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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.

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a stupid engineering joke
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.
slide3
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
But first
  • More on bacterial transcription and promoters and such
slide6
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
slide7
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
slide11
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

slide12
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
slide14
RNAP binds specific promoter sequences
  • Sigma factors recognize consensus

-10 and -35 sequences

slide15
RNA polymerase promoters

TTGACA

TATAAT

Deviation from consensus -10 , -35 sequence leads to

weaker gene expression

slide16
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
slide17
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

slide18
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)
slide19
glucose

cAMP

Crp

lac operon

no mRNA

Cofactor binding alters conformation

  • Crp binds cAMP, induces allosteric changes

glucose

cAMP

Crp

mRNA

slide20
Cooperative binding of Crp and RNAP

Binds more stably than either protein alone

slide21
Interaction of CAP-cAMP, RNA Pol and

DNA of lac control region

slide22
lac operon – activator and repressor

CAP = catabolite

activator protein

CRP = cAMP receptor

protein

slide25
cAMP signals low glucose

activator binding-site

slide29
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

slide31
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

slide32
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.

slide35
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
slide38
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
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 =
slide40
The synthetic biologists got together and decided on a reference promoter against which others would be measured.
  • Much like the standard meter.
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