Environment of the 5′-terminal nucleotide of the mRNA codon at the P and E sites of human ribosome: Crosslinking with pUUUGUU derivatives bearing a photoactivatable group at an uracil residue or 5′-phosphate

Abstract

Photoaffinity crosslinking was carried out between 80S ribosomes from human placenta and mRNA analogs, namely, derivatives of hexaribonucleotide pUUUGUU (comprising Phe and Val codons) with a perfluoroarylazido group at the C5 atom of the uracil residue at the first position, or at the 5′-terminal phosphate. Three types of ribosome complex with 5′-³²P-labeled derivatives of pUUUGUU were studied: (1) with Phe-tRNA^Phe and codon UUU at the P site; (2) with tRNA^Phe and codon UUU at the P site and PheVal-tRNA^Val and codon GUU at the A site; (3) with Val-tRNA^Val and codon GUU at the P site (codon UUU at the E site). Upon mild UV irradiation (>280 nm) of the complexes, the pUUUGUU derivatives were crosslinked to 18S rRNA and proteins in the ribosomal small subunit. In the absence of tRNA, no modification of ribosomes occurred. Nucleotides of 18S rRNA crosslinked to the mRNA analogs were identified using the reverse transcriptase analysis. It turned out that the photoactivatable group at the first nucleotide of codon pUUU at the P site is only crosslinked with G-1207 of 18S rRNA, whether this group is at the 5′-phosphate or the C5 atom of the uracil residue. If codon UUU is located at the E site, the pUUUGUU derivative with the photoactivatable group at the uracil residue modifies G-961 of 18S rRNA, which is for the first time found at the mRNA-binding center of 80S ribosomes.