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Eukaryotic Genomes
Eukaryotic Genomes
•
In eukaryotes, the DNA-protein complex, called chromatin is ordered
into higher structural levels than the DNA-protein complex in
prokaryotes
• Chromatin structure is based on successive levels of DNA packing
• Eukaryotic DNA is precisely combined with a large amount of
protein
• Eukaryotic chromosomes contain an enormous amount of DNA
relative to their condensed length
Nucleosomes: “Beads on a String”
•
Proteins called histones are responsible for the first level of DNA
packing in chromatin
•
Histones bind tightly to DNA and their association seems to remain
intact throughout the cell cycle
•
In electron micrographs unfolded chromatin has the appearance of
beads on a string
•
Each “bead” is a nucleosome - the basic unit of DNA packing
2 nm
DNA double helix
Histones
Histone
tails
Histone H1
Linker DNA
(“string”)
Nucleosome
(“bead”)
(a) Nucleosomes (10-nm fiber)
10 nm
Higher Levels of DNA Packing
• The next level of packing forms the 30-nm chromatin
fiber
30 nm
Nucleosome
(b) 30-nm fiber
• The 30-nm fiber, in turn forms looped domains,
making up a 300-nm fiber
Protein scaffold
Loops
300 nm
(c) Looped domains (300-nm fiber)
Scaffold
Chromatin Condensation
• In interphase cells most chromatin is in the highly
extended form called euchromatin
• In a mitotic chromosome the looped domains
themselves coil and fold forming the characteristic
metaphase chromosome
700 nm
1,400 nm
(d) Metaphase chromosome
Eukaryotic Gene Regulation
Signal
NUCLEUS
• In eukaryotes, gene regulation
is more complex
Chromatin
Chromatin modification:
DNA unpacking involving
histone acetylation and
DNA demethlation
DNA
Gene available
for transcription
• Many key stages of gene
expression can be regulated
in eukaryotic cells
Gene
Transcription
RNA
Primary transcript
Intron
RNA processing
Tail
Cap
• However, transcriptional
controls are still the primary
method of gene regulation
• But there are also
posttranscriptional controls.
Exon
mRNA in nucleus
Transport to cytoplasm
CYTOPLASM
mRNA in cytoplasm
Degradation
of mRNA
Translation
Polypetide
Cleavage
Chemical modification
Transport to cellular
destination
Active protein
Degradation of protein
Degraded protein
Eukaryotic Gene Regulation - Transcription
•
Chromatin modification
•
Histone modification
•
DNA methylation
•
Transcription factors and control Elements
• Activators
• Repressors
Eukaryotic Gene Regulation - Transcripton
• Coiling of DNA within the nucleus help regulate gene
transcription in eukaryotes.
• Studies have shown that transcription factors are unable to
bind to promoters located in regions of DNA that are coiled
around the histones of a nucleosome:
2 nm
DNA double helix
Histones
Histone
tails
Histone H1
Linker DNA
(“string”)
Nucleosome
(“bead”)
(a) Nucleosomes (10-nm fiber)
10 nm
Chromatin Modification
•
Chromatin-modifying enzymes provide initial control of gene expression
•
By making a region of DNA either more or less able to bind the transcription
machinery
•
Chemical modification of histone tails can affect the configuration of chromatin
and thus gene expression
Chromatin changes
Transcription
RNA processing
mRNA
degradation
Translation
Protein processing
and degradation
Histone
tails
DNA
double helix
Amino acids
available
for chemical
modification
(a) Histone tails protrude outward from a nucleosome
Histone Modification
• Histone acetylation (COCH3)seems to loosen
chromatin structure and thereby enhance transcription
Unacetylated histones
Figure 19.4 b
Acetylated histones
(b) Acetylation of histone tails promotes loose chromatin structure that
permits transcription
DNA Methylation
• Addition of methyl groups to certain bases
in DNA is associated with reduced transcription
Eukaryotic Gene Regulation – Control Elements
•
Associated with most eukaryotic genes are multiple control elements - segments of
noncoding DNA that help regulate transcription by binding certain proteins
•
Distal control elements, groups of which are called enhancers may be far from a gene
•
