Secondary Structure of Bacteriophage T4 Gene 60 mRNA: Implications for Translational Bypassing.

Abstract

Translational bypassing is a unique phenomenon of Bacteriophage T4 gene 60 in which the ribosome generates a single polypeptide chain from a discontinuous open reading frame. In about half of translational events, the ribosome skips over a 50 nucleotide segment in the open reading frame to generate a full-length subunit of a Type II DNA topoisomerase; the rest of the time the ribosome terminates at an in-frame stop codon at the 5’ edge of the untranslated region or coding gap. Mutational and computational analyses suggest a stable structure forms in the coding gap to induce bypassing, yet there is no direct evidence of structure in this region. We have probed the structure of gene 60 mRNA with Tb3+ ions and the selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE) reagent 1M7 and constructed a secondary structure model compatible with experimental data. Our model confirms the presence of a predicted UUCG-loop hairpin at the 5’ edge of the coding gap that aids in efficient translational bypassing. Contrary to several previously proposed models, however, the rest of the untranslated region is highly reactive to both probing reagents suggesting it possesses little structure. Furthermore, mutational analyses reveal that the untranslated region does base pair to any other region of gene 60 mRNA. The autonomous nature of the coding gap is consistent its role as a foreign genetic element inserted into gene 60 mRNA to inhibit cleavage by the MobA homing endonuclease. These results indicate that the 5’ hairpin may be the only structural element required to stimulate translational bypassing.