Crosslinking of mRNA Analogs Containing 4-Thiouridine Residues on the 3'- or 5'-Side of the Coding Triplet to the mRNA Binding Center of the Human Ribosome

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Abstract
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 GGGACC (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.

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