Paper Summary

BioE 10
Geunwon Jung

Regulation of transcription by unnatural amino acids

Chang C Liu, Lei Qi, Charles Yanofsky, and Adam P Arkin

Regulation of transcription of gene expression is essential to controlling and expressing biological
systems. However, as of today, there are only a few regulatory methods to control gene expressions. Chang C
Liu, Lei Qi, Charles Yanofsky and Adam P Arkin developed an original method to attenuate or activate
transcription in the cis-regulatory leader-peptide elements with genetically encoded unnatural amino acids
(UAA), which is not produced in the body.
For the study, two such elements were chosen: trp-operons and tna-operon regulatory regions. Trp-
operons wild-type function is to turn off the transcription of genes downstream of it in the presence of
tryptophan. On the other hand, for tna-operon, the exact opposite happens when sufficient tryptophan is present.
Therefore, these two systems serve as complements of each other.
To engineer a UAA-induced transcriptional OFF switch, the researchers first replaced the tryptophan
codon with a nonsense codon, UAG. DNA corresponding to this region encodes for super-folder green
fluorescent protein (GFP). After it is inserted in the vector to transform plasmid pCCL-006 (or with other
plasmid), E. Coli cells were then grown in rich media. (The mechanisms described here all assume the presence
of excess tryptophan.) In order to confirm the system is stable across individual cells, the researchers added a
few experiments. Such experiments included measuring fluorescence without background subtraction in various
AcF concentrations.
As a result, cells containing only pCCL-006 gene revealed high fluorescence regardless of the presence
of AcF, which means that RNA transcription continues, just as in the previous studies. However, when
researchers co-transformed the plasmid with both pCCL-006 and pEVOL-Tyr that actually encodes for the
UAG codon, cellular fluorescence conspicuously decreased, meaning that the rate of transcription dropped.
Finally, when plasmid was co-transformed with pCCL-006 and pEVOL-pAcF, which encodes for
acetylphenylalanine (AcF), a type of UAA, sixfold decrease in fluorescence indicating transcriptional
termination was observed upon the presence of AcF. And according to the additional tests performed, the switch
is titratable and stable across individual cells, because the attenuation displayed a standard concentration
dependence on AcF and unimodal population response was shown.
To engineer the ON-switch system, a similar method was applied. But this time, the researchers
mutated tna-operon instead of trp-operon, and pCCL-015/-016 substituted the previous plasmids, mainly to
minimize the structural difference between the two operons. Similar sets of experiments were done, but with
modifications suitable to tna-operon.
When the ON-switch system was implemented and E. Coli Top10 cells were transformed with pCCL-
015 and grown, it revealed a high fluorescence, thus a high rate of transcription. Similarly, E. Coli cells with
pCCL-016 and pEVOL-Tyr also showed high fluorescence regardless of the presence of AcF. However, E. Coli
cells co-transformed with both pCCL-016 and pEVOL-pAcF, the leader peptide was properly transcripted only
when AcF was aroundwith a 25-fold increase. With the additional sets of tests, it was confirmed that this ON
switch system, like the OFF one, is also titratable and stable across cells.
Furthermore, to examine the hypothesis that induction of these OFF and ON switches can be extended
to any genetically encodable UAA, the researchers chose three additional UAAspEVOL-pAzF, pEVOL-BoF,
and pEVOL-IFand perform the same test. Consequently, UAA-induced transcriptional switches were
uniformly observed in all cases, and it was concluded that these ON and OFF switches are general to any UAAs.
Based on these researchers work, there are many tasks that can be studied, including the regulation of
higher-order UAA-induced gene regulation, independent but homogenous control of multiple genes. Moreover,
many expect the strategy to be applied to the wide range of hosts, including yeast and mammalian cells.