Polysomes Bypass a 50 Nucleotide Coding Gap less Efficiently than Monosomes Due to Attenuation of a 5' mRNA Stem Loop and Enhanced Drop-off.

Abstract Efficient translational bypassing of a 50 nt non-coding gap in a phage T4 topoisomerase subunit gene (gp60) requires several recoding signals. Here we investigate the function of the mRNA stem loop 5′ of the take-off codon, as well as the importance of ribosome loading density on the mRNA for efficient bypassing. We show that polysomes are less efficient at mediating bypassing than monosomes, both in vitro and in vivo, due to their preventing formation of a stem loop 5′ of the take-off codon and allowing greater peptidyl-tRNA drop off. A ribosome profiling analysis of phage T4 infected E. coli yielded protected mRNA fragments within the normal size range derived from ribosomes stalled at the take-off codon. However, ribosomes at this position also yielded some 53 nucleotide fragments, 16 longer. These were due to protection of the nucleotides that form the 5′ stem loop. NMR shows that the 5′ stem loop is highly dynamic. The importance of different nucleotides in the 5′ stem loop is revealed by mutagenesis studies. These data highlight the significance of the 5′ stem loop for the 50 nt bypassing, and further enhance appreciation of relevance of the extent of ribosome loading for recoding.