1. Biology Sylvia S. Mader Michael Windelspecht
Chapter 13
Regulation of Gene Expression
Lecture Outline
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2. Outline 13.1 Prokaryotic Regulation 13.2 Eukaryotic Regulation
13.3 Genetic Mutations

3. 13.1 Prokaryotic Regulation
Bacteria don’t require same enzymes all the time
Enzymes produced as needed
Operon model: (François Jacob & Jacques Monod 1961) explains prokaryotic regulation of gene expression
Operon: group of structural & regulatory genes functioning as 1 unit

4. Prokaryotic Regulation
An operon consists of three components
Promoter
Short DNA sequence where RNA polymerase first attaches
Operon start, transcription begins
Operator
Short DNA sequence where active repressor binds
RNA polymerase can’t bind, no transcription
Structural Genes
>1 genes coding for enzymes/proteins of metabolic pathway
Transcribed as a block
Long segment of DNA
Regulatory gene- codes for repressor protein
located outside the operon
repressor protein controls whether operon is active or not

5. Prokaryotic Regulation
The trp Operon
Regulator genes code for inactive repressor
If tryptophan is absent:
Repressor unable to attach to operator (expression is “on”)
RNA polymerase binds to promoter
Enzymes for tryptophan synthesis are produced
If tryptophan is present:
Combine w/repressor protein as co-repressor
Repressor binds operator, RNA polymerase can’t bind to promoter
Repressor blocks synthesis of structural genes for tryptophan synthesis

6. transcription is prevented.
The trp Operon
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promoter
operator
regulator gene
structural genes
When the repressor
binds to the operator,
transcription is prevented.
active
repressor

7. The trp Operon regulator gene promoter operator structural genes DNA
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regulator gene
promoter
operator
structural genes
DNA
RNA polymerase 5′ 3′
mRNA X
mRNA
inactive
repressor
enzymes
a. Tryptophan absent. Enzymes needed to synthesize tryptophan are produced.
RNA polymerase cannot bind to promoter.
DNA
active repressor
mRNA
tryptophan
inactive
repressor
b. Tryptophan present. Presence of tryptophan prevents production of enzymes used to synthesize tryptophan.

8. Prokaryotic Regulation
The lac Operon
Regulator genes code for active repressor
If lactose is absent:
Repressor attaches to operator
RNA polymerase can’t bind to promoter
Expression is normally “off”
If lactose is present:
combines w/ repressor; can’t bind to operator
RNA polymerase binds to promoter
3 enzymes necessary for lactose catabolism are produced

9. The lac Operon a. b. regulatory gene promoter operator
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regulatory gene
promoter
operator
lactose metabolizing genes
DNA
mRNA
RNA polymerase
active repressor a.
RNA polymerase bound to promoter.
DNA x
mRNA
inactive
repressor
lactose
active
repressor
enzymes
mRNA b.

10. Prokaryotic Regulation
Further control of the lac operon
E. coli preferentially breaks down glucose
lac operon is maximally activated only in the absence of glucose
When glucose is absent
Cyclic AMP (cAMP) accumulates
cAMP binds to catabolite activator protein (CAP)
CAP + cAMP, binds to site near lac promoter  RNA polymerase better able to bind to promoter
structural genes of the lac operon expressed more efficiently 10

11. Prokaryotic Regulation
Further control of the lac operon
When glucose is present
little cAMP in cell
CAP is inactive
lac operon is not expressed maximally 11

12. Action of CAP a. Lactose present, glucose absent (cAMP level high)
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CAP binding site
promoter
operator
DNA
inactive CAP 12
a. Lactose present, glucose absent (cAMP level high)

13. Action of CAP a. Lactose present, glucose absent (cAMP level high)
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CAP binding site
promoter
operator
DNA
cAMP
inactive CAP 13
a. Lactose present, glucose absent (cAMP level high)

14. Action of CAP a. Lactose present, glucose absent (cAMP level high)
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CAP binding site
promoter
operator
DNA
cAMP
inactive CAP 14
a. Lactose present, glucose absent (cAMP level high)

15. Action of CAP a. Lactose present, glucose absent (cAMP level high)
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CAP binding site
promoter
operator
DNA
cAMP
active CAP
inactive CAP 15
a. Lactose present, glucose absent (cAMP level high)

16. Action of CAP a. Lactose present, glucose absent (cAMP level high)
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CAP binding site
promoter
operator
DNA
cAMP
active CAP
inactive CAP 16
a. Lactose present, glucose absent (cAMP level high)

17. Action of CAP a. Lactose present, glucose absent (cAMP level high)
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CAP binding site
promoter
operator
DNA
RNA polymerase binds
fully with promoter.
cAMP
active CAP
inactive CAP 17
a. Lactose present, glucose absent (cAMP level high)

18. Action of CAP 18 CAP binding site promoter operator DNA
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CAP binding site
promoter
operator
DNA
RNA polymerase binds
fully with promoter.
cAMP
active CAP
inactive CAP
a. Lactose present, glucose absent (cAMP level high)
CAP binding site
promoter
operator
DNA
RNA polymerase does
not bind fully with promoter.
inactive CAP 18
b. Lactose present, glucose present (cAMP level low)

