Messenger RNA release from ribosomes during 5′‐translational blockage by consecutive low‐usage arginine but not leucine codons in Escherichia coli

Abstract

SummaryIn '5Ј-translational blockage', significantly reduced yields of proteins are synthesized in Escherichia coli when consecutive low-usage codons are inserted near translation starts of messages (with reduced or no effect when these same codons are inserted downstream). We tested the hypothesis that ribosomes encountering these low-usage codons near the translation start prematurely release the mRNA. RNA from polysome gradients was fractionated into pools of polysomes and monosomes and a ribosome-free pool. New hybridization probes, called 'molecular beacons', and standard slot blots were used to detect test messages containing either consecutive lowusage AGG (arginine) or synonymous high-usage CGU insertions near the 5Ј end. The results show an approximately twofold increase in the ratio of free to bound mRNA when the low-usage codons were present in the message compared with when highusage codons were present. In contrast, there was no difference in the ratio of free to bound mRNA when consecutive low-usage CUA or high-usage CUG (leucine) codons were inserted or when the arginine codons were inserted near the 3Ј end. These data indicate that at least some mRNA is released from ribosomes during 5Ј-translational blockage by arginine but not leucine codons, and they support proposals that premature termination of translation can occur in some conditions in vivo in the absence of a stop codon.

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“…Over the last 20 years, experimental studies reported various phenotypic effects associated with modification of sequences codon usage: alteration of mRNA structure [32],[33], modification of translation efficiency [34],[35], and protein aggregation due to alteration of folding route and final tertiary structure [36]–[38]. Although a recent report has linked a phenotypic effect to the presence of two weakly used codons combined with a non-synonymous SNP [39], to the best of our knowledge no significant impact of a single rare codon has ever been described, most likely because it should involve a decrease in protein synthesis too weak to be measured accurately.…”