Arrangement of mRNA 3’ of the A Site Codon on the Human 80S Ribosome

Abstract

Positioning of mRNA 3’ of the A site codon was studied with the use of short mRNA analogues carrying a UUU triplet at the 5’-termini and a perfluorophenylazide group at either the N7 atom of the guanosine or the C5 atom of the uridine 3’ of the triplet. Modified nucleotides were directed to positions +7, +9 or +12 with respect to the first nucleotide of the P site codon by tRNAPhe cognate to the UUU triplet targeted to the P site. Mild UV-irradiation resulted in cross-linking of the mRNA analogues to the 18S rRNA and to 40S proteins, the yield of cross-linking depending on the nature of the mRNA nucleotide bearing the modified group and its position on the ribosome. In addition, the yield of cross-linking to the 18S rRNA decreased strongly when the modified nucleotide was moved from position +7 to position +12. All the mRNA analogues cross-linked to the 18S rRNA nucleotides that had been found earlier at the decoding site, namely, to the invariant dinucleotide A1824/A1825 and the variable A1823 in the 3’-minidomain of the 18S rRNA, and also to the invariant nucleotide C1698 in the 3’-minidomain and the conserved region 605-620 in the apical region of helix 18 in the 5’-domain. The results indicate that (i) the mRNA makes a sharp turn between positions +6 and +7, (ii) the triplet immediately 3’ of the A site codon neighbors the 18S rRNA and proteins, and (iii) the codon 3’ of the triplet mentioned is surrounded mainly by proteins.