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32 Gene regulation, continued Lecture Outline 11/21/05• Review the operon concept

– Repressible operons (e.g. trp)– Inducible operons (e.g. lac)

• Positive regulation of lac (CAP)• Practice applying the operon concept to

predict:– the phenotypes of mutants– The characteristics of other operons

• Gene regulation in prokaryotes vs eukaryotes

Genes of operon

Protein

Operator

Polypeptides that make upenzymes for tryptophan synthesis

Regulatorygene

RNA polymerase

Promoter

trp operon

5′

3′mRNA

trpDtrpE trpC trpB trpAtrpRDNA

mRNA

E D C B A

The trp operon:

Figure 18.21a

5′

Tryptophan absent -> repressor inactive -> transcription

One long mRNA codes severalpolypeptides, each with its own startand stop codonThe “operator” is a

particular sequence ofbases where therepressor can bind

DNA

mRNA

ProteinTryptophan

(corepressor)

Active repressor

No RNA made

Tryptophan present -> repressor active -> operon “off”.Figure 18.21b

Active repressor canbind to operator andblock transcription

Trp operon

2

Lac operonInducible operons are normally off

When lactose is present,repressor can no longerbind DNA. Transcriptionoccurs

Positive vs Negative GeneRegulation

• Both the trp and lac operons involve negativecontrol of genes– because the operons are switched off by the

active form of the repressor protein

• Some operons are also subject to positivecontrol– An activator protein is required to start

transcription.– E.g. catabolite activator protein (CAP)

Promoter

Operator

InactiveCAP

ActiveCAPcAMP

DNA

Inactive lacrepressor

lacl lacZ

Figure 18.23a

– In E. coli, glucose is always the preferred foodsource

– When glucose is scarce, the lac operon isactivated by the binding of CAP

Positive Gene Regulation- CAP

Active form ofCAP helps RNApolymerase bindto promoter, sotranscription canstart

ATP

GTP

cAMP

Proteinkinase A

Cellular responses

G-protein-linkedreceptor

AdenylylcyclaseG protein

First messenger(signal moleculesuch as epinephrine)

You’ve seencAMP used inother signalingpathways

•Enzyme adenylyladenylyl cyclasecyclase

3

• When glucose is abundant,– cAMP is used up– CAP detaches from the lac operon,– prevents RNA polymerase from binding to

the promoter

Inactive lacrepressor

InactiveCAP

DNA

RNApolymerasecan’t bind

Operator

lacl lacZ

Promoter

Figure 18.23b If it is busy phosphorylating glucose, it cannotactivate adenylate cyclase, so level of cAMP falls

Glucose transporter complex also activates adenylatecyclase

How do genetic switcheswork?

DNA binding proteins can be either repressorsor activators, depending on how they intereact

with RNA polymerase

This configuration helpsRNA polymerase bind

This configuration blocksRNA polymerase

Activator

Repressor

4

Dual control of the lac operon

off, because CAP not bound

off, because repressor activeand CAP not bound

off, because repressor active

Operon active

+ glucose+ lactose

+ glucose- lactose

- glucose- lactose

- glucose+ lactose

Glucose must be absent Lactose must be present

X-ray structure of CAP-cAMP bound to DNA

Many Operons use CAPlac, gal, mal, ara, etc.

CAP binds to RNA polymerase

mRNA 5'

DNA

mRNA

Protein

Allolactose(inducer)

Inactiverepressor

lacl lacz lacY lacA

RNApolymerase

Permease Transacetylaseβ-Galactosidase

5′

3′mRNA 5′

The Lac operon

Figure 18.22b What will happen if there is a deletion of the:+ lactose? - lactose?

• operator?• lac repressor gene?• CAP binding site?

5

Arabinoseis another sugar that E. coli can metabolize

• Will those genes be repressible orinducible?

• How might it be regulated?

Arabinose can bind to the repressor

Arginine is an essential amino acid.

• Will that pathway be repressible orinducible?

• How might argenine synthesis beregulated?

Galactose is yet another sugarthat E. coli can metabolize.

• Will those genes be repressible orinducible?

• How might gal be regulated?

O galEO galT galK

Gal repressor protein(galR)

Epimerase Transferase Kinase

P

Don’t memorizethese names- justthe general concept.

CAP

Galactose

Gene Regulation inProkaryotes and Eukarykotes

• Prokaryotes– Operons

• 27% of E. coli genes• (Housekeeping genes

not in operons)

– simultaneoustranscription andtranslation

• Eukaryotes– No operons, but they still

need to coordinateregulation

– More kinds of controlelements

– RNA processing– Chromatin remodeling

• Histones must be modifiedto loosen DNA

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Figure 19.3

Signal

NUCLEUSChromatin

Chromatin modification:

Gene

DNA Gene availablefor transcription

RNA ExonTranscription

Primary transcriptRNA processing

Transport to cytoplasm

Intron

Cap mRNA in nucleusTail

CYTOPLASMmRNA in cytoplasm

Degradationof mRNA

Translation

PolypetideCleavage

Chemical modificationTransport to cellular

destination

Active protein

Degradation of protein

Degraded protein

Nucleosome

30 nm

(b) 30-nm fiber

DNA Packing

Protein scaffold

300 nm

(c) Looped domains (300-nm fiber)

Loops

Scaffold

700 nm

1,400 nm

(d) Metaphase chromosome

Figure 19.2

Histone Modification

Figure 19.4a

Chromatin changes

Transcription

RNA processing

mRNA degradation

Translation

Protein processingand degradation

DNAdouble helix Amino acids

availablefor chemicalmodification

Histonetails

Histone acetylation loosensDNA to allow transcription

Figure 19.4 b

Unacetylated histones Acetylated histones