Regulatory molecules called activators can bind to regulatory enhancers to facilitate
transcription factors
Enhancer
(distal control elements)
Poly-A signal Termination
sequence
region
Proximal
control elements
Exon
Intron
Exon
Intron
Exon
DNA
Downstream
Upstream
Promoter
Chromatin changes
Primary RNA
transcript 5
(pre-mRNA)
Transcription
Exon
Intron
Exon
mRNA
degradation
Intron RNA
Cleared 3 end
of primary
transport
Coding segment
Translation
Protein processing
and degradation
Exo
n
RNA processing:
Cap and tail added;
introns excised and
exons spliced together
Transcription
RNA processing
Intron
Poly-A
signal
mRNA
G P P P
5 Cap 5 UTR
(untranslated
region)
Start
codon
Stop
codon
3 UTR Poly-A
(untranslated tail
region)
Transcription Factors
TRANSCRIPTION
•
•
•
•
To initiate transcription, eukaryotic
RNA polymerase requires the
assistance of proteins called
transcription factors
1 Eukaryotic promoters
DNA
RNA PROCESSING
Pre-mRNA
mRNA
Ribosome
TRANSLATION
Polypeptide
Promoter
5
3
A T A T T T
T
TATA box
General transcription factors are
essential for the transcription of all
protein-coding genes.
Only a few general transcription
factors independently bind to a
DNA sequence such as the TATA
box within the promoter.
Others in the initiation complex
are involved in protein-protein
interactions, binding each other
and RNA polymerase II.
3
5
T A T A A A A
Start point
Template
DNA strand
Several transcription
factors
2
Transcription
factors
5
3
3
5
3 Additional transcription
factors
RNA polymerase II
Transcription factors
5
3
3
5
5
RNA transcript
Transcription initiation complex
Regulation of Transcription Initiation
•
In eukaryotes, regulatory molecules called activators can bind to regulatory
enhancers
Distal control
element
Activators
Enhancer
1 Activator proteins bind
to distal control elements
grouped as an enhancer in
the DNA. This enhancer has
three binding sites.
Promoter
Gene
TATA
box
General
transcription
factors
DNA-bending
protein
Group of
Mediator proteins
2 A DNA-bending protein
brings the bound activators
closer to the promoter.
Other transcription factors,
mediator proteins, and RNA
polymerase are nearby.
RNA
Polymerase II
Chromatin changes
3 The activators bind to
certain general transcription
factors and mediator
proteins, helping them form
an active transcription
initiation complex on the promoter.
Transcription
RNA processing
mRNA
degradation
RNA
Polymerase II
Translation
Protein processing
and degradation
Transcription
Initiation complex
RNA synthesis
Combinatorial Control of Gene Activation
• A particular combination of control elements will be able to
activate transcription only when the appropriate activator
proteins are present
Enhancer Promoter
Albumin
gene
Control
elements
Crystallin
gene
Liver cell
nucleus
Available
activators
Albumin
gene
expressed
Crystallin gene
not expressed
(a) Liver cell
Lens cell
nucleus
Available
activators
Albumin
gene not
expressed
Crystallin gene
expressed
(b) Lens cell
Repressors
• Some specific transcription factors function as
repressors proteins inhibit expression of a particular
gene
• Eukaryotic repressors can cause inhibition of gene
expression by blocking the binding of activators to
their control elements or to components of the
transcription machinery or by turning off transcription
even in the presence of activators.
Post-transcriptional control
• Although less common than transcriptional control of
gene expression, various types of posttranscriptional
control may also occur in eukaryotes
• An increasing number of examples are being found of
regulatory mechanisms that operate at various stages
after transcription
•
mRNA splicing
•
mRNA degradation – miRNA
•
Protein degradation
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
Chromatin changes
Transcription
RNA processing
mRNA
degradation
Translation
Protein processing
and degradation
Exons
DNA
Primary
RNA
transcript
RNA splicing
mRNA
or
mRNA Degradation
• RNA interference by single-stranded microRNAs (miRNAs)
can lead to degradation of an mRNA or block its translation
1 The microRNA (miRNA)
precursor folds
back on itself,
held together
by hydrogen
bonds.
