1. Eukaryotic Genomes: Organization, Regulation, and Evolution
Chapter 19

2. The BIG Questions… How are genes turned on & off in eukaryotes?
How do cells with the same genes differentiate to perform completely different, specialized functions? 2

3. Organization

4. Prokaryote vs. eukaryote genome
Prokaryotes
small size of genome
circular molecule of naked DNA
DNA is readily available to RNA polymerase
control of transcription by regulatory proteins
operon system
most of DNA codes for protein or RNA
no introns, small amount of non-coding DNA
regulatory sequences: promoters, operators
prokaryotes use operons to regulate gene transcription, however eukaryotes do not.
since transcription & translation are fairly simultaneous there is little opportunity to regulate gene expression after transcription, so control of genes in prokaryotes really has to be done by turning transcription on or off. 4

5. Prokaryote vs. eukaryote genome
Eukaryotes
much greater size of genome
how does all that DNA fit into nucleus?
DNA packaged in chromatin fibers
regulates access to DNA by RNA polymerase
cell specialization
need to turn on & off large numbers of genes
most of DNA does not code for protein
97% “junk DNA” in humans 5

6. Points of control
The control of gene expression can occur at any step in the pathway from gene to functional protein
unpacking DNA
transcription
mRNA processing
mRNA transport
out of nucleus
through cytoplasm
protection from degradation
translation
protein processing
protein degradation 6

7. Why turn genes on & off? Specialization Development
each cell of a multicellular eukaryote expresses only a small fraction of its genes
Development
different genes needed at different points in life cycle of an organism
afterwards need to be turned off permanently
Responding to organism’s needs
homeostasis
cells of multicellular organisms must continually turn certain genes on & off in response to signals from their external & internal environment
A prokaryote has most of its genes turned on most of the time.
Whereas in a multicellular organism, each cell has most of its genes turned off.
A brain cell expresses many different proteins than a muscle cell. 7

8. DNA packing How do you fit all that DNA into nucleus?
DNA coiling & folding
double helix
nucleosomes
chromatin fiber
looped domains
chromosome
nucleosomes
“beads on a string”
1st level of DNA packing
histone proteins have high proportion of positively charged amino acids (arginine & lysine)
bind tightly to negatively charged DNA
from DNA double helix to condensed chromosome 8

9. Chromatin Structure Based on successive levels of DNA packing
Eukaryotic DNA is precisely combined with a large amount of protein
Chromatin changes during the course of the cell cycle
Eukaryotic chromosomes contain an enormous amount of DNA relative to their condensed length
Helps to regulate gene expression, condense and release and form chromosomes

10. Nucleosomes Proteins called histones
Are responsible for the first level of DNA packing in chromatin
Bind tightly to DNA
The association of DNA and histones
Seems to remain intact throughout the cell cycle
In electron micrographs
Unfolded chromatin has the appearance of beads on a string

11. Nucleosomes 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

12. Higher Levels of DNA Packing
Interactions between the histone tails of the nucleosomes
Causes the nucleosomes to coil around each other
Degree of packing of DNA regulates transcription
tightly packed = no transcription
= genes turned off
Nucleosome
30 nm

13. Regulation

14. Gene Regulation All organisms In each type of differentiated cell
Must regulate which genes are expressed at any given time
Each cell of a multi-cellular eukaryote
Expresses only a fraction of its genes
In each type of differentiated cell
A unique subset of genes is expressed
In interphase cells
Most chromatin is in the highly extended form called euchromatin
Genes within highly packed heterochromatin
Are usually not expressed

15. Gene Regulation Many key stages of gene expression
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
Many key stages of gene expression
Can be regulated in eukaryotic cells
Each gene is regulated in its own particular way or ways

16. Regulation of Chromatin Structure
Histone Modifications-
Chemical modification of histone tails
Affect the configuration of chromatin and thus gene expression
Histone acetylation
Addition of acetyl group (-COCH3) Seems to loosen chromatin structure and thereby enhance transcription
DNA Methylation- addition of a methyl group (– CH3) reducing transcription in some species

17. Transcription initiation
Control regions on DNA
promoter
nearby control sequence on DNA
binding of RNA polymerase & transcription factors
“base” rate of transcription
enhancers
distant control sequences on DNA
binding of activator proteins
“enhanced” rate (high level) of transcription

