Biology Reference
In-Depth Information
they rely on intricate RNA
DNA interactions, respectively. However,
there are two promising platforms for designing mutually orthogonal sets of transcription
factors where the rules are clearer. The first is based on engineering zinc finger protein
transcription factors (ZFP-TFs). ZFP-TFs use a zinc finger protein domain to recognize
specific DNA sequences and an effector domain to recruit transcriptional initiation
components, thus activating transcription from a cognate DNA sequence. The advantage of
ZFP-TFs from an engineering perspective is that the ZFPs are composed of an array of finger
domains, each of which recognizes an overlapping 4-bp DNA sequence.
27
Therefore,
modular engineering schemes have been developed where libraries of fingers are designed
to recognize specific three or four base motifs; the fingers can then be fused together into
polydactyl ZFPs and artificial ZFP-TFs that recognize custom promoter sequences.
28,29
In
theory, this design scheme facilitates the construction of an arbitrarily large number of
mutually orthogonal ZFP-TF/promoter pair libraries that can be used for transcriptional
circuit engineering. In combination with standard promoter sequences these should lead
to orthogonal and homogeneously acting families of transcriptional regulators.
RNA and protein
A related and more recent approach is the use of transcription activator-like effector proteins
(TALEs) to engineer synthetic transcription factors. TALEs are DNA-binding proteins that
contain tandem repeats of 33
34 amino acid segments that are responsible for DNA
recognition.
30
Each repeat utilizes two amino acids at position 12 and 13 of the repeat
domain to recognize a single nucleotide in the corresponding DNA binding site. Since there
is a code-like consistency in which amino acid pairs recognize single DNA bases, custom
TALEs that recognize custom DNA sequences can be engineered. As the natural function of
TALEs is to activate transcription, they become an ideal platform for engineering custom
transcription factors. Indeed, recent efforts have demonstrated the feasibility of this
approach. For example, a study by Zhang et al. generated a collection of 13 custom TALEs,
rationally designed to activate transcription at 13 cognate sequences in mammalian cells.
31
Over 77% (10 out of 13) of these TALEs activated expression of their target gene
with
66
10-fold induction, suggesting that this platform can indeed be a reliable method
for generating multiple specific transcription factor/promoter pairs. Engineering of synthetic
regulatory circuits, especially in mammalian cells, where well-characterized parts for
transcriptional regulation are few, will benefit from the continued establishment of TALEs
as a platform for custom transcription factor design.
.
As methods for the construction of transcriptional
expand, the ability to sense
custom extracellular signals must expand alongside so that regulatory circuits can be
interfaced with external signals. Towards this end, transcriptional regulators that themselves
sense external signals such as small molecules have been engineered. For example, Liu et al.
recently reported the construction of transcriptional regulators that attenuate or activate the
expression of controlled genes in response to specific unnatural amino acid inputs.
32
Though the engineering of molecular recognition between a transcriptional regulator and a
small molecule is often a time-consuming task, in this study, the authors took advantage of
the fact that the expanded genetic code field, whose goal is to add new amino acids to the
genetic code, already provides nearly 100 engineered synthetases that recognize custom
unnatural amino acids and charge them onto engineered tRNAs that suppress blank
codons.
33
Therefore, by placing blank codons into leader peptide elements that control the
transcription of bacterial operons, the authors automatically gained a large number of
transcriptional regulators that are triggered by specific unnatural amino acids.
wires
'
'
Another instance of sensing in transcriptional regulators was reported recently by Qi et al.,
who showed that the rational fusion of aptamer domains with various RNA control systems
could add a sensing function to their control.
34
In one example, the authors fused a
theophylline-specific aptamer to the pT181 RNA-based transcriptional attenuation system
(described above), and showed that the resulting fusion would be active only in the
