Key result
Close AUG codon spacing drives robust dicistronic expression whereas stepwise separation converts to monocistronic translation.
Why the study?
The mechanism by which closely spaced AUG initiation codons confer dicistronic character on eukaryotic mRNAs and support bicistronic expression was unclear.
Closely spaced AUG codons may represent an under-recognized strategy for bicistronic expression from eukaryotic mRNAs, suggesting 5'-3' ribosome scanning involves backward excursions averaging about 15 nucleotides.
Robust downstream AUG expression in dicistronic viral RNA warrants no clinical change; leaves open conservation in mammalian systems.
TYMV RNA supports the translation of two proteins, p69 and p206, from AUG initiation codons 7 nucleotides apart. We have studied the translation of this overlapping dicistronic mRNA with luciferase reporter RNAs electroporated into cowpea protoplasts and in toe-printing studies that map ribosomes stalled during initiation in wheat germ extracts. Agreement between these two assays indicates that the observed effects reflect ribosome initiation events. The robust expression from the downstream AUG206 codon was dependent on its closeness to the upstream AUG69 codon. Stepwise separation of these codons resulted in a gradual increase in upstream initiation and decrease in downstream initiation, and expression was converted from dicistronic to monocistronic. Selection by ribosomes for initiation between the nearby AUG codons was responsive to the sequence contexts that govern leaky scanning, but the normally strong position effect favoring upstream initiation was greatly diminished. Similar dicistronic expression was supported for RNAs with altered initiation sequences and for RNAs devoid of flanking viral sequences. Closely spaced AUG codons may thus represent an under-recognized strategy for bicistronic expression from eukaryotic mRNAs. The initiation behavior observed in these studies suggests that 5'-3' ribosome scanning involves backward excursions averaging about 15 nucleotides.
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Matsuda et al. (2006) studied TYMV RNA translation. Close spacing of AUG initiation codons vs. Stepwise separation of codons was evaluated on Translation expression (dicistronic vs monocistronic). Close spacing of AUG initiation codons supported robust dicistronic expression, whereas stepwise separation converted expression from dicistronic to monocistronic.
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