2+. Binding was stimulated 25-fold by the presence of elongation factor EFTu. Photoactivation of the p-azidophenacyl group by irradiation resulted in covalent linking of 6% of the noncovalently bound tRNA to the ribosomes. Covalent linking was dependent on the simultaneous presence of ribosomes, poly(U2,G),EFTu.GTP, required irradiation, and did not occur when S-(phenacyl)valyl-tRNA, a nonphotolyzable analog, replaced S-(p-azidophenacyl)valyl-tRNA. The attached tRNA was distributed approximately equally between both the 30S and 50S subunits. At the 30S subunit, 30% of the tRNA was bound to protein while 70% was linked to 16S RNA. At the 50S subunit, however, negligible binding to the 23S RNA was observed. More than 90% of the tRNA was attached to low molecular weight material according to sodium dodecyl sulfate-sucrose gradient analysis, and more than 87% of this fraction consisted of tRNA-protein complexes as assayed by phenol solubility and electrophoretic mobility before and after protease treatment. These results, in conjunction with our previous report (I. Schwartz and J Ofengand (1974), Proc. Natl. Acad. Sci. U.S.A. 71, 3951) which showed that covalent linking of this same tRNA derivative at the ribosomal P site resulted in attachment solely to the 16S RNA, demonstrate that 16S, but not 23S or 5S rRNA, is an important component of the tRNA binding site in the region of the 4-thiouridine residue and furthermore show that ribosomal A and P sites are topologically distinct.
D000345:Affinity Labels; D001665:Binding Sites; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D008722:Methods; D008958:Models, Molecular; D010445:Peptide Elongation Factors; D010777:Photochemistry; D012333:RNA, Messenger / Q000378:metabolism; D012335:RNA, Ribosomal / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate; D014316:Tritium; D014633:Valine
D000345:Affinity Labels; D010445:Peptide Elongation Factors; D012333:RNA, Messenger; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; D014316:Tritium; D012967:Sodium Dodecyl Sulfate; D012343:RNA, Transfer; D014633:Valine