Molecular Biology

Part III Regulation of Gene Expression B. Operons, Part 2

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The lac operon model: from structure to function

The lac model: from structure to function
How does lac work? How do we know?

What does lac look like in 3-D? Another operon: ara (a BAD operon!)

Map of the lac operon: defined by genetic mapping (e.g. interrupted mating and deletion analyses) and by footprinting. (n.b.: first published versions were wrong!)

Weaver 2005 Molecular Biology 3rd ed. Fig 7.16

Weaver 2005 Molecular Biology 3rd ed. Fig 7.3

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

Weaver 2005 Molecular Biology 3rd ed. Fig 7.5-c/d

nitrocellulose filter binding assay: -- filter binds protein -- protein binds to radiolabled DNA

Weaver 2005 Molecular Biology 3rd ed. Fig 7.6

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More filter-binding assay of lac repressor function: binding to mutant operator

wild-type operator

mutant operator

Conclusion: operator is DNA that works by binding to repressor protein

Weaver 2005 Molecular Biology 3rd ed. Fig 7.7

crystal of lac repressor protein

adding IPTG shatters crystal: indicates strong binding to repressor

A second control on lac operon: POSITIVE control

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

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Weaver 2005 Molecular Biology Fig. 7-17b

Weaver 2005 Molecular Biology 3rd ed. Fig 7.13

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Weaver 2005 Molecular Biology 3rd ed. Fig 7.10

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Revising the lac model: from 1-D to 3-D

X-ray diffraction studies

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Lewis et al. 1996 Science

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inducer binding site

inducer binding site

Lewis et al. 1996 Science

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Lewis et al. 1996 Science

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IPTG in inducer binding site

Lewis et al. 1996 Science

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conformation of helix-turn-helix binding portion changes with inducer Lewis et al. 1996 Science

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Lewis et al. 1996 Science

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ara operon
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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Ara operon competitive binding assay results Lobel & Schleif

Weaver 2005 Molecular Biology 3rd ed. Fig. 7-22

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trp operon
repressible by end product regulation by repressor at promoter
AND

Attenuation: regulation by termination

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First mechanism for regulating the trp operon similar to regulation of the lac operon: repressor binds to a promoter, inhibits transcription, etc.

Weaver 2005 Molecular Biology 3rd ed. Fig 7-27

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A second method for regulating the trp operon: action by the leader sequence

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

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Watson et al. 1987 Molecular 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

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Bacillus subtilis uses a different method of attenuation for the trp operon Weaver 2005 MolecularBiology 3rd ed. Fig 7-33
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mobility shift when TRAP is added

Anti-TRAP (AT) prevents TRAP binding to RNA, so no mobility shift

Valbuzzi & Yanofsky 2001 Science

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PAGE gel shows mobility of TRAP alone and with Trp or Anti-TRAP glutaraldehyde cross-links proteins together, so they move together on gel

Valbuzzi & Yanofsky 2001 Science

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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 action the term is much more recent than our understanding of the trp operon, for example
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Autogenous regulation
another kind of regulation at the level of translation

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Autogenous regulation: another example of a riboswitch

Watson et al. 1987 Molecular Biology of the Gene 4th ed. Fig. 16-28

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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.

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