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
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
• 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