EFFICIENCY OF T4 GENE-60 TRANSLATIONAL BYPASSING

Ribosomes translating bacteriophage T4 gene 60 mRNA bypass 50 noncodin g nucleotides from a takeoff site at codon 46 to a landing site just u pstream 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, w hich allow detection of all major products synthesized from the gene 6 0 mRNA, show that 50% of ribosomes bypass the coding gap while the res t 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 rel ied on comparing test constructs to constructs with a precise excision of the gap (gap deletion), Further analysis showed that these estimat es are distorted by differences between test and gap deletion function al mRNA levels, An internal translation initiation site at Metl2 of ge ne 60 (which eliminates part of the essential nascent peptide) also di storts these estimates, Together, these results support an efficiency estimate of similar to 50%, less than previously reported, This estima te suggests that bypassing efficiency is determined by the competition between reading signals and release factors and gives new insight int o the kinetics of bypassing signal action.