Efficiency of T4 Gene 60 Translational Bypassing

Abstract

Ribosomes translating bacteriophage T4 gene 60 mRNA bypass 50 noncoding nucleotides from a takeoff site at codon 46 to a landing site just upstream of codon 47. A key signal for efficient bypassing is contained within the nascent peptide synthesized prior to takeoff. Here we show that this signal is insensitive to the addition of coding information at its N terminus. In addition, analysis of amino-terminal fusions, which allow detection of all major products synthesized from the gene 60 mRNA, show that 50% of ribosomes bypass the coding gap while the rest either terminate at a UAG stop codon immediately following codon 46 or fail to resume coding. Bypassing efficiency estimates significantly lower than 50% were obtained with enzymatic reporter systems that relied on comparing test constructs to constructs with a precise excision of the gap (gap deletion). Further analysis showed that these estimates are distorted by differences between test and gap deletion functional mRNA levels. An internal translation initiation site at Met12 of gene 60 (which eliminates part of the essential nascent peptide) also distorts these estimates. Together, these results support an efficiency estimate of ~50%, less than previously reported. This estimate suggests that bypassing efficiency is determined by the competition between reading signals and release factors and gives new insight into the kinetics of bypassing signal action.