Functional topography of human ribosomes as studied by affinity labeling with reactive mRNA analogs.
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.
Karpova
G G
GG
Institute of Bioorganic Chemistry, Siberian Division of the Russian Academy of Sciences, Novosibirsk.
Graifer
D M
DM
Malygin
A A
AA
Mundus
D A
DA
Zenkova
M A
MA
Mamaev
S V
SV
D000345:Affinity Labels; D006801:Humans; D009690:Nucleic Acid Conformation; D009943:Organophosphorus Compounds; D012333:RNA, Messenger / Q000737:chemistry; D012337:RNA, Ribosomal, 18S / Q000737:chemistry; D012360:RNA, Transfer, Phe / Q000737:chemistry; D012270:Ribosomes / Q000737:chemistry*; D014500:Uracil Nucleotides
D000345:Affinity Labels; D009943:Organophosphorus Compounds; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S; D012360:RNA, Transfer, Phe; D014500:Uracil Nucleotides; C064973:4-(N-2-chloroethyl-N-methylamino)-benzylphosphamide hexauridylate