22 An enzyme
called Dicer moves
along the doublestranded RNA,
cutting it into
shorter segments.
3 One strand of
each short doublestranded RNA is
degraded; the other
strand (miRNA) then
associates with a
complex of proteins.
4 The bound
miRNA can base-pair
with any target
mRNA that contains
the complementary
sequence.
55 The miRNA-protein
complex prevents gene
expression either by
degrading the target
mRNA or by blocking
its translation.
Chromatin changes
Transcription
RNA processing
mRNA
degradation
Translation
Protein processing
and degradation
Protein
complex
Dicer
Degradation of mRNA
OR
miRNA
Target mRNA
Hydrogen
bond
Blockage of translation
Post translation
•
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
3 Enzymatic components of the
2 The ubiquitin-tagged protein
1 Multiple ubiquitin molChromatin changes
ecules are attached to a protein
by enzymes in the cytosol.
is recognized by a proteasome,
which unfolds the protein and
sequesters it within a central cavity.
proteasome cut the protein into
small peptides, which can be
further degraded by other
enzymes in the cytosol.
Transcription
RNA processing
mRNA
degradation
Proteasome
and ubiquitin
to be recycled
Ubiquitin
Translation
Proteasome
Protein processing
and degradation
Protein to
be degraded
Ubiquinated
protein
Protein entering a
proteasome
Protein
fragments
(peptides)
Cancer Biology
•
Mutations are changes in the genetic material of a cell
•
Mutations can occur during DNA replication, recombination, or repair
•
Cancer results from genetic changes that affect cell cycle control
•
Mutation of genes controlling cell division can lead to cancer
•
The gene regulation systems can go wrong due to
•
Chromosomal alterations - translocations
•
Point mutations - Carcinogens
•
Carcinogens are chemical or physical agents that interact with DNA to
cause mutations leading to cancer
•
•
Radiation - X-rays and ultraviolet light
•
Chemicals – arsenic, asbestos, benzene, ethanol, formaldehyde,
gasoline
Tumor viruses - transform cells into cancer cells through the integration
of viral nucleic acid into host cell DNA.
Cancer
•
Cancer results from genetic changes that affect cell cycle control
•
Cancer is the unregulated cell growth and division forming a cluster of
cells forming a tumor that constantly expands in size
•
Cells that leave the tumor, spread to other parts of the body, and form
new tumors are called metastases
Genes Associated with Cancer
•
The genes that normally regulate cell growth and division during the
cell cycle include genes for
•
Growth Factors
•
GF Receptors
•
Intracellular molecules of signaling pathways
•
Mutations altering any of these genes in somatic cells can lead to
cancer
•
Most human cancers result from mutations in one of two types of
growth-regulating genes:
•
Proto-oncogenes code for proteins involved in stimulating cell division
•
Tumor-suppressor genes code for proteins involved in inhibiting cell
division
Growth Factors and Cancer
• Proto-oncogenes code for proteins involved in stimulating cell
division (e.g. growth factors, growth factor receptors, cyclins)
• Mutated proto-oncogenes that stimulate a cell to divide when it
shouldn’t are called oncogenes (cancer-causing genes).
• Tumor-suppressor genes code for proteins involved in inhibiting cell
division
• Mutated tumor-suppressor genes that do not inhibit cell division
when they should can also cause cancer.
(c) Effects of mutations. Increased
cell division, possibly leading to
cancer, can result if the cell cycle is
overstimulated, as in (a), or not
inhibited when it normally would be,
as in (b).
EFFECTS OF MUTATIONS
Protein
overexpressed
Cell cycle
overstimulated
Protein absent
Increased cell
division
Cell cycle not
inhibited
Cancer
•
Proto-oncogenes are normal cellular genes that code for proteins that
stimulate normal cell growth and division
•
Oncogenes are cancer-causing genes
•
A DNA change that makes a proto-oncogene excessively active, converts it to
an oncogene, which may promote excessive cell division and cancer
Proto-oncogene
DNA
Translocation or transposition:
gene moved to new locus,
under new controls
Gene amplification:
multiple copies of the gene
New
promoter
Normal growth-stimulating
protein in excess
Normal growth-stimulating
protein in excess
Point mutation
within a control
element
Point mutation
within the gene
Oncogene
Oncogene
Normal growth-stimulating
protein in excess
Hyperactive or
degradationresistant protein
Ras protein
•
The Ras protein, encoded by the ras gene, is a G protein that relays a signal
from a growth factor receptor to a cascade of protein kinases
•
Many ras oncogenes have a mutation that leads to a hyperactive Ras protein
that issues signals on its own, resulting in excessive cell division
1
Growth
factor
MUTATION
Ras
GTP
3
(a) Cell cycle–stimulating pathway.
