Biology Reference
In-Depth Information
systems, gene expression is up-regulated by the synthetic PROTEON and PROTEOFF
transactivators, rather than controlled by derepression upon induction. Also, unlike the
TETON protein of the original TetOn system, multiple domains of the PROTEON and
PROTEOFF proteins must bind to their complementary operator sequences of the synthetic
promoter prior to recruiting RNA polymerase (RNApol) and activating transcription. With
these added degrees of complexity, molecular dynamic (MD) simulations were employed to
guide the experimental construction of both systems. First, the structures of the synthetic
promoter and the synthetic proteins were optimized and their geometrical constraints were
satisfied in the simulations. Next, the two systems were built experimentally and tested.
Operator sites from the tetracycline operon and the lux operon were integrated along with
10 RNApol binding sites, a transcriptional start site, an mRNA enhancer
sequence, and a strong RBS to construct the synthetic promoter as shown in
Figure 7.3
.In
parallel, the PROTEON and PROTEOFF proteins were built by linking the inducible DNA-
binding protein rTetR or TetR to the transactivator domain of LuxR with a flexible peptide
linker. The association between the synthetic promoter and PROTEON is also illustrated in
Figure 7.3
.
35 and
aTc-dependent gene expression control is achieved with both the proTeOn and proTeOff
systems as depicted in
Figure 7.4
. For proTeOn, in the absence of aTc, the inducible DNA
binding domain (rTetR) does not bind its operator (
tetO
)and the PROTEON protein does
not up-regulate the target gene. Upon activation with aTc, rTetR binds the inducer,
undergoes a conformational change, and binds
tetO
. This binding brings the LuxR
transactivator domain near its operator site allowing it to bind the
luxbox
and up-regulate
gene transcription through RNApol recruitment to the promoter.
In contrast, for proTeOff, in the absence of aTc, its inducible DNA binding domain (TetR)
binds
tetO.
This binding brings its LuxR transactivator domain close to its operator site,
permitting it to bind
luxbox
and up-regulate transcription through RNApol recruitment. After
127
(A)
5' - actctatcattg
actctatcattgatagagt
tetO
actta
acataagc
UP element
acctgtaggatcgtacaggt
luxbox
ttagcgaagaaaatggtt
tgttatagt
-10
cgaa
t
txn
aaa
cctcgagttatctcgagtgagatattgttgacg
mRNA stability sequence
cacc
aaggaggaa
RBS
acagct
atg
tln
agtaaa - 3'
start
start
(B)
Linker
Linker
rTetR/
TetR
rTetR/
TetR
LuxR
Δ
N
tetO
luxbox
luxbox
FIGURE 7.3
proTeOn and proTeOff systems
'
design. (A) Synthetic promoter sequence. Both PROTEON and PROTEOFF bind and recruit
RNApol to this synthetic promoter sequence. Moving from 5
0
to 3
0
: the rTetR/TetR protein domain binds tetO, RNApol binds
the UP element and the 10 region, LuxR binds the luxbox, the mRNA stability sequence stabilizes the mRNA transcript,
and ribosomes bind the RBS of this resulting mRNA message. (B) proTeOn and proTeOff molecular model. Both proTeOn and
proTeOff are designed to assemble as shown. The inducible DNA binding domain (rTetR or TetR in blue) binds the tetO
operator (purple), and the transcription activator domain (LuxR in orange) binds the luxbox (red). The two domains bind their
operators along the same face of the DNA double helix and are connected (TetR/rTetR
'
s C-terminus to LuxR
'
s N-terminus) by
a linker peptide (green).
