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 (s(4)U) upstream or downstream of the GAC codon. In the presence of tRNA(Asp), the GAC codon of the mRNAs was targeted to the ribosomal P site thus placing s(4)U 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 s(4)U 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 s(4)U. Cross-links of mRNAs containing s(4)U 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 s(4)U in positions -3 to -1 confirmed that tRNA(Asp) actually phased the mRNA on the ribosome.