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

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• 作者：Bulygin ; Konstantin N. ; Matasova ; Natalie B. ; Graifer ; Dmitri M. ; Veniyaminova ; Aliya G. ; et. al.
• 关键词：Human ribosome ; Decoding site ; Ribosomal protein ; Cross-linking ; Oligoribonucleotide ; Derivative
• 刊名：Biochimica et Biophysica Acta (BBA)/Gene Structure and Expression
• 出版年：1997
• 出版时间：April 10, 1997
• 年：1997
• 卷：1351
• 期：3
• 页码：325-332
• 全文大小：676 K

文摘

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 pAUGU_n (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-tRNA^Phe. 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 pAUGU_n were cross-linked to proteins S3 and S3a in comparable extent; 3′-derivative of AUGU₃pC modified protein S3a preferentially.