19. 13.2 Eukaryotic Regulation
A variety of mechanisms
5 primary levels of control:
Nuclear levels
Chromatin Structure
Transcriptional Control
Posttranscriptional Control
Cytoplasmic levels
4. Translational Control
5. Posttranslational Control

20. Control of Gene Expression in Eukaryotic Cells
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histones
Chromatin
structure
Transcriptional control 3
pre-
mRNA 5
intron
exon
Posttranscriptional control
mRNA 5 3
nuclear pore
nuclear envelope
Translational
control
polypeptide chain
Posttranslational
control
plasma
membrane
functional protein

21. Eukaryotic Regulation
1. Chromatin Structure
Chromatin= Eukaryotic DNA + histone proteins
Nucleosomes
DNA wound around 8 molecules of histone proteins
Looks like beads on a string
levels of chromatin packing determined by degree of nucleosome coiling

22. Chromatin Structure Regulates Gene Expression
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nucleolus
heterochromatin
euchromatin
nucleosome
inaccessible
promoter
1 µm
a. Darkly stained heterochromatin and lightly stained euchromatin
chromatin remodeling complex
H2B
histone protein
H2A H4
histone
tail H3
accessible
promoter
DNA H1
DNA to be transcribed
b. A nucleosome
c. DNA unpacking
(a): © Dennis Kunkel Microscopy,Inc./Visuals Unlimited

23. Eukaryotic Regulation
1. Chromatin Structure cont.
Euchromatin
Loosely coiled DNA
Transcriptionally active
Heterochromatin
Tightly packed DNA
Transcriptionally inactive
Barr Body
Females have 2 X chromosomes, only one is active
other X chromosome is tightly packed & is inactive
inactive X chromosome is a Barr body

24. X-Inactivation in Mammalian Females
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Coats of tortoiseshell
cats have patches
of orange and black.
active X chromosome
allele for
orange color
inactive X
Barr bodies
cell division
inactive X
allele for
black color
active X chromosome
Females have two
X chromosomes.
One X chromosome is inactivated in
each cell. Which one is by chance.
© Chanan Photo 2004

25. Eukaryotic Regulation
2. Transcriptional Control- most critical level!
Transcription controlled by transcription factors (proteins)
Transcription activator bind to enhancer DNA
Regions of DNA where factors that regulate transcription can also bind
Transcription factors are always present in the cell, but most likely have to be activated before they will bind to DNA

26. Eukaryotic Transcription Factors
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DNA
promoter
gene
enhancer
transcription
activator
transcription
factor complex
mediator proteins
RNA polymerase
mRNA transcription

27. Eukaryotic Regulation
3. Posttranscriptional control operates on primary mRNA transcript
Alter pre-mRNA transcript:
Excision of introns can vary
Splicing of exons can vary
Determines type of mature transcript that leaves nucleus
Control speed of mRNA transport from nucleus
Affect # of transcripts arriving at rough ER & amount of gene produced

28. Alternative Processing of pre-mRNA
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intron
exon
intron
exon 5 A B C D E 3 5 A B C D E 3
cap
pre-mRNA
poly-A
tail
cap
pre-mRNA
poly-A
tail
RNA
splicing
RNA
splicing
intron
intron C A B C D E A B D E
mRNA
mRNA
protein product 1
protein product 2 a. b.

29. Eukaryotic Regulation
4. Translational Control – Determines degree of mRNA translated into protein
Features of mRNA affect whether translation occurs and how long mRNA is active
Presence of 5′ cap
Length of poly-A tail on 3′ end
MicroRNAs (miRNAs) regulate translation by causing destruction of mRNAs before they are translated

30. Eukaryotic Regulation
5. Posttranslational Control – Affects activity of protein product
accomplished by regulating:
Activation
Degradation rate 30

31. 13.3 Gene Mutations gene mutation- permanent change in DNA sequence
gene mutation effects
No effect  Complete protein inactivation
Germ-line mutations occur in sex cells
Somatic mutations occur in body cells

32. Gene Mutation Causes 1. Spontaneous mutations
Chemical changes in DNA leads to mispairing during replication
Movement of transposons from one location to another
Replication Errors- rare!
DNA polymerase
Proofreads new strands
Generally corrects errors
Overall mutation rate= 1 in 1,000,000,000 nucleotide pairs replicated

33. Gene Mutation Causes 2. Induced mutations Caused by mutagens
Many mutagens are carcinogens (cancer-causing)
Chemical Mutagens- food additives!
Ultraviolet Radiation- the sun, x-rays, gamma rays, can repair damage (DNA repair enzymes)
Organic Chemicals- tobacco smoke  cancers!!

