Covalent cross-linking of tRNAGly1 to the ribosomal P site via the dihydrouridine loop.
The dihydrouracil residue at position 20 of Escherichia coli tRNAGly1 has been replaced by the photoaffinity reagent, N-(4-azido-2-nitrophenyl)glycyl hydrazide (AGH). The location of the substituent was confirmed by the susceptibility of the modified tRNA to cleavage with aniline. When N-acetylglycyl-tRNAGly1 derivatized with AGH was bound noncovalently to the P site of E. coli 70 S ribosomes, 5-6% on average was photochemically cross-linked to the ribosomal particles in a reaction requiring poly(G,U), irradiation and the presence of the AGH label in the tRNA. Approximately two-thirds of the covalently attached tRNA was associated with 16 S RNA in the 30 S subunit. This material was judged to be in the P site by the criterion of puromycin reactivity. As partial RNAase digestion of the tRNA-16 S RNA complex produced labeled fragments from both 5' and 3' segments of the rRNA, there appeared to be more than one site of cross-linking in the 30 S subunit. The small amount of N-acetylglycyl-tRNAGly1 associated with the 50 S subunit was also linked mainly to rRNA, but it was not puromycin-reactive.
D000345:Affinity Labels / Q000378:metabolism; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D009690:Nucleic Acid Conformation; D010777:Photochemistry; D011691:Puromycin / Q000494:pharmacology; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D014529:Uridine / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D012346:RNA, Transfer, Amino Acyl; D011691:Puromycin; C046007:N-(4-azido-2-nitrophenyl)glycyl hydrazide; D012260:Ribonucleases; D014529:Uridine