FUNCTIONAL TOPOGRAPHY OF HUMAN RIBOSOMES AS STUDIED BY AFFINITY LABELING WITH REACTIVE MESSENGER-RNA ANALOGS

Derivatives of 5'-P-32 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[P-32]pC and (Up)11U[P-32]pC bearing 4-(N-2-chloroethyl-N-methyl-amino)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 P-32-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.