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
Environmental change DNA RNA Turn gene(s) on/off protein Proteins to deal with new environment Transcriptional Control • Very important to: • express genes when needed • repress genes when not needed • 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 ββ’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 s70 s54 sS sS sF s32 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 • Repressor binding prevents RNAP binding promoter • An activating transcription factor found to be • required for full lac operon expression: CAP (or Crp)
glucose cAMP Crp lac operon no mRNA Cofactor binding alters conformation • Crp binds cAMP, induces allosteric changes glucose cAMP Crp 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
cAMP signals low glucose activator binding-site
lac operon off low
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
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.
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
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?