10.5 Conclusions and Discussion 207RNA–protein interactions occur throughout the lifetime of a transcript and
play critical roles in the degradation process. As results with the bicistronic 5′
UTR design [101] highlighted in Section 10.3.3 suggest, some RNA–protein
interactions can effectively inhibit other RNA–protein interactions. Although
this cross-inhibition of RNA–protein interactions sometimes confounds system
behavior, this principle could be exploited as a TSC mechanism. For example,
sequence motifs that recruit protective RNA-binding proteins to the 5′ and 3′
UTRs could insulate transcripts from ribonuclease binding. Pentatricopeptide
repeat (PPR) proteins, a family of single-stranded RNA-binding proteins found
in plants [128], would be a good candidate for this application. PPR proteins are
similar to the DNA-binding transcription activator-like effector (TALE) proteins
[129] in that each protein has an RNA-binding domain, the target specificity of
which is governed by a series of two-amino-acid repeats, where each repeat cor-
responds to a target nucleotide. The sequence motif code with which a class of
PPR proteins binds RNA has recently been uncovered [130]. PPR proteins have
been implicated in controlling transcript stability in maize chloroplasts by bind-
ing to the 5′ and 3′ ends of mRNA [131], and a PPR protein was shown to limit
5 ′ → 3 ′ and 3′ → 5 ′ degradation in vitro when its binding site was introduced into
an mRNA [132].
Similarly to the PPR proteins, RNA has been found to bind the 3′ UTR of a
transcript and enhance its stability by offering protection against exoribonucle-
ase activity. GadY is an asRNA with complementarity to the 3′ UTR of the gadX
gene in E. coli [56] and is probably one of many such asRNA.
Though the biochemical details involving polyadenylation are not yet fully
understood in bacteria, its ubiquity [133] and use in contexts such as in the glmS
gene [63] for increasing degradation highlight potential utility for engineering
TSC. As E. coli has only 3′ → 5 ′ exoribonucleases, degradation from the 3′ end is
an essential part of rendering a transcript nonfunctional. Adding poly(A) tails of
varying lengths – perhaps in an inducible manner similar to a riboswitch or
riboregulator – to transcripts could function to reliably control and enhance
3 ′ end degradation by PNPase.
10.5 Conclusions and Discussion
Knowledge of RNA degradation in bacteria has progressed substantially since
the advent of synthetic biology. Key components and processes that account for
bulk mRNA turnover, translation effects, sRNA action, and polyadenylation
have become well understood. With this know-how and the continuing efforts of
the RNA-based engineering community, TSC is positioned to become an even
more powerful method for programming functions in synthetic biological
systems. A forward-engineering approach that harnesses understanding of
biochemical mechanisms to build predictive models for generating desired out-
puts [7] is now possible, with a number of mechanisms to up- and downregulate
transcript half-life. Building on existing RNA device engineering efforts, inspira-
tion from natural mechanisms can point to new ways of regulating stability; and
as RNA device engineering matures, more complex and wholly synthetic devices