Control of Gene Expression

1
Control of Gene Expression

• AP Chap 18

2

• Prokaryotes and eukaryotes alter gene
  expression in response to their changing
  environment.

Clustering of genes producing mRNAs for proteins
(enzymes) in a pathway make the control easier
and more efficient.
3
Bacteria often respond to environmental change by
regulating transcription

• Natural selection has favored bacteria that
  produce only the products needed by that cell.

We are very conservative!
4
How can cells regulate their production of
enzymes for metabolic processes?

• by feedback inhibition or
• by gene regulation

5
Fig. 18-2
Precursor
Feedback inhibition
trpE gene
Enzyme 1
trpD gene
Regulation of gene expression
trpC gene
Enzyme 2
trpB gene
Enzyme 3
trpA gene
Tryptophan
(b) Regulation of enzyme production
(a) Regulation of enzyme activity
6

• Gene expression in bacteria is controlled by
  the operon model.
• The operon model works on a feedback process.
• The operon model for gene regulation was first
  described by Jacob and Monod in 1961.

7
Jacob and Monod
8
OPERONS

• An operon is the entire stretch of DNA that
  includes the promoter, the operator, and the
  genes that they control. The regulator may be
  located away from the operon unit.
• DNA
• regulator promoter operator genes
• Remember Clustering of genes for proteins
  (enzymes) in a pathway make the control easier
  and more efficient.

9

• regulator promoter operator genes
  

STOP
RNA polymerase binds here
Makes a repressor which binds to operator and
stops/starts transcription
GO
10
Induction System inducible operonthink,
induceto turn on

• System initially off
• The system is off because an active repressor is
  bound to the operator.

11
Fig. 18-4a
Regulatory gene
Promoter
Operator
DNA
lacI
lacZ
No RNA made
3?
mRNA
RNA polymerase
5?
Active repressor
Protein
(a) Lactose absent, repressor active, operon off
12

• If the lacI gene is deleted, how will
  transcription of the lac operon be affected?
• Transcription will always be turned off
• Transcription will always be turned on.
• No effect will be observed.
• B

13

• The presence of an inducer (usually a substrate
  that needs to be broken down) turns it on.
• The inducer binds to the repressor and makes it
  inactive so transcription can occur.
• The inducer acts as an allosteric effector and
  changes the shape of the repressor.
• Ex- Lac (lactose) operon used to produce enzymes
  to break down lactose (milk sugar).
• The inducer in the lac operon is lactose (more
  specifically allolactose).

14
Lactose present, repressor inactive, operon ON
Fig. 18-4b
lac operon
DNA
lacY
lacZ
lacA
lacI
RNA polymerase
3?
mRNA
mRNA 5?
5?
Permease
Transacetylase
?-Galactosidase
Protein
Inactive repressor
Allolactose (inducer)
(b) Lactose present, repressor inactive, operon on
VCAC Molecular Processes Lac Operon First Look
http//highered.mheducation.com/sites/0072995246/s
tudent_view0/chapter7/the_lac_operon.html
15

• A mutation in the lacI gene leads to the
  inability of the repressor to bind the inducer.
  In the presence of lactose, how will this
  mutation affect transcription of the lac operon?
• A) Transcription will always be turned off.
• B) Transcription will always be turned on.

A, the repressor will always be active and bound
to the operator.
16
Repressible System

• System initially ON, transcription ongoing and
  making a product
• Operator can be turned off by a repressor which
  is made active by being activated by a
  corepressor molecule (usually the end product)
• The product acts as a corepressor inhibiting
  further synthesis of enzymes involved in the
  process.

17

• Ex tryptophan operon trytophan (product) acts
  as a corepressor inhibiting further synthesis of
  enzymes involved in the process

18
Tryptophan absent, repressor inactive, operon ON
Fig. 18-3a
trp operon
Promoter
Promoter
Genes of operon
DNA
trpD
trpA
trpR
trpE
trpC
trpB
Operator
Regulatory gene
Stop codon
Start codon
3?
mRNA 5?
RNA polymerase
mRNA
5?
A
B
C
D
Protein
Inactive repressor
Polypeptide subunits that make up enzymes for
tryptophan synthesis
E
(a) Tryptophan absent, repressor inactive, operon
on
19
Tryptophan present, repressor active, operon OFF
Fig. 18-3b-1
DNA
No RNA made
mRNA
Protein
Active repressor
(repressor)
Tryptophan (corepressor)
(b) Tryptophan present, repressor active, operon
off
http//highered.mheducation.com/sites/0072995246/s
tudent_view0/chapter7/the_trp_operon.html
20
INDUCIBLE REPRESSIBLE OFF ON
turned on by turned off by
inducer (substrate) corepressor (product)
used in catabolic used in anabolic
pathways pathways Both
use allosteric effectors and are
NEGATIVE CONTROL.
21
Positive Gene Regulation

