Chapter 18-Gene Expression

1
Chapter 18-Gene Expression

• Prokaryotes and eukaryotes alter gene expression
  in response to their changing environment
• In multicellular eukaryotes, gene expression
  regulates development and is responsible for
  differences in cell types
• RNA molecules play many roles in regulating gene
  expression in eukaryotes

2
Bacteria often respond to environmental change by
regulating transcription

• Natural selection has favored bacteria that
  produce only the products needed by that cell
• A cell can regulate the production of enzymes by
  feedback inhibition or by gene regulation
• Gene expression in bacteria is controlled by the
  operon model

3
Figure 18.2
Precursor
Feedbackinhibition
trpE gene
Enzyme 1
trpD gene
Regulationof geneexpression
Enzyme 2
trpC gene
?
trpB gene
?
Enzyme 3
trpA gene
Tryptophan
(b)
(a)
Regulation of enzymeactivity
Regulation of enzymeproduction
4
Operons The Basic Concept

• A cluster of functionally related genes can be
  under coordinated control by a single on-off
  switch
• The regulatory switch is a segment of DNA
  called an operator usually positioned within the
  promoter
• An operon is the entire stretch of DNA that
  includes the operator, the promoter, and the
  genes that they control

5

• The operon can be switched off by a repressor
• The repressor binds to the operator and blocks
  RNA polymeraseno transcription
• The repressor is the product of a separate
  regulatory gene
• The repressor can be in an active or inactive
  form
• A corepressor cooperates with a repressor protein
  to switch an operon off
• For example, E. coli can synthesize the amino
  acid tryptophan

6

• By default the trp operon is on and the genes for
  tryptophan synthesis are transcribed
• When tryptophan is present, it binds to the trp
  repressor protein, which turns the operon off
• The repressor is active only in the presence of
  its corepressor tryptophan thus the trp operon
  is turned off (repressed) if tryptophan levels
  are high

7
Figure 18.3
trp operon
Promoter
Promoter
Genes of operon
DNA
trpE
trpD
trpC
trpA
trpR
trpB
Operator
Regulatory gene
RNApolymerase
Start codon
Stop codon
3?
mRNA 5?
mRNA
5?
E
D
C
B
A
Protein
Inactive repressor
Polypeptide subunits that make upenzymes for
tryptophan synthesis
(a) Tryptophan absent, repressor inactive, operon
on
DNA
No RNAmade
mRNA
Protein
Activerepressor
Tryptophan (corepressor)
(b) Tryptophan present, repressor active, operon
off
8
Repressible and Inducible Operons Two Types of
Negative Gene Regulation

• A repressible operon is one that 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

9

• The lac operon is an inducible operon and
  contains genes that code for enzymes used in the
  hydrolysis and metabolism of lactose
• By itself, the lac repressor is active and
  switches the lac operon off
• A molecule called an inducer inactivates the
  repressor to turn the lac operon on

10
Figure 18.4
Regulatorygene
Promoter
Operator
DNA
DNA
lacZ
lacI
NoRNAmade
3?
mRNA
RNApolymerase
5?
Activerepressor
Protein
(a) Lactose absent, repressor active, operon off
lac operon
lacI
lacZ
lacY
lacA
DNA
RNA polymerase
3?
mRNA
mRNA 5?
5?
?-Galactosidase
Permease
Transacetylase
Protein
Inactiverepressor
Allolactose(inducer)
(b) Lactose present, repressor inactive, operon on
11

• Inducible enzymes usually function in catabolic
  pathways their synthesis is induced by a
  chemical signal
• Repressible enzymes usually function in anabolic
  pathways their synthesis is repressed by high
  levels of the end product
• 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 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 (cAMP)
• Activated CAP attaches to the promoter of the lac
  operon and increases the affinity of RNA
  polymerase, thus accelerating transcription

13

• 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

14
Figure 18.5
Promoter
DNA
lacZ
lacI
Operator
CAP-binding site
RNApolymerasebinds andtranscribes
ActiveCAP
cAMP
Inactive lacrepressor
InactiveCAP
Allolactose
(a)
Lactose present, glucose scarce (cAMP level
high)abundant lac mRNA synthesized
Promoter
DNA
lacZ
lacI
Operator
CAP-binding site
RNApolymerase lesslikely to bind
InactiveCAP
Inactive lacrepressor
Lactose present, glucose present (cAMP level
low)little lac mRNA synthesized
(b)
15
Eukaryotic gene expression

• All organisms must regulate which genes are
  expressed at any given time
• Almost all the cells in an organism are
  genetically identical.
• Differences between cell types result from
  differential gene expression, the expression of
  different genes by cells with the same genome.
• Errors in gene expression can lead to diseases
  including cancer.
• Gene expression is regulated at many stages.

