1. Eukaryotic Genomes

2. 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

3. 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
10 nm
DNA double helix
Histone
tails
His-
tones
Linker DNA
(“string”)
Nucleosome
(“bead”)
Histone H1
(a) Nucleosomes (10-nm fiber)

4. Higher Levels of DNA Packing
The next level of packing forms the 30-nm chromatin fiber
The 30-nm fiber, in turn forms looped domains, making up a 300-nm fiber
Nucleosome
30 nm
(b) 30-nm fiber
Protein scaffold
300 nm
(c) Looped domains (300-nm fiber)
Loops
Scaffold

5. 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

6. Eukaryotic Gene Regulation
Signal
NUCLEUS
Chromatin
Chromatin modification:
DNA unpacking involving
histone acetylation and
DNA demethlation
Gene
DNA
Gene available
for transcription
RNA
Exon
Transcription
Primary transcript
RNA processing
Transport to cytoplasm
Intron
Cap
mRNA in nucleus
Tail
CYTOPLASM
mRNA in cytoplasm
Degradation
of mRNA
Translation
Polypetide
Cleavage
Chemical modification
Transport to cellular
destination
Active protein
Degradation of protein
Degraded protein
In eukaryotes, gene regulation is more complex
Many key stages of gene expression can be regulated in eukaryotic cells
However, transcriptional controls are still the primary method of gene regulation
But there are also posttranscriptional controls.

7. Eukaryotic Gene Regulation - Transcription
Chromatin modification
Histone modification
DNA methylation
Transcription factors and control Elements
Activators
Repressors

8. 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
10 nm
DNA double helix
Histone
tails
His-
tones
Linker DNA
(“string”)
Nucleosome
(“bead”)
Histone H1
(a) Nucleosomes (10-nm fiber)

9. 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
(a) Histone tails protrude outward from a nucleosome
Chromatin changes
Transcription
RNA processing
mRNA
degradation
Translation
Protein processing
and degradation
DNA
double helix
Amino acids
available
for chemical
modification
Histone
tails

10. Histone Modification
Histone acetylation (COCH3)seems to loosen chromatin structure and thereby enhance transcription
Figure 19.4 b
(b) Acetylation of histone tails promotes loose chromatin structure that permits transcription
Unacetylated histones
Acetylated histones

11. DNA Methylation
Addition of methyl groups to certain bases in DNA is associated with reduced transcription

12. 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)
Proximal
control elements
DNA
Upstream
Promoter
Exon
Intron
Poly-A signal
sequence
Termination
region
Transcription
Downstream
Poly-A
signal
Primary RNA
transcript
(pre-mRNA) 5
Intron RNA
RNA processing:
Cap and tail added;
introns excised and
exons spliced together
Coding segment P G
mRNA
5 Cap
5 UTR
(untranslated
region)
Start
codon
Stop
3 UTR
tail
Chromatin changes
RNA processing
degradation
Translation
Protein processing
and degradation
Cleared 3 end
of primary
transport

13. Transcription Factors
RNA PROCESSING
TRANSLATION
DNA
Pre-mRNA
mRNA
Ribosome
Polypeptide T A
TATA box
Start point
Template
DNA strand 5 3
Transcription
factors
Promoter
RNA polymerase II
Transcription factors
RNA transcript
Transcription initiation complex
Eukaryotic promoters 1
Several transcription 2
Additional transcription 3
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.
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.

14. Regulation of Transcription Initiation
In eukaryotes, regulatory molecules called activators can bind to regulatory enhancers
Distal control
element
Activators
Enhancer
Promoter
Gene
TATA
box
General
transcription
factors
DNA-bending
protein
Group of
Mediator proteins
RNA
Polymerase II
RNA synthesis
Transcription
Initiation complex
Chromatin changes
RNA processing
degradation
mRNA
Translation
Protein processing
and degradation
A DNA-bending protein
brings the bound activators
closer to the promoter.
Other transcription factors,
mediator proteins, and RNA
polymerase are nearby. 2
Activator proteins bind
to distal control elements
grouped as an enhancer in
the DNA. This enhancer has
three binding sites. 1
The activators bind to
certain general transcription
factors and mediator
proteins, helping them form
an active transcription
initiation complex on the promoter. 3

15. 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
Control
elements
Albumin
gene
Crystallin
Liver cell
nucleus
Lens cell
Available
activators
expressed
gene not
Crystallin gene
not expressed
(a)
(b)

16. 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.

