1. Regulation of Prokaryotic and Eukaryotic Gene Expression

2. This metabolic control occurs on two levels:
A bacterium can tune its metabolism to the changing environment and food sources
This metabolic control occurs on two levels:
Adjusting activity of metabolic enzymes
Regulating genes that encode metabolic enzymes

3. LE 18-20 Regulation of enzyme activity Regulation of enzyme production
Precursor
Feedback
inhibition
Enzyme 1
Gene 1
Enzyme 2
Gene 2
Regulation
of gene
expression
Enzyme 3
Gene 3
Enzyme 4
Gene 4
Enzyme 5
Gene 5
Tryptophan

4. Operons: The Basic Concept
In bacteria, genes are often clustered into operons, composed of
An operator, an “on-off” switch
A promoter
Genes for metabolic enzymes

5. Polypeptides that make up enzymes for tryptophan synthesis
LE 18-21a
trp operon
Promoter
Promoter
Genes of operon
DNA
trpR
trpE
trpD
trpC
trpB
trpA
Operator
Regulatory
gene
RNA
polymerase
Start codon
Stop codon 3¢
mRNA 5¢
mRNA 5¢ E D C B A
Protein
Inactive
repressor
Polypeptides that make up
enzymes for tryptophan synthesis
Tryptophan absent, repressor inactive, operon on

6. LE 18-21b_1 DNA mRNA Protein Active repressor Tryptophan (corepressor)
Tryptophan present, repressor active, operon off

7. LE 18-21b_2 DNA No RNA made mRNA Protein Active repressor Tryptophan
(corepressor)
Tryptophan present, repressor active, operon off

8. Two Types of Negative Gene Regulation
A repressible operon
Is usually on
binding of a repressor to the operator shuts off transcription
The trp operon is a repressible operon
An inducible operon
Is one that is usually off
a molecule called an inducer inactivates the repressor and turns on transcription
the lac operon is an inducible operon, which contains genes coding for enzymes in hydrolysis and metabolism of lactose

9. LE 18-22a Regulatory gene Promoter Operator DNA lacl lacZ No RNA made3¢
mRNA
RNA
polymerase 5¢
Active
repressor
Protein
Lactose absent, repressor active, operon off

10. LE 18-22b lac operon DNA lacl lacZ lacY lacA RNA polymerase 3¢ mRNA5¢
Permease
Transacetylase
Protein
-Galactosidase
Inactive
repressor
Allolactose
(inducer)
Lactose present, repressor inactive, operon on

11. Inducible enzymes usually function in catabolic (“breakdown”) pathways
Explain to a neighbor why this makes sense.
Repressible enzymes usually function in anabolic (“synthesis”) pathways
Regulation of the trp and lac operons involves negative control of genes because operons are switched off by the active form of the repressor

12. Positive Gene Regulation
Some operons are also subject to positive control through a stimulatory activator protein, such as catabolite activator protein (CAP)
When glucose (a preferred food source of E. coli ) is scarce, the lac operon is activated by the binding of CAP
When glucose levels increase, CAP detaches from the lac operon, turning it off

13. Lactose present, glucose scarce (cAMP level high): abundant lac
LE 18-23a
Promoter
DNA
lacl
lacZ
RNA
polymerase
can bind
and transcribe
CAP-binding site
Operator
Active
CAP
cAMP
Inactive lac
repressor
Inactive
CAP
Lactose present, glucose scarce (cAMP level high): abundant lac
mRNA synthesized

14. Lactose present, glucose present (cAMP level low): little lac
LE 18-23b
Promoter
DNA
lacl
lacZ
CAP-binding site
Operator
RNA
polymerase
can’t bind
Inactive
CAP
Inactive lac
repressor
Lactose present, glucose present (cAMP level low): little lac
mRNA synthesized

15. Summarize… Explain to a neighbor why bacteria regulate gene expression
Give an example of how bacteria regulate gene expression

16. Eukaryotic Gene Regulation

17. How to prevent expression?
Every cell in a multi-cellular eukaryote does not express all its genes, all the time (usually only 3-5%)
Long-term control of gene expression in tissue = differentiation
How to prevent expression?
Regulation at transcription
Regulation after transcription

19. Chromatin Regulation Chromatin remodeling allows transcription
Chromatin = DNA + proteins
Chromatin coiled around histones = nucleosomes
Allows DNA to be packed into nucleus, but also physically regulates expression by making regions ‘available’ or not

21. Chromatin regulation can be small-scale (gene) or large scale (chromosome)
Non-expressed = heterochromatin (condensed)
Expressed = euchromatin (relaxed)

22. Changes to Chromatin (DNA)
Methylation
Methylating (adding methyl groups) to DNA bases, keeping them “tight” and “closed” – inaccessible to transcription.
Histone Acetylation
“Acetylating” histones (adding acetyl groups) promotes loose chromatin and permits transcription

24. Transcription Regulation
What we know from prokaryotes:
Several related genes can be transcribed together (ie. lac operon)
Need RNA Polymerase to recognize a promoter region
Why eukaryotes are different:
Genes are nearly always transcribed individually
3 RNA Polymerases occur, requiring multiple proteins to initiate transcription

25. Transcription Regulation Con’t
Typical prokaryotic promoter:
recognition sequence + TATA box ->
RNA Polymerase -> transcription
Typical eukaryotic promoter:
recognition sequence + TATA box + transcription factors -> RNA Polymerase -> transcription

27. RNA polymerase interacts w/promoter, regulator sequences, & enhancer sequences to begin transcription
Regulator proteins bind to regulator sequences to activate transcription
Found prior to promoter
Enhancer sequences bind activator proteins
Typically far from the gene
Silencer sequences stop transcription if they bind with repressor proteins

28. Now, Can You:
Explain why gene expression control is necessary in a eukaryotic cell?
Describe how expression is regulated in before & during transcription?
Tell me what differentiation is? Euchromatin? A silencer sequence?
Explain how gene expression regulation is different in eukaryotes/prokaryotes?

29. Post-Transcription Regulation
Have mRNA variation
Alternative splicing: shuffling exons
Allows various proteins to be produced in different tissues from the same gene
Change the lifespan of mRNA
Produce micro RNA that will damage mRNA, preventing translation
Edit RNA & change the polypeptide produced
Insert or alter the genetic code

30. Translation Regulation
mRNA present in cytosol does not necessarily get translated into proteins
Control the rate of translation to regulate gene expression
How?
Modify the 5’ cap
Feedback regulation (build up of products = less translation)

31. Translation Regulation Con’t
Modify the lifespan of proteins:
Attach ubiquitin = target for breakdown via proteasome (woodchipper)

32. So…
What are the ways that a cell can regulate gene expression AFTER transcription?
How can the process of RNA splicing allow one pre-mRNA to produce 5 different proteins in 5 different tissues?
And…

33. Can you accurately fill in this table?