Abstract
Analysis of the Escherichia coli transcriptome identified a unique subset of messenger RNAs (mRNAs) that contain a conventional untranslated leader and Shine-Dalgarno (SD) sequence upstream of the gene’s start codon while also containing an AUG triplet at the mRNA’s 5’- terminus (5’-uAUG). Fusion of the coding sequence specified by the 5’-terminal putative AUG start codon to a lacZ reporter gene, as well as primer extension inhibition assays, reveal that the majority of the 5’-terminal upstream open reading frames (5’-uORFs) tested support some level of lacZ translation, indicating that these mRNAs can function both as leaderless and canonical SD-leadered mRNAs. Although some of the uORFs were expressed at low levels, others were expressed at levels close to that of the respective downstream genes and as high as the naturally leaderless cI mRNA of bacteriophage λ. These 5’-terminal uORFs potentially encode peptides of varying lengths, but their functions, if any, are unknown. In an effort to determine whether expression from the 5’-terminal uORFs impact expression of the immediately downstream cistron, we examined expression from the downstream coding sequence after mutations were introduced that inhibit efficient 5’-uORF translation. These mutations were found to affect expression from the downstream cistrons to varying degrees, suggesting that some 5’-uORFs may play roles in downstream regulation. Since the 5’-uAUGs found on these conventionally leadered mRNAs can function to bind ribosomes and initiate translation, this indicates that canonical mRNAs containing 5’-uAUGs should be examined for their potential to function also as leaderless mRNAs.
Highlights
Translation initiation is the rate-limiting step in protein synthesis and requires ribosomes to recognize and bind messenger RNAs
The results presented here indicate that AUG triplets at the 5’-termini of SD-led messenger RNAs (mRNAs) can function as start codons and have the ability to bind ribosomes and be translated as leaderless mRNAs
Using β-galactosidase and toeprint assays, we have shown that many 5’-terminal upstream open reading frame (5’-upstream open reading frame (uORF)) were translated at biologically relevant levels and bound by 70S ribosomes, with some appearing to play a role in expression of the downstream coding sequence (CDS)
Summary
Translation initiation is the rate-limiting step in protein synthesis and requires ribosomes to recognize and bind messenger RNAs (mRNA). Addition of a 5’-terminal AUG to an internal segment of lacZ mRNA makes it competent to form ternary complexes with 70S ribosomes and initiator tRNA [11], suggesting that a 5’-terminal AUG might be a sufficient signal for ribosomes to identify a leaderless mRNA. We utilized the RegulonDB database [17] to conduct an in silico search of E. coli for canonical, SD-led mRNAs that contain 5’-terminal AUG triplets These 5’-terminal upstream AUGs (5’-uAUGs) were assayed for their ability to act as initiation codons for translation of the putative 5’-terminal upstream open reading frame (5’-uORF). Our results suggest that certain 5’-uAUGs and their defined ORFs impact regulation of downstream expression
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