Synthetic Biology Parts, Devices and Applications

194 10 Programming Gene Expression by Engineering Transcript Stability Control and Processing in Bacteria

functional RNA that changes conformation upon ligand binding to dynamically

sequester or present an RBS [42], has been discovered that also uncovers RNase E

cleavage sites when bound to a target metabolite, lysine. As a result, lysine bind-

ing reduces the rate of translation initiation from the RBS and decreases

transcript half-life, further reducing protein expression [28]. More generally,

riboswitches are thought to decrease transcript stability in the RBS-sequestering

state by precluding ribosome binding [28] (also see Section 10.1.3).

Like the antisense oligos, small RNA (sRNA) can create base pairing in the 5′

UTR and thus limit binding by ribosomes or RNase E. sRNA, an abundant form

of regulatory noncoding RNA (ncRNA) in bacteria [43], is typically tens to hun-

dreds of bases long. They usually function to enhance or repress ribosome bind-

ing by base pairing, via a short (10–20-bp) seed region, with the target mRNA at

the Shine–Dalgarno (SD) and/or start codon regions of an mRNA [6, 44, 45]. In

many cases, sRNA target binding is mediated by the RNA chaperone activity of

the Hfq protein [46, 47]. Hfq deletion studies point to the importance of Hfq in

sRNA action [47–49] and stability, with increased sRNA degradation by PNPase

in stationary-phase Hfq-strains [50]. Hfq has been found to coprecipitate with

nearly half of the known sRNAs of Salmonella [51] and with a quarter of the then

known sRNAs in E. coli [52]. Interactions between some sRNA and mRNA can

occur in the absence of Hfq, however [48, 53].

Hairpin stabilized

Hairpin prevents 5′ entry

5 ′

5 ′

5 ′^5 ′

R

R R

5 ′-PPP

5 ′-HO

–3′

–3′ –3′

–3′

–3′

+

E

E

E

+

–3′

Direct entry

possible

(a)

(b)

(c)

Ribozyme-mediated 5′ cleavage

Ribozyme

cleavage

No 5′-PP

removal

by RppH

RppH Direct entry

possible

Riboregulator- / riboswitch-controlled RBS access

RBS

sequestered

Tr ans-activating

RNA binding

of riboregulator

Small molecule

ligand binding

of riboswitch

RBS released

Ligand-bound

aptamer

RBS released

Figure 10.3 Examples of engineered transcript stability control. (a) Synthetic secondary

structure hairpins within the 5′ UTR can increase half-life by preventing RNase E 5′ entry;

direct entry by RNase E is still possible. (b) 5′ ribozyme-mediated transcript cleavage creates a

5 ′-OH not recognized by RppH and therefore cannot become a 5′-P for RNase E to bind;

degradation of these processed transcripts occurs through RNase E direct entry.

(c) Riboregulators and riboswitches typically work by cis-RNA sequestration of the RBS, which

can be relieved by either trans-RNA binding to the cis-RN or ligand binding to the cis-RNA.

These binding events free the RBS from the cis-RNA and therefore allow translation.