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

protein

Environmental change

Turn gene(s) on/off

Proteins to deal with new environment

Very important to:1. express genes when needed2. repress genes when not needed3. 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 Transcription factors Promoter DNA

RNAP binding sites Operator – repressor binding Other TF binding sites

Start site of txn is +1

α α β β’σ

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

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

Sigma subunit Type of gene controlled # of genes controlled

RpoD Growth/housekeeping ~1000

RpoN N2; stress response ~15

RpoS Stationary phase, virulence ~100

RpoH Heat shock ~40

RpoF Flagella-chemotaxis ~40

RpoE ? ~5

FecI Ferric citrate transport ~5

Extreme heat shock, unfolded proteins

s70

s54

sS

sS

sF

s32

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

changes glucose

cAMP

Crp

lac operon

no mRNA

cAMP

Crp

glucose

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 receptorprotein

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

•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

araPBAD

Control of the ara Operon II - Positive

•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.

araPBAD

Control can also happen at the Ribosome binding site

What about the terminator?

• Termination sequence has 2 features:Series of U residuesGC-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?