Interaction of mRNA with the Escherichia coli ribosome: accessibility of phosphorothioate-containing mRNA bound to ribosomes for iodine cleavage.

The contacts of phosphate groups in mRNAs with ribosomes were studied. Two mRNAs were used: one mRNA contained in the middle two defined codons to construct the pre- and the post-translocational states, the other was a sequence around the initiation site of the natural cro-mRNA. Phosphorothioate nucleotides were randomly incorporated at a few A, G, U or C positions during in vitro transcription. Iodine can cleave the thioated positions if they are not shielded by ribosomal components. Only a few minor differences in iodine cleavage of ribosome bound and non-bound mRNA were observed: the nucleotide two positions upstream of the decoding codons (i.e. those codons involved in codon-anticodon interactions) showed a reduced accessibility for iodine and the nucleotide immediately following the decoding codons an enhanced accessibility in both elongating states. In initiating ribosomes where the mRNA contained a strong Shine-Dalgarno sequence, at least five phosphates were additionally slightly protected covering the Shine-Dalgarno sequence and nucleotides downstream including the initiator AUG in the P site (Al, G3, G-2, G-5 and A-7). The low contact levels of the phosphates in the mRNA with the elongating ribosome strikingly contrast with the pronounced contact patterns previously described for tRNAs. The data obtained in this study, as well as results of previous studies, suggest that mRNA regions downstream and upstream of decoding codons form only weak contacts with ribosomal components and that the mRNA thus is mainly fixed by codon-anticodon interaction on the elongating ribosome.