regulation operons

8/13/2019 Regulation Operons

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Molecular Biology

Part III

Regulation of Gene ExpressionB. Operons, Part 2


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The lac operon model: from

structure to function

The lacmodel: from structure to function

Howdoes lacwork?

How do we know?

What does laclook like in 3-D?

Another operon: ara(a BAD operon!)


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Weaver 2005 Molecular Biology 3rd ed. Fig 7.16

Map of the lacoperon: defined by genetic mapping (e.g. interrupted mating and

deletion analyses) and by footprinting. (n.b.: first published versions werewrong!)


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4Weaver 2005 Molecular Biology 3rd ed. Fig 7.3


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5Weaver 2005 Molecular Biology 3rd ed. Fig 7.5-a/b


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Weaver 2005

Molecular Biology

3rded. Fig 7.6

nitrocellulose filterbinding assay:

-- filter binds protein

-- protein binds to

radiolabled DNA


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8Weaver 2005 Molecular Biology 3rd ed. Fig 7.7

More filter-binding assay of lacrepressor function: binding to mutant operator

wild-type operator

mutant operator

Conclusion:

operator is DNA thatworks by binding to

repressor protein


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crystal of lac

repressor

protein

adding IPTG

shatters crystal:

indicates strong

binding to repressor


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10Weaver 2005 Molecular BiologyFig. 7-17b

CAP-cAMP complex binding to laccontrol region (as a dimer)

A second control on lacoperon: POSITIVE control


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Revising the lacmodel: from 1-D to

3-D

X-ray diffraction studies


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Lewis et al. 1996

Science


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15Lewis et al. 1996 Science

inducer

binding

site

inducer

binding

site


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Lewis et al. 1996

Science


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Lewis et al. 1996

Science

IPTG ininducer binding

site


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Lewis et al. 1996

Science

conformation of

helix-turn-helix binding

portion changes

with inducer


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Lewis et al. 1996

Science


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araoperon

positive control (by 2 activator proteins:

C and CAP

inducible (by arabinose)

also is REPRESSED by C!


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Note that protein binding to

these supercoiled DNA

minicircles makes them

smaller, so electrophoretic

mobility is increased


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27Weaver 2005 Molecular Biology3rded. Fig. 7-22

Araoperon competitive binding assay results Lobel & Schleif


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trp operon

repressible by end product

regulation by repressor at promoterAND

Attenuation: regulation by termination


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Weaver 2005 Molecular

Biology3rded. Fig 7-27

Firstmechanism for

regulating the trp

operonsimilar to

regulation of the lac

operon: repressor binds

to a promoter, inhibitstranscription, etc.


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31Watson et al. 1987 Molecular Biology of the Gene 4th ed. Fig. 16-22

A secondmethod for regulating the trpoperon: action by the leader sequence


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Watson et al. 1987Molecular Biology

of the Gene 4th ed.

Fig. 16-23


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Watson et al. 1987 Molecular Biology of the Gene 4th ed.


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Animation of trp function!!

http://faculty.plattsburgh.edu/donald.slish/

Attenuation.html
http://faculty.plattsburgh.edu/donald.slish/Attenuation.htmlhttp://faculty.plattsburgh.edu/donald.slish/Attenuation.htmlhttp://faculty.plattsburgh.edu/donald.slish/Attenuation.htmlhttp://faculty.plattsburgh.edu/donald.slish/Attenuation.html


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Weaver 2005 MolecularBiology3rd ed. Fig 7-33

Baci l lus subt i l isuses a different method of attenuation for the trpoperon


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Valbuzzi & Yanofsky 2001 Science

mobility shift when

TRAP is added

Anti-TRAP (AT)

prevents TRAP

binding to RNA,

so no mobility

shift


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37Valbuzzi & Yanofsky 2001 Science

PAGE gel shows

mobility of TRAPalone and with

Trp or Anti-TRAP

glutaraldehyde

cross-links proteins

together, so theymove together

on gel


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trp: a model riboswitch

riboswitch refers to the broad class of

mechanisms using RNA to regulate gene

expression, typically by modulating the

level of translation

riboswitches have very different

mechanisms of actionthe term is much

more recent than our understanding of thetrpoperon, for example


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Autogenous regulation

another kind of regulation at the level of

translation


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40Watson et al. 1987 Molecular Biology of the Gene 4th

ed. Fig. 16-28

Autogenous regulation: another example of a riboswitch


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Final general rule:

In prokaryotes, mRNA is made and used

only when needed: turnover can be very

rapid, as the cell acclimates to the slings

and arrows of outrageous fortune.

regulation operons

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