16
Figure 18.6
Signal
NUCLEUS
Chromatin
Chromatin modificationDNA unpacking
involvinghistone acetylation andDNA
demethylation
DNA
Gene availablefor transcription
Gene
Transcription
Exon
RNA
Primary transcript
Intron
RNA processing
Tail
mRNA in nucleus
Cap
Transport to cytoplasm
CYTOPLASM
mRNA in cytoplasm
Translation
Degradationof mRNA
Polypeptide
Protein processing, suchas cleavage and
chemical modification
Active protein
Degradationof protein
Transport to cellulardestination
Cellular function (suchas enzymatic
activity,structural support)
17
Regulation of Chromatin Structure

• Genes within highly packed heterochromatin are
  usually not expressed
• Chemical modifications to histones and DNA of
  chromatin influence both chromatin structure and
  gene expression
• The histone code hypothesis proposes that
  specific combinations of modifications, as well
  as the order in which they occur, help determine
  chromatin configuration and influence
  transcription

18
Histone Modifications

• In histone acetylation, acetyl groups are
  attached to positively charged lysines in histone
  tails
• This loosens chromatin structure, thereby
  promoting the initiation of transcription
• The addition of methyl groups (methylation) can
  condense chromatin the addition of phosphate
  groups (phosphorylation) next to a methylated
  amino acid can loosen chromatin

19
Figure 18.7
Histone tails
DNA double helix
Amino acidsavailablefor chemicalmodification
Nucleosome(end view)
(a) Histone tails protrude outward from a
nucleosome
Unacetylated histones
Acetylated histones
(b)
Acetylation of histone tails promotes loose
chromatinstructure that permits transcription
20
DNA Methylation

• DNA methylation, the addition of methyl groups to
  certain bases in DNA, is associated with reduced
  transcription in some species
• DNA methylation can cause long-term inactivation
  of genes in cellular differentiation
• In genomic imprinting, methylation regulates
  expression of either the maternal or paternal
  alleles of certain genes at the start of
  development

21
Organization of a Typical Eukaryotic Gene

• Associated with most eukaryotic genes are
  multiple control elements, segments of noncoding
  DNA that serve as binding sites for transcription
  factors that help regulate transcription
• Control elements and the transcription factors
  they bind are critical to the precise regulation
  of gene expression in different cell types

22
Figure 18.8-3
Enhancer(distal controlelements)
Proximalcontrolelements
Poly-Asignalsequence
Transcriptionterminationregion
Transcriptionstart site
Exon
Intron
Exon
Exon
Intron
DNA
Upstream
Downstream
Promoter
Poly-Asignal
Transcription
Exon
Intron
Intron
Exon
Exon
Primary RNAtranscript(pre-mRNA)
Cleaved3? end ofprimarytranscript
5?
RNA processing
Intron RNA
Coding segment
mRNA
3?
P
P
G
P
AAA ??? AAA
Startcodon
Stopcodon
Poly-Atail
5? Cap
5? UTR
3? UTR
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The Roles of Transcription Factors

• To initiate transcription, eukaryotic RNA
  polymerase requires the assistance of proteins
  called transcription factors
• General transcription factors are essential for
  the transcription of all protein-coding genes
• In eukaryotes, high levels of transcription of
  particular genes depend on control elements
  interacting with specific transcription factors

24
Enhancers and Specific Transcription Factors

• Proximal control elements are located close to
  the promoter
• Distal control elements, groupings of which are
  called enhancers, may be far away from a gene or
  even located in an intron
• An activator is a protein that binds to an
  enhancer and stimulates transcription of a gene
• Activators have two domains, one that binds DNA
  and a second that activates transcription
• Bound activators facilitate a sequence of
  protein-protein interactions that result in
  transcription of a given gene

