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.
• The physicist immersed the ball in a beaker
full of water and measured the volume of the
displaced fluid. The mathematician measured
the diameter and calculated a triple integral.
The engineer looked it up in his Red Rubber
Ball Volume Table.
• Basically, engineers want to know the
characteristics of their parts and devices.
• They list these characteristics in tables, books
and files.
• Want a beam that can hold 1000 lbs? Look it
up.
But first
• More on bacterial transcription and promoters
and such
Genome Network Project, Nature Genetics, 2009
Transcriptional Control
DNA
RNA
Environmental change
Turn gene(s) on/off
protein
Proteins to deal with
new environment
Very important to:
1. express genes when needed
2. repress genes when not needed
3. Conserve energy resources; avoid expressing unnecessary/detrimental genes
RNA Structures Vary


RNA more like proteins than DNA:
structured domains connected by more flexible
domains, leading to different functions
e.g. ribozymes – catalytic RNA
Initiation
RNA polymerase α α β β’σ
 Transcription factors
 Promoter DNA
 RNAP binding sites
 Operator – repressor binding
 Other TF binding sites
Start site of txn is +1

Initiation

RNA polymerase
 4 core subunits
 Sigma factor (σ)–
determines promoter
specificity
 Core + σ = holoenzyme
 Binds promoter sequence
 Catalyzes “open complex” and
transcription of DNA to RNA
RNAP binds specific promoter
sequences
Sigma factors recognize consensus
-10 and -35 sequences

RNA polymerase promoters
TTGACA
TATAAT
Deviation from consensus -10 , -35 sequence leads to
weaker gene expression
Bacterial sigma factors






Sigma factors are “transcription factors”
Different sigma factors bind RNAP and recognize
specific -10 ,-35 sequences
Helps melt DNA to expose transcriptional start site
Most bacteria have major and alternate sigma factors
Promote broad changes in gene expression
 E. coli 7 sigma factors
 B. subtilis 18 sigma factors
Generally, bacteria that live in more varied
environments have more sigma factors
Sigma factors
Sigma subunit
Type of gene controlled
s70 RpoD
Growth/housekeeping
s54 RpoN
N2; stress response
~15
sS
RpoS
Stationary phase, virulence
~100
sS
RpoH
Heat shock
~40
sF
RpoF
Flagella-chemotaxis
~40
Extreme ?heat shock, unfolded proteins
~5
Ferric citrate transport
~5
s32 RpoE
FecI
# of genes controlled
~1000
E. coli can choose between 7 sigma factors and about 350
transcription factors to fine tune its transcriptional output
An Rev Micro Vol. 57: 441-466 T. M. Gruber
Lac operon control
• Repressor binding prevents RNAP binding promoter
• An activating transcription factor found to be
required for full lac operon expression: CAP (or Crp)
Cofactor binding alters conformation

Crp binds cAMP, induces allosteric
glucose
changes glucose
cAMP
Crp
cAMP
Crp
lac operon
no mRNA
mRNA
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
activator protein
CRP = cAMP receptor
protein
Cis-acting sequence is activator (or CAP)
binding site.
cAMP signals low glucose
activator binding-site
lac operon off
low
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
metabolizing enzymes
•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
araPBAD
•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
another.
•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
araPBAD
•When arabinose is present, it binds to AraC and changes
AraC conformation
•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
to Pc.
•Active transcription occurs.
Control can also happen at the
Ribosome binding site
What about the terminator?
• Termination sequence has 2 features:
Series of U residues
GC-rich self-complimenting region
• GC-rich sequences bind forming stem-loop
• Stem-loop causes RNAP to pause
• U residues unstable, permit release of RNA chain
One type of characterization is
Tuning
• Some promoters bind RNAPs better so they
are stronger
• Some RBSs make mRNA that bind better to
the ribosome so they are stronger
• And some are weaker…
Tuning
• By mixing and matching promoters and RBS
parts we can have genetic devices that work at
various levels
•
•
•
•
Weak Promoter + weak RBS = weak device
Strong Promoter + strong RBS = strong device
Weak Promoter + strong RBS =
Medium Promoter + medium RBS =
• The synthetic biologists got together and
decided on a reference promoter against
which others would be measured.
• Much like the standard meter.
Why would synthetic biologists want
to be able to tune a system/device?