Translational recoding signals: Expanding the synthetic biology toolbox

Abstract

Innovation follows discovery. If the 20th century was a golden age of discovery in the biomolecular biosciences, the current century may be remembered by the explosion of beneficial devices and therapies conceived by the bioengineers of the era. Much as the development of solid-state electronic components made possible the information revolution, the rational combining of millions of basic molecular control modules will enable the development of highly sophisticated biomachines that will make today's smartphones appear rudimentary. The molecular toolbox is already well-stocked, particularly in our ability to manipulate DNA, control transcription, generate functionally novel hybrid proteins, and expand the genetic code to include unnatural amino acids. This review focuses on how RNA-based regulatory modules that direct alternative readings of the genetic code can be employed as basic circuit components to expand our ability to control gene expression.Since the elucidation of the genetic code in the early 1960s (1), the common perception has been that 1) all codons encode identical information in all organisms, and 2) coding sequences are "fixed" (i.e. ribosomes must maintain translational reading frame to faithfully decode all of the information contained in mRNAs). Shortly thereafter, however, bacteriophage geneticists identified bacterial mutants capable of suppressing stop codons in viral mRNAs (2), which led to the discovery of tRNAs (called suppressor tRNAs) in both bacteria (3) and eukaryotes (4) that are capable of reassigning the meaning of "stop codons" to encode specific amino acids. In the next decade, there were additional discoveries of cis-acting mRNA elements that direct ribosomes to reassign the meanings of codons or induce ribosomes to shift into alternative reading frames. This general phenomenon is collectively referred to as "translational recoding," which is defined as instances in which "the rules for decoding are temporarily altered through the action of specific signals built into the mRNA sequences" (5).In a unique and thought-provoking research article, Anzalone et al. (6) exploited RNA recoding elements to create a gene expression control circuit. They employed a directed evolutionary approach in vitro to identify RNA aptamers that stim-ulated the activity of a translational recoding element. These aptamers were then exploited to construct single-mRNA logic gates that were applied to control a new module governing cell death. This work marked a seminal milestone in the effort to integrate translational reprogramming modules into the synthetic biology toolbox. Importantly, these synthetic elements enabled manipulation of gene expression during the elongation phase of translation, thus addressing a critical gap in the synthetic biology toolbox for regulating gene expression in synthetic biology.

Origin, Evolution and Stability of Overlapping Genes in Viruses: A Systematic Review

“…Indeed, non-canonical translational strategies such as leaky scanning, ribosomal frameshifting and alternative initiation are essential for expression of overlapping genes [41][42][43][44][45]156]. Therefore, detection of overlapping genes in eukaryotes may further improve our knowledge of gene expression by translational recoding [157].…”

Exploring the synthetic biology potential of bacteriophages for engineering non-model bacteria

“…These viral frameshifting events could inspire synthetic programmed frameshift to generate signal splitters in genetic circuitry: e.g. the expression of one coding sequence results in two enzymes with different functioning 102 . The translational bypassing phenomenon has so far only been observed in coliphage T4, where the mRNA of gp60 contains a stretch of 50 non-coding nucleotides, but is still translated into a single polypeptide.…”

Translational recoding: canonical translation mechanisms reinterpreted

“…Manipulation of the frameshifting efficiency opens new perspectives in developing antiviral therapies and controlling gene expression of cellular mRNAs (120). An intriguing example is the interferon-stimulated cellular protein Shiftless.…”