34. The Ames Test For Mutagenicity
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Suspected
chemical
mutagen
Control
bacterial
strain
(requires
histidine)
bacterial
strain
(requires
histidine)
Plate onto petri plates
that lack histidine.
bacterial
growth
Incubate overnight
Mutation occurred
Mutation did not occur

35. Gene Mutations Point Mutations Frameshift Mutations
Involve change in 1 DNA nucleotide
Change 1 codon to different codon
Effects on protein vary:
Nonfunctional
Reduced functionality
Unaffected
Frameshift Mutations
1-2 nucleotides inserted or deleted from DNA
Protein always rendered nonfunctional
Normal : THE CAT ATE THE RAT
After deletion: THE ATA TET HER AT
After insertion: THE CCA TAT ETH ERA T

36. Point Mutations in Hemoglobin
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No mutation A C A C G T G G G T G A G G T C T C C T C
Val
His
Leu
Thr
Pro
Glu
Glu
b. Normal red blood cell
c. Sickled red blood cell
b, c: © Stan Flegler/Visuals Unlimited.

37. Point Mutations in Hemoglobin
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No mutation C A C G T G G A G T G A G G T C T C C T C
Val
His
Leu
Thr
Pro
Glu
Glu
His
His
(normal protein) C A C G T A G A G T G A G G T C T C C T C
Val
His
Leu
Thr
Pro
Glu
Glu
b. Normal red blood cell
c. Sickled red blood cell
b, c: © Stan Flegler/Visuals Unlimited. 37

38. Point Mutations in Hemoglobin
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3 5
No mutation C A C G T G G A G T G A G G T C T C C T C
Val
His
Leu
Thr
Pro
Glu
Glu
His
His
(normal protein) C A C G T A G A G T G A G G T C T C C T C
Val
His
Leu
Thr
Pro
Glu
Glu
b. Normal red blood cell
c. Sickled red blood cell
Glu
Val
(abnormal protein) C A C G T G G A G T G A G G T C A C C T C
Val
His
Leu
Thr
Pro
Val
Glu
b, c: © Stan Flegler/Visuals Unlimited. 38

39. Point Mutations in Hemoglobin
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3 5
No mutation C A C G T G G A G T G A G G T C T C C T C
Val
His
Leu
Thr
Pro
Glu
Glu
His
His
(normal protein) C A C G T A G A G T G A G G T C T C C T C
Val
His
Leu
Thr
Pro
Glu
Glu
b. Normal red blood cell
c. Sickled red blood cell
Glu
Val
(abnormal protein) C A C G T G G A G T G A G G T C A C C T C
Val
His
Leu
Thr
Pro
Val
Glu
Glu
Stop
(incomplete protein) C A C G T G G A G T G A G G T A T C C T C
Val
His
Leu
Thr
Pro
Stop a.
b, c: © Stan Flegler/Visuals Unlimited. 39

40. Gene Mutations
Cancer development involves series of accumulating mutations
Proto-oncogenes – Stimulate cell division (gas pedal)
Mutated proto-oncogenes  oncogenes that’re always active
Tumor suppressor genes – inhibit cell division (brakes)
Mutations in oncogene & tumor suppressor genes:
Stimulate cell cycle uncontrollably
Lead to tumor formation

41. Cell Signaling Pathway That Stimulates a Mutated Tumor Suppressor Gene
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receptor
plasma
membrane
nucleus
mutated tumor suppressor gene

42. Cell Signaling Pathway That Stimulates a Mutated Tumor Suppressor Gene
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inhibiting growth factor
receptor
plasma
membrane
nucleus
mutated tumor suppressor gene 42

43. Cell Signaling Pathway That Stimulates a Mutated Tumor Suppressor Gene
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inhibiting growth factor
receptor
plasma
membrane
signal
transducers
nucleus
mutated tumor suppressor gene 43

44. Cell Signaling Pathway That Stimulates a Mutated Tumor Suppressor Gene
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inhibiting growth factor
receptor
plasma
membrane
signal
transducers
transcription factor
nucleus
mutated tumor suppressor gene 44

45. Cell Signaling Pathway That Stimulates a Mutated Tumor Suppressor Gene
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inhibiting growth factor
receptor
plasma
membrane
signal
transducers
transcription factor
protein that is
unable to inhibit
the cell cycle
or promote
apoptosis
nucleus
mutated tumor suppressor gene 45

46. Cell Signaling Pathway That Stimulates an Oncogene
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receptor
plasma
membrane
cytoplasm
nucleus
oncogene

47. Cell Signaling Pathway That Stimulates an Oncogene
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stimulating growth factor
receptor
plasma
membrane
cytoplasm
nucleus
oncogene 47

48. Cell Signaling Pathway That Stimulates an Oncogene
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stimulating growth factor
receptor
plasma
membrane
signal
transducers
cytoplasm
nucleus
oncogene 48

49. Cell Signaling Pathway That Stimulates an Oncogene
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stimulating growth factor
receptor
plasma
membrane
signal
transducers
cytoplasm
transcription factor
nucleus
oncogene 49

50. Cell Signaling Pathway That Stimulates an Oncogene
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stimulating growth factor
receptor
plasma
membrane
signal
transducers
cytoplasm
transcription factor
protein that
overstimulates
the cell cycle
nucleus
oncogene 50