18. Post-Transcriptional Regulation

19. Associated with most eukaryotic genes are multiple control elements
Segments of noncoding DNA that help regulate transcription by binding certain proteins
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

20. Transcription Factors
To initiate transcription
Eukaryotic RNA polymerase requires the assistance of proteins called transcription factors
Only when the complete initiation complex is assembled can the polymerase produce the complimentary strand
Some specific transcription factors function as repressors
To inhibit expression of a particular gene

21. Post- Transcriptional Regulation
Chromatin changes
Transcription
RNA processing
mRNA
degradation
Translation
Protein processing
and degradation
Exons
DNA
Primary
RNA
transcript
RNA splicing or
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

22. Post-Transcriptional Regulation
mRNA Degradation
The life span of mRNA molecules in the cytoplasm
Is an important factor in determining the protein synthesis in a cell
Determines how long the mRNA will last in the cytoplasm and how many times the mRNA will be read
mRNA can last from hours to weeks

23. mRNA Degradation Small RNAs mRNA double-stranded RNA sRNA + mRNA
microRNA (miRNA)
single stranded that binds to mRNA that fold back on themselves.
a dicer cuts the double stranded RNA into short fragments. One strand is degraded and the other either degrades or blocks translation
RNA interference (RNAi)
double stranded RNA injected into cell turns off a gene
Small interfering RNAs (siRNA)
bind to mRNA
create sections of double-stranded mRNA
“death” tag for mRNA
triggers degradation of mRNA
cause gene “silencing”
even though post-transcriptional control, still turns off a gene
Small RNAs
mRNA
double-stranded RNA
sRNA + mRNA
mRNA degraded
functionally turns gene off 23

24. Initiation of Translation
The initiation of translation of selected mRNAs
Can be blocked by regulatory proteins that bind to specific sequences or structures of the mRNA
Prevent ribosome from attaching to the mRNA
Translation of all the mRNAs in a cell may be regulated simultaneously
Plays a role in the translation of mRNA’s stored in egg cells

25. Post- Translational Regulation
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 (can breakdown all proteins into 7-9 amino acid fragments)
Mutations in proteasomes can lead to cancer

26. 6 4 5 1 3 2 1. transcription -DNA packing -transcription factors
2. mRNA processing
-splicing
3. mRNA transport
out of nucleus
-breakdown by sRNA
4. mRNA transport in cytoplasm
-protection by 5’ cap & poly-A tail
5. translation
-factors which block start of translation
6. post-translation
-protein processing
-protein degradation
-ubiquitin, proteasome 6
post-translation 4 5
translation
mRNA transport
in cytoplasm 1
transcription 3
mRNA transport out of nucleus 2
mRNA processing 26

27. Cancer

28. Cancer
Cancer results from genetic changes that affect cell cycle control
The gene regulation systems that go wrong during cancer play important roles in embryonic development
The genes that normally regulate cell growth and division during the cell cycle
Include genes for growth factors, their receptors, and the intracellular molecules of signaling pathways

29. Cancer Oncogenes Proto-oncogenes Are cancer-causing genes
Are normal cellular genes that code for proteins that stimulate normal cell growth and division
An oncogene arises from a genetic change in a proto-oncogene that either increases the amount of protein produced or in the activity of the protein molecule

30. Mutations that change proto-oncogenes into oncogenes
Cancer cells are often found to contain chromosomes that have been broken and rejoined incorrectly, translocating fragments from one chromosome to another. If the translocated proto-oncogene ends up near an active promotor, it may increase transcription, making it an oncogene.
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

31. Tumor Suppression Tumor-suppressor genes (p53 is common)
Encode proteins that inhibit abnormal cell division
Mutation in these may contribute to the onset of cancer
Code for proteins that:
Repair damaged DNA
Control adhesion of cells to each other
Inhibit the cell cycle

32. Cancer Development Normal cells are converted to cancer cells
By the accumulation of multiple mutations affecting proto-oncogenes and tumor-suppressor genes
A multistep model for the development of colorectal cancer
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)

33. Other Promoters of Cancer
Certain viruses
Promote cancer by integration of viral DNA into a cell’s genome
Individuals who inherit a mutant oncogene or tumor-suppressor allele
Have an increased risk of developing certain types of cancer