Another engineering principle: Characterization. A stupid engineering joke:. A physicist, a mathematician and an engineer were each asked to establish the volume of a red rubber ball.
Another engineering principle:Characterization.
Genome Network Project, Nature Genetics, 2009
Turn gene(s) on/off
Proteins to deal with
RNA Structures Vary
structured domains connected by more flexible domains, leading to different functions
Start site of txn is +1
α α ββ’σ
RNAP binds specific promoter sequences
-10 and -35 sequences
RNA polymerase promoters
Deviation from consensus -10 , -35 sequence leads to
weaker gene expression
Bacterial sigma factors
Extreme heat shock, unfolded proteins
E. coli can choose between 7 sigma factors and about 350
transcription factors to fine tune its transcriptional output
An Rev Micro Vol. 57: 441-466T. M. Gruber
Lac operon control
Cofactor binding alters conformation
Cooperative binding of Crp and RNAP
Binds more stably than either protein alone
Interaction of CAP-cAMP, RNA Pol and
DNA of lac control region
lac operon – activator and repressor
CAP = catabolite
CRP = cAMP receptor
Cis-acting sequence is activator (or CAP) binding site.
cAMP signals low glucose
lac operon off
lac operon very weakly on
lac operon fully induced
The ara Operon
•another example of operon that has both positive
and negative regulation
•araB, A, and D encode the 3 arabinose
•araC encodes the control protein AraC which is both
a positive regulator (in the presence of arabinose) and
a negative regulator (in the absence of arabinose).
•cAMP-CAP complex also acts as a positive regulator
Organization of the ara operon
Control of the ara Operon I - Negative
•When arabinose is absent, the AraC protein acts as
a negative regulator.
•AraC acts as a dimer, and causes the DNA to loop.
Looping brings the I1 and O2 sites in proximity to one
•One AraC monomer binds to I1 and a second monomer
binds to O2.
•Binding of AraC prevents RNA Pol from binding to
the PBAD promoter
Control of the ara Operon II - Positive
•When arabinose is present, it binds to AraC and changes
•An arabinose-AraC dimer complex binds preferentially
to I1 and I2, and NOT to O2 which causes ‘opening’
of the loop. This allows RNA Pol to bind to PBAD.
•If glucose levels are low, cAMP-CAP complex binds
•Active transcription occurs.
What about the terminator?
Why would synthetic biologists want to be able to tune a system/device?