• Some operons are also subject to positive control
  through a stimulatory protein, such as catabolite
  activator protein (CAP), an activator of
  transcription
• When glucose (a preferred food source of E. coli)
  is scarce, CAP is activated by binding with
  cyclic AMP
• Activated CAP attaches to the promoter of the lac
  operon and increases the affinity of RNA
  polymerase, thus accelerating transcription

22
Fig. 18-5
Promoter
Operator
DNA
lacI
lacZ
RNA polymerase binds and transcribes
CAP-binding site
Active CAP
cAMP
Inactive lac repressor
Inactive CAP
Allolactose
(a) Lactose present, glucose scarce (cAMP level
high) abundant lac mRNA synthesized
Promoter
Operator
DNA
lacI
lacZ
CAP-binding site
RNA polymerase less likely to bind
Inactive CAP
Inactive lac repressor
(b) Lactose present, glucose present (cAMP level
low) little lac mRNA synthesized
23

• When glucose levels increase, CAP detaches from
  the lac operon, and transcription returns to a
  normal rate
• CAP helps regulate other operons that encode
  enzymes used in catabolic pathways

24

• The lac operon responds to lactose, while
  sensing the levels of available glucose.

Lactose Glucose Lac mRNA transcription
absent high off
present low on
What if lactose is high and glucose is high, will
lac mRNA transcription be off or on?
25
Control of Gene Expression in Eukaryotes
26

• In response to environmental signals
• More complicated than prokaryotes due to
  specialized cells. No operons in eukaryotes.
• Essential for development and cell specialization
  in multicellular organisms
• RNA is important in eukaryotic gene expression.

27

• All cells contain the same DNA so controlling
  gene expression is essential.
• Human cells only 20 genes expressed only 1.5
  code for proteins!
• Commonly occurs at level of transcription hence,
  
• gene expression transcription of DNA

http//www.dnalc.org/resources/3d/09-how-much-dna-
codes-for-protein.html
28
But, eukaryotic gene expression can be regulated
at any stage
lt Agt
29
Fig. 18-6
Signal
NUCLEUS
Chromatin
Chromatin modification
DNA
Gene available for transcription
In the nucleus
Gene
Transcription
Exon
RNA
Primary transcript
Intron
RNA processing
Tail
mRNA in nucleus
Cap
Transport to cytoplasm
CYTOPLASM
mRNA in cytoplasm
Translation
Degradation of mRNA
Polypeptide
In the cytoplasm
Protein processing
Active protein
Degradation of protein
Transport to cellular destination
Cellular function
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Fig. 18-6a
Signal
NUCLEUS
Chromatin
Chromatin modification
DNA
Gene available for transcription
Gene
Transcription
Exon
RNA
Primary transcript
Intron
RNA processing
Tail
mRNA in nucleus
Cap
Transport to cytoplasm
CYTOPLASM
31
Fig. 18-6b
CYTOPLASM
mRNA in cytoplasm
Translation
Degradation of mRNA
Polypeptide
Protein processing
Active protein
Degradation of protein
Transport to cellular destination
Cellular function
32

• 1) Chromatin Modification
• Heterochromatin tightly wound DNA so genes not
  expressed
• Euchromatin DNA spread out, genes can be
  expressed

33

• Chemical modification
• by histone acetylation which keeps chromatin
  spread out and
• methylation (CH3) of DNA which keeps DNA tightly
  packed and so inhibits transcription.

34
Fig. 18-7
Histone tails
http//www.dnalc.org/resources/3d/08-how-dna-is-pa
ckaged-advanced.html
Amino acids available for chemical modification
DNA double helix
(a) Histone tails protrude outward from a
nucleosome
Histone acetylation
Acetylated histones
Unacetylated histones
(b) Acetylation of histone tails promotes loose
chromatin structure that permits transcription
35
The effect of methylated DNA
Methylated DNA inhibits transcription.
36

• Epigenetic inheritance not involve DNA sequence
  but inherited defects in chromatin modification
  enzymes

There may be more to inheritance than genes
alone. New clues reveal that a second epigenetic
chemical code sits on top of our regular DNA and
controls how our genes are expressed.
37
Epigenetic effects in mice
http//www.youtube.com/watch?vOOiCu5kzGxg
NOVA A Tale of Two Mice
38
(No Transcript)
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• The mice were identical (so same genes). Why are
  they different?
• What chemical modification occurred in the
  normal mouse?
• What happened when mice were fed BPA?
• What happened when mice were feed nutrients (soy)
  containing methylated molecules?

