Lecture 2 - MBLG2x71 - 09 March 2012

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University of Sydney LibraryElectronic Item

MBLG2x71

Author of lecture: Professor A.S. WeissTitle of lecture: L2. Transcriptional Regulation I

MBLG2x719 March 2012

Transcriptional Regulation I

Professor Tony Weiss

Models of Gene Expression: the lac operon. Uses of the lac operonin Molecular Biology,blue/white colour selection

Pages 553-554, 559-561 Watson et al. Molecular Biology of the Gene 6th Edn.

With thanks to: R. Benjamin, UNT USA


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Gene expression responds to

environmental conditions

-10 copies per cell, some enzymes forglycolysis present at 100,000 copies/cell

Genes whose products are presentlyunneeded or at acceptable levels are

turned off

Mechanisms to achieve proper mix ofgene expression are varied

Lactose metabolism in E. coli

Jacob and Monod (1946) studied as models stem man others followed

Enzyme -galactosidase only expressed

when lactose present in the medium Enzyme said to be expressed in

inducible fashion with lactose as inducer

Identified cis-acting elements (operator,promoter) and trans-acting factors


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LactoseHydrolysis

Enzyme encoded bylacZgene

Glucose andgalactose products

Enz me cleaves

broad range of -galactosides

Including syntheticanalogs such as X-gal

Bacterial Genes and Operons

expressed as a unit on a single mRNA

Operon Polycistronic mRNA


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E. coli lacoperon

separately from the lacoperon

Encodes lacrepressor

Low level of expression

lacoperonexpression

lacoperon

encodes

polycistronicmRNA giving

rise to 3differentenzymes


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Structural Genes of lac

Operon

Structural genes encode the primarystructure of the enzymes/proteins

For lacoperon these are lacZ, lacYand lacA

Enzymes encoded are -galactosidase, lactosepermease and transacetylase, respectively


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lacOperon Expression


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In absence of inducer, repressor binds to operatorand blocks RNAP from binding to promoter


Binding of inducer to repressor causes conformational changein protein, preventing interaction with operator

RNA polymerase binds to promoter and expresses operon


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Gratuitous Inducers

allolactose for the pure at heart) But other -galactosides also work

Isopropylthiogalactoside (IPTG) alsoacts as an inducer but is not

Shows induction does not involveinteraction with the actual enzyme beingsynthesized


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Isopropylthiogalactoside (IPTG)

inducer

Not metabolised

Level remainsconstant


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The lacpromoter is quite weak

if lactose if available (and glucose isnt available) cellscould get very hungry!

Solution: when cells are hungry:

make the lacpromoter work more effectively

also called CRP (cAMP receptor protein)


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(CAP and CRP are the same)

Not enough glucose

CAP binds cAMP then binds the promoter


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With glucose

CAP needs cAMP to enable it to bind promoter

(conformation is incorrect for binding)


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Xgal is incrediblyuseful!

and theres a movie

www.youtube.com/watch?v=oBwtxdI1zvk

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Lecture 2 - MBLG2x71 - 09 March 2012