Protein environment of mRNA at the decoding site of 80S ribosomes from human placenta as revealed from affinity labeling with mRNA analogs – derivatives of oligoribonucleotides

摘要

Affinity labeling of 80S ribosomes from human placenta has been studied using various mRNA analogs, namely, 2′,3′-O-[4-(N-2-chloroethyl)-N-methylamino]benzylidene derivatives of oligoribonucleotides (Up)n?1U[32P]pC (n=3, 6 or 12) and AUGU3[32P]pC as well as ([4-(N-2-chloroethyl)-N-methylamino]benzylmethyl-[5′-32P]-phosphamide derivatives of pAUGUn (n=3 or 6). Labeling of 80S ribosomes with the derivatives of oligouridylates was carried out in complexes obtained nonenzymatically in the presence of saturating amounts of Phe-tRNAPhe. Complexes with derivatives bearing AUG codon were obtained using a fractionated lysate from rabbit reticulocytes which contained protein translation factors and was deprived from endogeneous ribosomes and mRNAs. In all cases, 40S subunits were labeled preferentially. Within the subunits, both 18S rRNA and proteins were found to be modified. Sites of cross-linking in 18S rRNA have been identified earlier. Here, it is shown that the main targets of cross-linking among the ribosomal proteins were S3 and S3a (with minor modification of S26) for the 3′-derivatives of (Up)5UpC and (Up)11UpC. For the same derivative of (Up)2UpC, the reverse modification pattern was observed. 5′-derivatives of pAUGUn were cross-linked to proteins S3 and S3a in comparable extent; 3′-derivative of AUGU3pC modified protein S3a preferentially.