Synthetic Biology Parts, Devices and Applications

206 10 Programming Gene Expression by Engineering Transcript Stability Control and Processing in Bacteria

kinetic folding algorithms that should be broadly useful for analyzing and engi-

neering TSC mechanisms [89].

10.4.2 Unused Mechanisms Found in Nature

Despite more than a decade of progress, there are naturally occurring mecha-

nisms for controlling transcript stability that have yet to be exploited for engi-

neering gene expression. In the following, we enumerate several promising

mechanisms that could become part of a toolkit to predictably control transcript

stability.

The lysC riboswitch, recently described by Caron et al. (Figure 10.5b) [28],

contains two RNase E cleavage sites that are exposed, while the RBS is simultane-

ously sequestered, in the presence of the ligand lysine. In a synthetic context,

rational introduction of these sites into existing riboswitch designs could enhance

their function by decreasing transcript half-life in the presence of ligand.

An sRNA titration system has been observed by two different groups in

Salmonella (Figure 10.5a) [126, 127]. This system functions by relieving sRNA-

based repression by expressing a decoy mRNA to shunt away sRNA targeting an

mRNA, leading to rapid degradation of the sRNA. Such a system could allow for

quick removal of engineered sRNA-based repression that rapidly activates gene

expression.

Decoy RNA attenuates sRNA-

mediated mRNA degradation

sRNA binds

Decoy RNAs

Hfq

5 ′

5 ′

5 ′

5 ′

R R

R

R R R

R

–3′

–3′

–3′

–3′

–3′

–3′

5 ′

5 ′

No sRNA-mediated degradation;

translation from cognate mRNA

possible

IysC riboswitch control of RBS and

nuclease target site accessibility

E

E

E

E

Lysine binding

sequesters RBS and

reveals two RNase E

binding sites

Slow direct

entry

possible

RNase E binds revealed

UCUUCC ssRNA sites

(a) (b)

Direct entry

possible

+

+

+

+

Figure 10.5 Examples of unutilized transcript stability control mechanisms. (a) In naturally

occurring biological systems, decoy RNAs attenuate sRNA-mediated mRNA degradation by

shunting sRNAs away from cognate transcripts, increasing message stability and the level of

genetic expression. (b) The lysC transcript of E. coli contains a riboswitch that controls access

to the RBS and RNase E target sites, showing the functional integration of multiple TSC

mechanisms.