17. 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

18. 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 or

19. Blockage of translation
mRNA Degradation
RNA interference by single-stranded microRNAs (miRNAs) can lead to degradation of an mRNA or block its translation 5
Chromatin changes
Transcription
RNA processing
mRNA
degradation
Translation
Protein processing
and degradation
Degradation of mRNA OR
Blockage of translation
Target mRNA
miRNA
Protein
complex
Dicer
Hydrogen
bond
The micro-
RNA (miRNA)
precursor folds
back on itself,
held together
by hydrogen
bonds. 1 2
An enzyme
called Dicer moves
along the double-
stranded RNA,
cutting it into
shorter segments.
One strand of
each short double-
stranded RNA is
degraded; the other
strand (miRNA) then
associates with a
complex of proteins. 3
The bound
miRNA can base-pair
with any target
mRNA that contains
the complementary
sequence. 4
The miRNA-protein
complex prevents gene
expression either by
degrading the target
mRNA or by blocking
its translation.

20. 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
Chromatin changes
Transcription
RNA processing
mRNA
degradation
Translation
Protein processing
and degradation
Ubiquitin
Protein to
be degraded
Ubiquinated
protein
Proteasome
and ubiquitin
to be recycled
Protein
fragments
(peptides)
Protein entering a
proteasome
Multiple ubiquitin mol-
ecules are attached to a protein
by enzymes in the cytosol. 1
The ubiquitin-tagged protein
is recognized by a proteasome,
which unfolds the protein and
sequesters it within a central cavity. 2
Enzymatic components of the
proteasome cut the protein into
small peptides, which can be
further degraded by other
enzymes in the cytosol. 3

21. 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.

22. 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

23. 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

24. 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.
EFFECTS OF MUTATIONS
Protein
overexpressed
Cell cycle
overstimulated
Increased cell
division
Cell cycle not
inhibited
Protein absent
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).
(c)

25. 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
Point mutation
within a control
element
within the gene
Oncogene
Normal growth-stimulating
protein in excess
Hyperactive or
degradation-
resistant protein
New
promoter

26. 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
(a) Cell cycle–stimulating pathway.
This pathway is triggered by a growth
factor that binds to its receptor in the
plasma membrane. The signal is relayed to
a G protein called Ras. Like all G proteins, Ras
is active when GTP is bound to it. Ras passes
the signal to a series of protein kinases.
The last kinase activates 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. 1 2 4 3 5
GTP
Ras
Hyperactive
Ras protein
(product of
oncogene)
issues signals
on its own
NUCLEUS
Gene expression
Protein that
stimulates
the cell cycle P
MUTATION
DNA
G protein 3
Protein kinases
(phosphorylation
cascade) 4 2
Receptor
Transcription
factor (activator) 5

27. 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. UV
light
DNA
Defective or
missing
transcription
factor, such as
p53, cannot
activate
MUTATION
Protein that
inhibits
the cell cycle
pathway, DNA damage is an intracellular
signal that is passed via protein kinases
and leads to activation of 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.
(b) Cell cycle–inhibiting pathway. In this 1 3 2
Protein kinases 2 3
Active
form
of p53
DNA damage
in genome 1

28. CANCER IS CAUSED BY MUTATIONS IN SEVERAL GENES
More than one somatic mutation is generally needed to produce a full-fledged 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
Normal
epithelium
Hyperpro-
liferative
Early
benign
polyp
Intermediate
benign polyp
Late
Carcinoma
Metastasis
APC
Oncogene
K-ras
DCC
p53
Other
mutations
Loss of APC
Mutation of K-ras
And DCC
Mutation
of p53

29. 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
Small benign
growth (polyp)
Larger benign
growth (adenoma)
Malignant tumor
(carcinoma)
2 Activation of
ras oncogene
3 Loss of
tumor-
suppressor
gene DCC
4 Loss of
tumor-suppressor
gene p53
5 Additional
mutations
1 Loss of tumor-
gene APC (or
other)
Colorectal cancer - A multistep model

30. 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

31. Cancer
Since cancer-causing mutations accumulate over time, cancer risk increases with age.