Protein S3 in the Human 80S Ribosome Adjoins mRNA 3′ of the A-Site Codon
M. V. Molotkov1, D. M. Graifer1, E. A. Popugaeva1, K. N. Bulygin1, M. I. Meschaninova1, A. G. Ven’yaminova1 and G. G. Karpova1 #
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1Institute of Chemical Biology and Fundamental Medicine, Siberian Division, Russian Academy of Sciences, pr. Akademika Lavrent’eva 8, Novosibirsk, 630090, Russia
Received: June 5, 2006; in final form: June 15, 2006
Abstract. The protein environment of mRNA 3′ of the A-site codon (the decoding site) in the human 80S ribosome was studied using a set of oligoribonucleotide derivatives bearing a UUU triplet at the 5′-end and a perfluoroarylazide group at one of the nucleotide residues 3′ of this triplet. Analogues of mRNA were phased into the ribosome using binding at the tRNAPhe P-site, which recognizes the UUU codon. Mild UV irradiation of ribosome complexes with tRNAPhe and mRNA analogues resulted in the predominant crosslinking of the analogues with the 40S subunit components, mainly with proteins and, to a lesser extent, with rRNA. Among the 40S subunit ribosomal proteins, the S3 protein was the main target for modification in all cases. In addition, minor crosslinking with the S2 protein was observed. The crosslinking with the S3 and S2 proteins occurred both in ternary complexes and in the absence of tRNA. Within ternary complexes, crosslinking with S15 protein was also found, its efficiency considerably falling when the modified nucleotide was moved from positions +5 to +12 relative to the first codon nucleotide in the P-site. In some cases, crosslinking with the S30 protein was observed; it was most efficient for the derivative containing a photoreactive group at the +7 adenosine residue. The results indicate that the S3 protein in the human ribosome plays a key role in the formation of the mRNA binding site 3′ of the codon in the decoding site.
Key words: 80S ribosome, mRNA analogue, mRNA-binding site, photoaffinity crosslinking, ribosomal proteins
Russian Journal of Bioorganic Chemistry 2007, 33 (4): 399-409