Characterization of Ribosomal Bypassing through a 50-Nt mRNA Untranslated Gap

Abstract

The prokaryotic ribosome bypasses a 50-nt untranslated region in the gene60 mRNA of bacteriophage T4, with a ∼50% bypassing efficiency. Multiple cis-acting signals are required for efficient ribosome bypassing. How the conformations of the ribosome and the mRNA change in response to these elements remains elusive.We have developed an in vitro translation assay to assess the effects of these elements. Using complementary DNA-oligos, we can mask the mRNA sequences and the structural elements, which allow us to determine whether the ribosome is actually “scanning” through the region after take-off. We can also tune the concentrations of each translation factor, including Release Factor 1 (RF1) that recognizes the UAG stop codon at the 5'-junction of the gap. Interestingly, the ribosome bypassing efficiency did not exceed ∼50% even in the complete absence of RF1. Additionally, the drop in the protein yield under such condition may reflect the inability for the ribosome to be liberated by RF1 and consequently a lower ribosome turn-over rate. These observations indicate the existence of another drop-off pathway independent of the RF-mediated termination.The bypassing efficiency seems to be affected by ribosome loading rate, and that multiple unexpected ribosome drop-off products were found when bypassing was blocked by the DNA-oligos. The possibility arises that both the upstream ribosomes and the stable mRNA hairpin could push the ribosome at the take-off site to slide through the untranslated gap after the tRNA:mRNA interaction is weakened by the nascent peptide signal. Thus, the directionality of sliding could be provided without additional energy expenditure during bypassing.