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Figure 18.9
Activationdomain
DNA-bindingdomain
DNA
26
Figure 18.10-3
Promoter
Activators
Gene
DNA
Distal controlelement
TATA box
Enhancer
Generaltranscriptionfactors
DNA-bendingprotein
Group of mediator proteins
RNApolymerase II
RNApolymerase II
Transcriptioninitiation complex
RNA synthesis
27
Figure 18.11
Enhancer
Promoter
Controlelements
Albumin gene
Crystallingene
LENS CELLNUCLEUS
LIVER CELLNUCLEUS
Availableactivators
Availableactivators
Albumin genenot expressed
Albumin geneexpressed
Crystallin genenot expressed
Crystallin geneexpressed
(a) Liver cell
(b) Lens cell
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RNA Processing

• In alternative RNA splicing, different mRNA
  molecules are produced from the same primary
  transcript, depending on which RNA segments are
  treated as exons and which as introns
• Significantly expands the eukaryote genome and
  greatly multiplies the number of human proteins
  that can be made.

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Figure 18.13
Exons
DNA
4
1
2
3
5
Troponin T gene
PrimaryRNAtranscript
3
5
1
2
4
RNA splicing
or
mRNA
2
3
5
5
1
1
2
4
30
Mechanisms of Post-Transcriptional Regulation

• Transcription alone does not account for gene
  expression
• Regulatory mechanisms can operate at various
  stages after transcription
• Such mechanisms allow a cell to fine-tune gene
  expression rapidly in response to environmental
  changes

31
Figure 18.6
Signal
NUCLEUS
Chromatin
Chromatin modificationDNA unpacking
involvinghistone acetylation andDNA
demethylation
DNA
Gene availablefor transcription
Gene
Transcription
Exon
RNA
Primary transcript
Intron
RNA processing
Tail
mRNA in nucleus
Cap
Transport to cytoplasm
CYTOPLASM
mRNA in cytoplasm
Translation
Degradationof mRNA
Polypeptide
Protein processing, suchas cleavage and
chemical modification
Active protein
Degradationof protein
Transport to cellulardestination
Cellular function (suchas enzymatic
activity,structural support)
32
1. mRNA Degradation

• The life span of mRNA molecules in the cytoplasm
  is a key to determining protein synthesis
• Eukaryotic mRNA is more long lived than
  prokaryotic mRNA
• Nucleotide sequences that influence the lifespan
  of mRNA in eukaryotes reside in the untranslated
  region (UTR) at the 3? end of the molecule

33
2. Initiation of Translation

• The initiation of translation of selected mRNAs
  can be blocked by regulatory proteins that bind
  to sequences or structures of the mRNA
• Alternatively, translation of all mRNAs in a
  cell may be regulated simultaneously
• For example, translation initiation factors are
  simultaneously activated in an egg following
  fertilization

34
3-4. Protein Processing and Degradation

• After translation, various types of protein
  processing, including cleavage and the addition
  of chemical groups, are subject to control
• Proteasomes are giant protein complexes that bind
  protein molecules and degrade them

35
Figure 18.14
Proteasomeand ubiquitinto be recycled
Ubiquitin
Proteasome
Ubiquitinatedprotein
Proteinfragments(peptides)
Protein tobe degraded
Protein enteringa proteasome
36
Concept 18.3 Noncoding RNAs play multiple roles
in controlling gene expression

• Only a small fraction of DNA codes for proteins,
  and a very small fraction of the
  non-protein-coding DNA consists of genes for RNA
  such as rRNA and tRNA
• A significant amount of the genome may be
  transcribed into noncoding RNAs (ncRNAs)
• Noncoding RNAs regulate gene expression at two
  points mRNA translation and chromatin
  configuration

37
Effects on mRNAs by MicroRNAs and Small
Interfering RNAs

• MicroRNAs (miRNAs) are small single-stranded RNA
  molecules that can bind to mRNA
• These can degrade mRNA or block its translation

