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

3mRNA

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 several polypeptides, each with its own start and stop codon

The “operator” is a particular sequence of bases where the repressor can bind

DNA

mRNA

Protein

Tryptophan(corepressor)

Active repressor

No RNA made

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

Active repressor can bind to operator and block transcription

Trp operon

Lac operonInducible operons are normally off

When lactose is present, repressor can no longer bind DNA. Transcription occurs

Positive vs Negative Gene Regulation

• Both the trp and lac operons involve negative control of genes– because the operons are switched off by the active form of the

repressor protein

• Some operons are also subject to positive control– 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 food source

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

Positive Gene Regulation- CAP

Active form of CAP helps RNA polymerase bind to promoter, so transcription can start

ATP

GTP

cAMP

Proteinkinase A

Cellular responses

G-protein-linkedreceptor

Adenylylcyclase

G protein

First messenger(signal moleculesuch as epinephrine)

You’ve seen cAMP used in other signaling pathways

•Enzyme adenylyladenylyl cyclasecyclase

• 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 cannot activate adenylate cyclase, so level of cAMP falls

Glucose transporter complex also activates adenylate cyclase

How do genetic switches work?

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

with RNA polymerase

This configuration helps RNA polymerase bind

This configuration blocks RNA polymerase

Activator

Repressor

Dual control of the lac operon

off, because CAP not bound

off, because repressor active and 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 Transacetylase-Galactosidase

5

3mRNA 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?

Arabinose is another sugar that E. coli can metabolize

• Will those genes be repressible or inducible?

• How might it be regulated?

Arabinose can bind to the repressor

Arginine is an essential amino acid.

• Will that pathway be repressible or inducible?

• How might argenine synthesis be regulated?

Galactose is yet another sugar that E. coli can metabolize.

• Will those genes be repressible or inducible?

• How might gal be regulated?

O galEO galT galK

Gal repressor protein(galR)

Epimerase Transferase Kinase

P

Don’t memorize these names- just the general concept.

CAP

Galactose

Gene Regulation in Prokaryotes and Eukarykotes

• Prokaryotes– Operons

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

not in operons)

– simultaneous transcription and translation

• Eukaryotes– No operons, but they still

need to coordinate regulation

– More kinds of control elements

– RNA processing– Chromatin remodeling

• Histones must be modified to loosen DNA

Figure 19.3

Signal

NUCLEUSChromatin

Chromatin modification:

Gene

DNA Gene availablefor transcription

RNA ExonTranscription

Primary transcript

RNA 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

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 loosens DNA to allow transcription

Figure 19.4 b

Unacetylated histones Acetylated histones