40
How do environmental factors affect gene
expression?

• Mechanisms may involve DNA methylation and
  histone acetylation
• Diet, chemicals, metals, and stress are known to
  affect DNA methylation.
• Other enzymes have been identified for
  demethylation, phosphorylation, and many others.

41

• For example, methylation of cytosine(s) in the
  promoter region could prevent the binding of
  transcription factors or create binding sites for
  complexes that deacetylate neighboring histones
  that in turn compact the chromatin, encouraging a
  gene to become silent.
• A similar mechanism is now recognized in a number
  of cancers. There is also indirect evidence to
  suggest that methylation could apply to a number
  of complex diseases, including schizophrenia.

42
2) Transcription Level

• Regulation of transcription initiation
• DNA control elements (enhancers) located away
  from the gene bind specific transcription factors
  (tfs) at the gene.
• Bending of DNA is necessary to enable activators
  in the enhancers to contact tfs at the promoter,
  initiating transcription.

43
Fig. 18-9-1
Promoter
Activators
Gene
DNA
Distal control element
Enhancer
TATA box
44
Fig. 18-9-2
Promoter
Activators
Gene
DNA
Distal control element
Enhancer
TATA box
General transcription factors
DNA-bending protein
Group of mediator proteins
45
Fig. 18-9-3
Promoter
Activators
Gene
DNA
Distal control element
Enhancer
TATA box
General transcription factors
DNA-bending protein
Bending of DNA enables activators to contact
proteins at the promoter, initiating
transcription.
Group of mediator proteins
RNA polymerase II
RNA polymerase II
Transcription initiation complex
RNA synthesis
46
Specific enhancers control the expression of
genes.
Fig. 18-UN7
47
Fig. 18-10
Enhancer
Promoter
Albumin gene
Control elements
Crystallin gene
LENS CELL NUCLEUS
LIVER CELL NUCLEUS
Available activators
Available activators
Albumin gene not expressed
Albumin gene expressed
Crystallin gene not expressed
Crystallin gene expressed
(b) Lens cell
(a) Liver cell
48
Post-transcriptional Control

• RNA processing
• Translation
• mRNA degradation
• Protein Processing and Degradation

49
RNA Processing

• Alternative RNA splicing produce different
  proteins

50
Fig. 18-11
Exons
DNA
Troponin T gene
Primary RNA transcript
RNA splicing
or
mRNA
51
mRNA Degradation

• mRNA can last a long time and be subject to
  various intron splicing
• The mRNA life span is determined in part by
  sequences in the leader and trailer regions

52
Translation

• Initiation of translation can be controlled via
  regulation of initiation factors

53
Protein Processing and Degradation

• Alteration of polypeptide - can be cut, groups
  (lipids, sugars) added, or transported to target
  locations
• Selective degradation of proteins the protein
  ubiquitin is added to proteins for degradation.
  Proteasomes (like garbage disposals) recognize
  them and destroy them.

54
Fig. 18-12
Proteasome and ubiquitin to be recycled
Ubiquitin
Proteasome
Ubiquitinated protein
Protein fragments (peptides)
Protein to be degraded
Protein entering a proteasome
55
Remember

• Prokaryotic gene control several genes
  controlled by one promoter in operon systems,
  mainly controlled at transcription level.
• Eukaryotic gene control one gene controlled by
  one promoter, no operators but have specific
  enhancers, can be controlled at any level.

56
(No Transcript)
57
How important is RNA?
Noncoding RNAs play multiple roles in
controlling gene expression.
58
RNAis RNA Interference molecules

• They are noncoding RNAs that regulate (interfere
  with) gene expression at three points
• 1. chromatin modification
• 2. block translation
• 3. mRNA degradation
• Were used originally by cells to fiend off
  viruses

epigenetics
http//www.teachersdomain.org/asset/lsps07_vid_rna
i/
59
Types of RNAis

• MicroRNAs (miRNAs) small single-stranded RNAs
  that interfere with mRNA and translation
• An estimated 1/3 of human genes are
  regulated by miRNAs.
• 2. Small interfering RNAs (siRNAs)
  double-stranded RNA formed when cells cut up
  intruding RNA. siRNAs are involved in formation
  of heterochromatin as well as alter translation.

