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

Abstract Derivatives of 5′- 32 P labeled (pU) 3 an (pU) 6 bearing 4-(N-2-chloroethyl-N-methylamino)benzylmethylamine residue attached to 5′-phosphate via phoshamide bond and (Up) 5 U[ 32 P]pC and (Up) 11 U[ 32 P]pC bearing 4-(N-2-chlroethyl-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 32 P-labeled pAUG and pAUGU 3 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 pAUGU 3 derivatives and within 976–1164 for (pU) 3 and pAUG ones. The sites of 18S rRNA modification with the derivatives of (Up) 5 UpC and (Up) 11 UpC 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.