CROSS-LINKING OF MESSENGER-RNA ANALOGS CONTAINING 4-THIOURIDINE RESIDUES ON THE 3'-SIDE OR 5'-SIDE OF THE CODING TRIPLET TO THE MESSENGER-RNA BINDING CENTER OF THE HUMAN RIBOSOME

The interaction between mRNA and 18S rRNA within complexes of human placenta 80S ribosomes has been investigated by photochemical cross-linking experiments using mRNA analogues substituted with 4-thiouridine at specific locations. mRNA analogues 51 or 54 nucleotides long were prepared from synthetic DNA templates. These mRNA analogues contained either the sequence GGG-ACC (coding for glycine and threonine, respectively) or the single triplet GGG together with 2-4 4-thiouridine residues located at various positions with respect to the coding triplets. The products of cross-linking of the mRNA analogues to 18S rRNA within different model complexes without tRNA or in the presence of cognate tRNAs were analyzed by reverse transcription. Two cross-linking sites in the 18S rRNA were detected. The first site, U630, was cross-linked by mRNA 8' (s4U at +20, +22, +24, and +26), mRNA 9e' (s4U at -16, -18, and -20), and mRNA 10 (s4U at +4, +6, -1, and -3) but, unexpectedly, not with either mRNA 10b (s4U at +4 and +6) or mRNA 10c (s4U at -1 and -3). The second site, U1111/A1112, was cross-linked by mRNA 10 and mRNA 10c but not by any of the other mRNA analogues. There is significant tRNA dependence on cross-linking only for mRNA analogue 9e'. Both of the sites detected correspond to sites of mRNA cross-linking in Escherichia coli 16S rRNA.