60
Fig. 18-13
Hairpin
miRNA
Hydrogen bond
Dicer
miRNA
miRNA- protein complex
3?
5?
(a) Primary miRNA transcript
Long RNA precursors fold on themselves and look
like hairpins. The hairpins are cut off and an
enzyme called dicer trims the ends. One strand
becomes a microRNA (miRNA).
Translation blocked
mRNA degraded
(b) Generation and function of miRNAs
61
Small Interfering RNAs (siRNAs)

• Small pieces of double-stranded RNA formed when
  cells cut up intrudingRNA.
• siRNAs are involved in formation of
  heterochromatin as well as alter translation.

62

• Double stranded RNA is introduced into a cell and
  gets chopped up by the enzyme dicer to form
  siRNA.
• siRNA binds to its corresponding mRNA which is
  then cut rendering it inactive.

63

• siRNAs and miRNAs are similar but form from
  different RNA precursors
• Both interfere with gene expression

64
Practical use of RNAis

• RNA interference (RNAi), is being explored by
  researchers as a therapeutic approach to treating
  a host of diseases. A genetic malfunction is
  causing a patient to lose her vision because of
  the over-production of blood vessels in her eyes.
  To treat this genetic malfunctioning, scientists
  attempt to manipulate the mechanism so that genes
  that normally trigger production of blood vessels
  instead do the opposite.

http//www.teachersdomain.org/asset/lsps07_vid_rna
itherapy/
65

• Cancer notes are NOT on the test!

66
CANCER AND GENE EXPRESSIONCancer results from
genetic changes that affect cell cycle control

• Cancer can be caused by mutations to genes that
  regulate cell growth and division
• - mutagens are chemicals, X-rays, tumor
  viruses in animals

67
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68
Fig. 18-21c
EFFECTS OF MUTATIONS
Protein overexpressed
Protein absent
Cell cycle not inhibited
Increased cell division
Cell cycle overstimulated
(c) Effects of mutations
69
Oncogenes and Proto-Oncogenes

• Oncogenes are cancer-causing genes
• Proto-oncogenes are the corresponding normal
  cellular genes that are responsible for normal
  cell growth and division

70
Conversion of a proto-oncogene to an oncogene can
lead to abnormal stimulation of the cell cycle

• Amplification of normal growth-stimulating gene
• Translocation of growth gene under control of a
  more active promoter
• Point mutation in control element or gene itself
  to make a hyperactive or degradation resistant
  growth protein.

71
Fig. 18-20
Proto-oncogene
DNA
Point mutation
Gene amplification
Translocation or transposition
within the gene
within a control element
New promoter
Oncogene
Oncogene
Normal growth- stimulating protein in excess
Normal growth-stimulating protein in excess
Normal growth- stimulating protein in excess
Hyperactive or degradation- resistant protein
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Tumor-Suppressor Genes

• help prevent uncontrolled cell growth
• Tumor-suppressor proteins
• Repair damaged DNA
• Control cell adhesion
• Inhibit the cell cycle (ras and p53)
• Activate suicide genes in apoptosis if DNA cant
  be repaired (p53 protein)

73
How do cancer genes work?

• 30 cancers ras proto-oncogene gene is mutated
• Ras gene codes for a protein that stimulates
  the production of a cell cycle protein
• 50 cancers p53 gene mutated codes for a
  transcription factor for growth-inhibiting
  proteins. These proteins bind to a p21 gene
  whose product binds to CDKs and halt cell cycle.
  It can also activate DNA repair genes or
  suicide genes if DNA cant be repaired.

74
p53 gene and DNA repair
Fig. 18-21b
Protein kinases
2
MUTATION
Defective or missing transcription factor,
such as p53, cannot activate transcription
Active form of p53
3
UV light
DNA damage in genome
1
DNA
Protein that inhibits the cell cycle
(b) Cell cycleinhibiting pathway
75
P53 and suicide genes
76
Multistep Model of Cancer Development

• More than one somatic mutation is needed
• Both alleles must be defective
• In some, genes for telomerase becomes activated
  and cells divided continually

77
Inherited Predisposition and Other Factors
Contributing to Cancer

• Individuals can inherit oncogenes or mutant
  alleles of tumor-suppressor genes
• Inherited mutations in the tumor-suppressor gene
  are common in individuals with colorectal cancer
• Mutations in the BRCA1 or BRCA2 gene are found in
  at least half of inherited breast cancers

78

• Even if you have cancer genes, it does not mean
  you will have cancer.
• Genes can be modified by siRNAs, epigenesis, and
  the environment