Functional topography of human ribosomes as studied by affinity labeling with reactive mRNA analogs

Biochimie. 1992 Apr;74(4):373-80. doi: 10.1016/0300-9084(92)90115-u.

Abstract

Derivatives of 5'-32P labeled (pU)3 an (pU)6 bearing 4-(N-2-chloroethyl-N-methylamino)benzylmethylamine residue attached to 5'-phosphate via phosphamide bond and (Up)5U[32P]pC and (Up)11U[32P]pC bearing 4-(N-2-chloroethyl-N-methylamino)benzyl residue attached to 3'-end via benzylidene bond were applied for the affinity labeling of 80S ribosomes from human placenta in the presence of a cognate tRNA. The derivatives of 32P-labeled pAUG and pAUGU3 analogous to the 5'-phosphamides of (pU)n were used for affinity labeling of 40S subunits in the presence of ternary complex eIF-2.GTP.Met-tRNA(f). The sites of the reagents' attachment to 18S ribosomal RNA were identified by blot-hybridization of the modified 18S rRNA with restriction fragments of the corresponding rDNA. They were found to be located within positions 976-1057 for (pU)6 and pAUGU3 derivatives and within 976-1164 for (pU)3 and pAUG ones. The sites of 18S rRNA modification with the derivatives of (Up)5UpC and (Up)11UpC were found within positions 1610-1869 at 3'-end of the molecule. All the sites identified here are located presumably within highly conserved parts of the eukaryotic small subunit rRNA secondary structure.

MeSH terms

  • Affinity Labels
  • Humans
  • Nucleic Acid Conformation
  • Organophosphorus Compounds
  • RNA, Messenger / chemistry
  • RNA, Ribosomal, 18S / chemistry
  • RNA, Transfer, Phe / chemistry
  • Ribosomes / chemistry*
  • Uracil Nucleotides

Substances

  • Affinity Labels
  • Organophosphorus Compounds
  • RNA, Messenger
  • RNA, Ribosomal, 18S
  • RNA, Transfer, Phe
  • Uracil Nucleotides
  • 4-(N-2-chloroethyl-N-methylamino)-benzylphosphamide hexauridylate