Synthetic Biology Parts, Devices and Applications

10.1 An Introduction to Transcript Control 193

transcripts ribosome-less at known RNase E cleavage sites showed decreased
transcript stability [25, 41].
The complex interplay of other transcript-related mechanisms that affect tran-
script degradation is even less well understood. Ribosomal pausing can lead to
cleavage by unknown ribonuclease activity. The subcellular localization of indi-
vidual transcripts also affects ribosomal occupancy, which in turn affects RNA
degradation [37].

10.1.4 Structural and Noncoding RNA-Mediated Transcript Control

In most cases, RNase E must bind the transcript – either at the 5′ end or at a
single- stranded interior region – to initiate cleavage and begin degradation. This
implies that anything affecting RNase E binding will also affect transcript stabil-
ity. Ribosomes, as explained before, are one such factor. RNA secondary struc-
tures, or other stable base pairings that prevent access to the 5′ end or internal
RNase E sites, are therefore expected to reduce RNase E binding and increase
transcript stability (Figures 10.2b and 10.3a).
Antisense oligos that base-pair near the 5′ end of a transcript were shown to
lower the rate of RNase E cleavage [19], and studies of naturally long-lived mRNA
in E. coli have pointed to 5′ hairpins as a means of precluding RNase E docking
and conferring transcript stability [31] [32]. A naturally occurring riboswitch, or

Ribosome-dense

transcript

Ribosome

Slow direct

entry

Slow 5′ entry possible

5 ′

5 ′

5 ′

5 ′

5 ′ 5 ′

5 ′

degradation

Monomers

(a) (b) (c)

Monomers

–3′

–3′

–3′

3 ′

5 ′

degradation

3 ′

–3′ AAA–3′

5 ′

+ +

+ +

–3′

+

+

+

+

3 ′

E

E

Hfq

E

E

E

R

R

R

PNP PNP

PNP

PNP

R

R

Exposed transcript (low ribosome

density) degrades more quickly

Slow 5′ entry

possible Fast direct

entry

Hfq-mediated sRNA or

asRNA binding

RNase E

cleavage

PNPase poly(A)

binding

3 ′ addition of

poly(A) foothold

by PAP I

Figure 10.2 Naturally occurring transcript stability control mechanisms. (a) mRNAs that are
highly occupied by translating ribosomes RNA will have occluded sites for RNase E 5′ entry
and direct entry, leading to relatively long transcript half-life and high levels of gene
expression. Exposed transcripts (i.e., with lower ribosome density), such as mRNAs transcribed
with bacteriophage polymerases with fast elongation rates, are more susceptible to RNase E
attack due to a lack of occluding ribosomes. (b) sRNA and asRNA operate through Hfq-
mediated binding to the RBS (green box) and/or start codon region of a target mRNA, which
prevents ribosome docking and likely recruits RNase E to the transcript. (c) The addition of
poly(A) tails to a transcript, usually by poly(A) polymerase (PAP I), creates a foothold for
binding by polynucleotide phosphorylase (PNPase), a 3′ → 5 ′ exoribonuclease.