38
Figure 18.15
Hairpin
Hydrogenbond
miRNA
Dicer
5?
3?
(a) Primary miRNA transcript
miRNA
miRNA-proteincomplex
mRNA degraded
Translation blocked
(b) Generation and function of miRNAs
39

• The phenomenon of inhibition of gene expression
  by RNA molecules is called RNA interference
  (RNAi)
• RNAi is caused by small interfering RNAs (siRNAs)
• siRNAs and miRNAs are similar but form from
  different RNA precursors

40
Chromatin Remodeling and Effects on Transcription
by ncRNAs

• In some yeasts siRNAs play a role in
  heterochromatin formation and can block large
  regions of the chromosome
• Small ncRNAs called piwi-associated RNAs (piRNAs)
  induce heterochromatin, blocking the expression
  of parasitic DNA elements in the genome, known as
  transposons
• RNA-based mechanisms may also block transcription
  of single genes

41
Cancer and Gene Regulation

• Oncogenes are cancer-causing genes.
• Proto-oncogenes are the corresponding normal
  cellular genes that are responsible for normal
  cell growth and division.
• Conversion of a proto-oncogene to an oncogene can
  lead to abnormal stimulation of the cell cycle.

42
Cancer Gene Regulation

• Gene regulation systems go wrong by one of the
  following
• Movement of genes within a genome
  (translocation)
• Amplification of proto-oncogenes
• Point mutations in a control element or in the
  proto-oncogenes

43

Proto-Oncogene to Oncogene
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
44
Tumor-Suppressor Genes

• Tumor-suppressor genes help prevent uncontrolled
  cell growth.
• Mutations that decrease protein products of
  tumor-suppressor genes may contribute to cancer
  onset.
• Tumor-suppressor proteins
• Repair damaged DNA control cell adhesion.
• Inhibit the cell cycle in the cell-signaling
  pathway.

45
The Multistep Model of Cancer Development

• Multiple mutations are generally needed for
  full-fledged cancer thus the incidence increases
  with age.
• At the DNA level, a cancerous cell is usually
  characterized by at least one active oncogene and
  the mutation of several tumor-suppressor genes.

46
Multi-Step Model of Cancer Development
Colon
EFFECTS OF MUTATIONS
Loss of tumor-suppressor gene p53
4
Loss of tumor- suppressor gene APC (or other)
Activation of ras oncogene
1
2
Colon wall
Loss of tumor-suppressor gene DCC
Additional mutations
5
3
Malignant tumor (carcinoma)
Larger benign growth (adenoma)
Small benign growth (polyp)
Normal colon epithelial cells
47
Cancer Signaling

• A clear example of external signals is
  density-dependent inhibition, in which crowded
  cells stop dividing
• Most animal cells also exhibit anchorage
  dependence, in which they must be attached to a
  substratum in order to divide
• Cancer cells exhibit neither density-dependent
  inhibition nor anchorage dependence

48
Figure 12.19
Anchorage dependence
Density-dependent inhibition
Density-dependent inhibition
20 ?m
20 ?m
(a) Normal mammalian cells
(b) Cancer cells
49
Cancer Cells Cell cycle control

• Cancer cells do not respond normally to the
  bodys control mechanisms
• Cancer cells may not need growth factors to grow
  and divide
• They may make their own growth factor
• They may convey a growth factors signal without
  the presence of the growth factor
• They may have an abnormal cell cycle control
  system

50

• A normal cell is converted to a cancerous cell by
  a process called transformation
• Cancer cells that are not eliminated by the
  immune system, form tumors, masses of abnormal
  cells within otherwise normal tissue
• If abnormal cells remain at the original site,
  the lump is called a benign tumor
• Malignant tumors invade surrounding tissues and
  can metastasize, exporting cancer cells to other
  parts of the body, where they may form additional
  tumors

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Figure 12.20
Lymph vessel
Tumor
Bloodvessel
Cancercell
Glandulartissue
Metastatictumor
A tumor growsfrom a singlecancer cell.
Cancer cells invade neighboringtissue.
Cancer cells spreadthrough lymph andblood
vessels to other parts of the body.
Cancer cells may survive and establisha new
tumor in another part of the body.
4
3
2
1
52

• Recent advances in understanding the cell cycle
  and cell cycle signaling have led to advances in
  cancer treatment