31 Gene regulation in bacteria. Lecture Outline 11/18/05 Finish up from last time: Transposable elements (“jumping genes”) Gene Regulation in Bacteria.

31 Gene regulation in bacteria

Lecture Outline 11/18/05• Finish up from last time:

• Transposable elements (“jumping genes”)

• Gene Regulation in Bacteria – Transcriptional control– Cells adjust to their environment by turning genes on

and off

• The operon concept– Repressors, Inducers, Operators, Promoters

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

Transposable elements• Normal and ubiquitous

– Prokaryotes-• Genes transpose to/from cell’s

chromosome, plasmid, or a phage chromosome.

– Eukaryotes-• Genes transpose to/from same or a

different chromosome.

• Cause genetic changes – Chromosome breaks– Duplications – Knock-out genes

I’ll talk about 2 kinds:

• Insertion sequences• Ac/Ds elements in corn

• A third major class: Retrotransposons– Uses RNA intermediate and reverse transcriptase– Most Important class in mammalian genomes

Insertion sequence (IS) elements:

• Simplest type of transposable element – Found in bacterial chromosomes and plasmids.– Encode only genes for mobilization and insertion.

Inverted terminal repeats

Integration of an Insertion Element

Don’t worry about the details, just the concept

Staggered cut at target site

Insert IS element

Fill in the gaps

IS element carries transposase gene

Transposase recognizes terminal repeats

TransposonsHave additional genes, such as those for antibiotic

resistance• (examples Tn3 (ampicillin), Tn10 (tetracycline)

Figure 18.19b

Inverted repeats Transposase gene

Insertion sequence

Insertion sequence

Antibioticresistance gene

Transposon

5

3

5

3

Barbara McClintock’s discovery of transposons in corn:

•Kernel color alleles/traits were “unstable”.

•McClintock concluded transposon called “Ds” inserted into the “C” gene for colored kernels

Nobel prize, 1983

Transposon effects on corn kernel color.

Ac activates Ds

Two transposable elements in different sites Normal gene for

purple kernels

Ds element inserts into color gene and inactivates it

Ac can make transposaseDs can move, but lacks enzyme

One method for Conservative Transposition

“Cut and Paste” Transposable element is cut out by transposase and inserts in another location.

No increase in the number of transposable elements- just a change in position

From Griffiths, Intro to Genetic Analysis

One method for replicative transposition

From Griffiths, Intro to Genetic Analysis

Gene regulation in bacteria

But ALL organisms must adjust to changes in their environment and all have evolved numerous control mechanisms.

E.coli bacteria eat whatever we eat!

Regulation of metabolism occurs at two levels:

– Adjusting the activity of metabolic enzymes already present

– Regulating the genes encoding the metabolic enzymes

Figure 18.20a, b

(a) Regulation of enzyme activity

Enzyme 1

Enzyme 2

Enzyme 3

Enzyme 4

Enzyme 5

Regulationof geneexpression

Feedbackinhibition

Tryptophan

Precursor

(b) Regulation of enzyme production

Gene 2

Gene 1

Gene 3

Gene 4

Gene 5

–

–

Types of Regulated Genes

• Constitutive genes are always expressed– Tend to be vital for basic cell functions (often called

“housekeeping genes”)

• Inducible genes are normally off, but can be turned on when substrate is present

• Common for catabolic enzymes (i.e. for the utilization of particular resources)

• Repressible genes are normally on, but can be turned off when the end product is abundant

• Common for anabolic enzymes

In bacteria, genes are often clustered into operons

Operons have:1. Several genes for metabolic enzymes

2. One promoter

3. An operator, or control site(“on-off” switch)

4. A separate gene that makes a repressor or activator protein that binds to the operator

R O 1 2 3PP

The trp Operon

5 genes: E, D, C, B, A

Same order as enzymes for trp synthesis

Controlled by a single promoter and operator

More Terminology

• Repressors and Activators are proteins that bind to DNA and control transcription.

• Co-repressors and Inducers: small “effector” molecules that bind to repressors or activators

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: regulated synthesis of repressible enzymes

Figure 18.21a

5

Tryptophan absent -> repressor inactive -> operon “on”

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

Tryptophan changes the shape of the repressor protein so it can bind DNA

• The lac operon: regulated synthesis of inducible enzymes

Figure 18.22a

DNA

mRNA

ProteinActiverepressor

RNApolymerase

NoRNAmade

lacZlacl

Regulatorygene

Operator

Promoter

Lactose absent, repressor active, operon off. The lac repressor is innately active, and inthe absence of lactose it switches off the operon by binding to the operator.

(a)

5

3

mRNA 5'

DNA

mRNA

Protein

Allolactose(inducer)

Inactiverepressor

lacl lacz lacY lacA

RNApolymerase

Permease Transacetylase-Galactosidase

5

3

(b) Lactose present, repressor inactive, operon on. Allolactose, an isomer of lactose, derepresses the operon by inactivating the repressor. In this way, the enzymes for lactose utilization are induced.

mRNA 5

lac operon

Figure 18.22b

Positive 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– Via a stimulatory activator protein, such as catabolite

activator protein (CAP)

Promoter

Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized.If glucose is scarce, the high level of cAMP activates CAP, and the lac operon produces large amounts of mRNA for the lactose pathway.

(a)

CAP-binding site OperatorRNApolymerasecan bindand transcribe

InactiveCAP

ActiveCAPcAMP

DNA

Inactive lacrepressor

lacl lacZ

Figure 18.23a

– In E. coli, when glucose is always the preferred food source

– When glucose is scarce, the lac operon is activated by the binding of the catabolite activator protein (CAP)

Positive Gene Regulation- CAP

• When glucose is abundant,– CAP detaches from the lac operon, which

prevents RNA polymerase from binding to the promoter

Figure 18.23b(b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized.

When glucose is present, cAMP is scarce, and CAP is unable to stimulate transcription.

Inactive lacrepressor

InactiveCAP

DNA

RNApolymerasecan’t bind

Operator

lacl lacZ

CAP-binding site

Promoter