Transition of the mRNA sequence downstream from the initiation codon into a single-stranded conformation is strongly promoted by binding of the initiator tRNA

Balakin, Andrew; Skripkin, Eugene; Shatsky, Ivan; Bogdanov, Alexey

Using an RNA footprinting technique, accessible sites on the mRNA initiation region bound to the ribosome have been determined. Chemical probing experiments have been done both in the presence and absence of the initiator tRNA with dimethyl sulfate, kethoxal and carbodiimide as reagent probes. As an mRNA, a mini-mRNA containing the initiation region of bacteriophage λ gene cro has been used. This region is characterized by a long single-stranded Shine-Dalgarno (SD) sequence followed by two hairpin structures of which the first one comprises in its loop the initiation codon. As compared to a free mRNA, the only nucleotides additionally protected in the binary mRNA-ribosome complex have been those which belong to the S-D sequence and the initiation codon. The protection of other nucleotides has not changed. Addition of the initiator RNA results in the modification of nucleotides in the stems of the downstream hairpin structures of the initiation region. This reflects their transition into a single-stranded conformation promoted by tRNA. A possible implication of these findings for the decoding process is discussed.