This pathway is triggered by 1 a growth
factor that binds to 2 its receptor in the
plasma membrane. The signal is relayed to 3
a G protein called Ras. Like all G proteins, Ras
is active when GTP is bound to it. Ras passes
the signal to 4 a series of protein kinases.
The last kinase activates 5 a transcription
activator that turns on one or more genes
for proteins that stimulate the cell cycle. If a
mutation makes Ras or any other pathway
component abnormally active, excessive cell
division and cancer may result.
P
Ras
P
P
P
P
P
GTP
4
2
Receptor
G protein
Hyperactive
Ras protein
(product of
oncogene)
issues signals
on its own
Protein kinases
(phosphorylation
cascade)
NUCLEUS
5
Transcription
factor (activator)
DNA
Gene expression
Protein that
stimulates
the cell cycle
p53 Gene - Tumor-suppressor Gene
•
p53 inhibits cell division when DNA is damaged by stimulating
transcription of p21. The p21 protein then binds to cyclins and
prevents them from binding with Cdk
•
Abnormal p53 fails to stop division in cells with damaged DNA. If
genetic damage accumulates as the cell continues to divide, the cell
can turn cancerous.
(b) Cell cycle–inhibiting pathway. In this
pathway, 1 DNA damage is an intracellular
signal that is passed via 2 protein kinases
and leads to activation of 3 p53. Activated
p53 promotes transcription of the gene for a
protein that inhibits the cell cycle. The
resulting suppression of cell division ensures
that the damaged DNA is not replicated.
Mutations causing deficiencies in any
pathway component can contribute to the
development of cancer.
2
Protein kinases
MUTATION
3
UV
light
1
Active
form
of p53
DNA damage
in genome
DNA
Protein that
inhibits
the cell cycle
Defective or
missing
transcription
factor, such as
p53, cannot
activate
transcription
Cancer
•
More than one somatic mutation is generally needed to produce a fullfledged cancer cell
•
About a half dozen DNA changes must occur for a cell to become fully
cancerous
•
These changes usually include at least one active oncogene and mutation
or loss of several tumor-suppressor genes
CANCER IS CAUSED BY MUTATIONS IN SEVERAL GENES
Tumor
suppressor
APC
K-ras
Loss of APC
Normal
epithelium
Tumor
suppressor
Oncogene
Hyperproliferative
epithelium
DCC
Mutation of K-ras
And DCC
Early
benign
polyp
Intermediate
benign polyp
Tumor
suppressor
p53
Mutation
of p53
Late
benign
polyp
Carcinoma
Other
mutations
Metastasis
Multistep Model of Cancer Development
• Colorectal cancer, with 135,000 new cases and 60,000 deaths in
the United States each year, illustrates a multistep cancer path
Colon
Colon wall
Normal colon
epithelial cells
•Colorectal cancer - A multistep model
1 Loss of tumorsuppressor
gene APC (or
other)
4 Loss of
tumor-suppressor
gene p53
2 Activation of
ras oncogene
Small benign
growth (polyp)
3 Loss of
tumorsuppressor
gene DCC
Larger benign
growth (adenoma)
5 Additional
mutations
Malignant tumor
(carcinoma)
Inherited Predisposition to Cancer
• The fact that multiple genetic changes are required to produce a
cancer cell helps explain the predispositions to cancer that run
in some families
• Individuals who inherit a mutant oncogene or tumor-suppressor
allele have an increased risk of developing certain types of
cancer
Cancer
• Since cancer-causing mutations accumulate
over time, cancer risk increases with age.