Sites of 18S rRNA contacting mRNA 3′ and 5′ of the P site codon in human ribosome: A cross-linking study with mRNAs carrying 4-thiouridines at specific positions

Long synthetic mRNAs were used to study the positioning of the E site codon, the 2nd and 3rd nucleotides of the A site bound codon and a nucleotide 3′ of this codon with respect to the 18S rRNA in the human 80S ribosome. The mRNAs contained a GAC triplet coding for Asp and a single 4-thiouridine residue (s4U) upstream or downstream of the GAC codon. In the presence of tRNAAsp, the GAC codon of the mRNAs was targeted to the ribosomal P site thus placing s4U in one of the following positions − 3, − 2, − 1, + 5, + 6 or + 7 with respect to the first nucleotide of the P site bound codon. It was found that mRNAs that bore s4U in positions + 5 to + 7 cross-linked to the 18S rRNA nucleotides C1696, C1698 and 1820–1825, the distribution of cross-links among these targets depending on the position of s4U. Cross-links of mRNAs containing s4U in positions − 3 to − 1 were found in the region 1699–1704 of the 18S rRNA. In the absence of tRNA, all mRNAs cross-linked only to C1696 and C1698. Absence of the cross-linked nucleotides C1696 and C1698 in the case of mRNAs containing s4U in positions − 3 to − 1 confirmed that tRNAAsp actually phased the mRNA on the ribosome.