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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.



But first
But first their parts and devices.

  • More on bacterial transcription and promoters and such



Environmental change their parts and devices.

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 their parts and devices.

  • RNA more like proteins than DNA:

    structured domains connected by more flexible domains, leading to different functions

  • e.g. ribozymes – catalytic RNA


Initiation their parts and devices.

  • 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 their parts and devices.

  • 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 their parts and devices.

  • Sigma factors recognize consensus

    -10 and -35 sequences


RNA polymerase promoters their parts and devices.

TTGACA

TATAAT

Deviation from consensus -10 , -35 sequence leads to

weaker gene expression


Bacterial sigma factors their parts and devices.

  • 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 their parts and devices.

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 their parts and devices. 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 their parts and devices.

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 their parts and devices.

Binds more stably than either protein alone


Interaction of CAP-cAMP, RNA Pol and their parts and devices.

DNA of lac control region


lac their parts and devices. operon – activator and repressor

CAP = catabolite

activator protein

CRP = cAMP receptor

protein



cAMP signals low glucose their parts and devices.

activator binding-site


lac their parts and devices. operon off

low


lac their parts and devices. operon very weakly on


lac their parts and devices. operon fully induced


The their parts and devices. 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 their parts and devices. ara operon


Control of the their parts and devices. 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 their parts and devices. 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? their parts and devices.

  • 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
One type of characterization is their parts and devices. Tuning



Tuning
Tuning their parts and devices.

  • 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 =




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