1. Control of Eukaryotic Genes
Chapter 19:
Control of Eukaryotic Genes

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?
Differential gene expressions
200 different cell types

3. 1. 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

4. Nucleosomes “Beads on a string” 1st level of DNA packing
8 histone molecules
Nucleosomes
“Beads on a string”
1st level of DNA packing
histone proteins
8 protein molecules
positively charged amino acids
bind tightly to negatively charged DNA
Histones leave the DNA transiently during DNA replication and stay with the DNA during transcription.
DNA packing movie

5. DNA packing as gene control
Degree of packing of DNA regulates transcription
tightly wrapped around histones
no transcription
genes turned off
Heterochromatin (Interphase)
darker DNA (H) = tightly packed
euchromatin
lighter DNA (E) = loosely packed H E

6. Points of control
The control of gene expression can occur at any step in the pathway from gene to functional protein
1. packing/unpacking DNA
2. Transcription (most common)
3. mRNA processing
4. mRNA transport
5. translation
6. protein processing
7. protein degradation
A typical human cell expresses about 20% of its genes at any given time.
About 200 different cell types.
Only about 1.5% of our DNA codes for proteins, a small fraction codes for RNA products such as rRNA and tRNA, and the rest is UTRs.

7. (a) Histone tails protrude outward from a nucleosome
Histone Modification
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
DNA
double helix
Amino acids (N-terminus)
available
for chemical
modification
Histone
tails
Figure 19.4a
(a) Histone tails protrude outward from a nucleosome

8. Unacetylated histones
Histone acetylation
Acetylation of histones unwinds DNA
loosely wrapped around histones
enables transcription
genes turned on
attachment of acetyl groups (–COCH3) to postive charged lysines
Neutralized (+) charged tails no longer bind to neighboring nucleosomes
transcription factors have easier access to genes
The enzymes that acetylate or deacetylate are closely associated with the transcription factors that allow for the binding of the transcription macherinery.
(b) Acetylation of histone tails promotes loose chromatin structure that permits transcription
Unacetylated histones
Acetylated histones

9. DNA methylation Methylation of DNA blocks transcription factors
no transcription
 genes turned off
attachment of methyl groups (–CH3) to cytosine
C = cytosine
nearly permanent inactivation of genes
ex. inactivated mammalian X chromosome = Barr body
Ex. Epigenetic inheritance
Inheritance of traits by mechanisms not involving the nucleotide sequence
Certain proteins that bind to methylated DNA recruit histone deacytlation enzymes – dual mechanism for turning off genes.
IT has been found in mice and plants that deficient methylating enzyme leads to abnormal embryonic development.
Methylation patterns are passted on and for every replication of DNA the same genes get methylated

10. Regulation of Transcription Initiation
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

11. 2. Transcription initiation
Noncoding control regions on DNA
promoter
nearby control sequence on DNA
binding of RNA polymerase & transcription factors
proximal control elements
UTR located close to the promoter
enhancer
distant control sequences on DNA
binding of activator proteins
“enhanced” rate (high level) of transcription
Distal control elements can be thousands of nucleotides upsteam or downstream of a gene or even within an intron.

12. Organization of a Typical Eukaryotic Gene
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
Figure 19.5

13. Model for Enhancer action
Enhancer DNA sequences
distant control sequences
Activator proteins
bind to enhancer sequence & stimulates transcription
Silencer (repressor) proteins
bind to enhancer sequence & block gene transcription
Much of molecular biology research is trying to understand this: the regulation of transcription.
Silencer proteins are, in essence, blocking the positive effect of activator proteins, preventing high level of transcription.
Specific transcription factors – activators or repressors.
Turning on Gene movie

14. Transcription complex
Activator Proteins
• regulatory proteins bind to DNA at distant enhancer sites
• increase the rate of transcription
Enhancer Sites
regulatory sites on DNA distant from gene
Enhancer
Activator
Activator
Activator
Coactivator B F E
RNA polymerase II A
TFIID H
Coding region
T A T A
Core promoter
and initiation complex
Initiation Complex at Promoter Site binding site of RNA polymerase

15. Combinatorial Control of Gene Activation
Enhancer
Promoter
Control
elements
Albumin
gene
Crystallin
Liver cell
nucleus
Lens cell
Available
activators
expressed
gene not
Crystallin gene
not expressed
(a)
(b)
A particular combination of control elements
Will be able to activate transcription only when the appropriate activator proteins are present

16. Coordinately Controlled Genes
Unlike the genes of a prokaryotic operon
Coordinately controlled eukaryotic genes each have a promoter and control elements
The same regulatory sequences
Are common to all the genes of a group, enabling recognition by the same specific transcription factors

17. 3. Post-transcriptional control
Alternative RNA splicing
Different mRNA molecules produced from the same primary transcript
Depends on which RNA segments are treated as introns and exons

18. 4. Regulation of mRNA degradation
Life span of mRNA determines amount of protein synthesis
mRNA can last from hours to weeks
Ex. Long lived hemoglobin & short lived growth factor
Determined by sequences towards the 3’ end UTR
Enzymatic shortening of poly A tail  removal of 5’ cap  nuclease degrades mRNA
RNA processing movie

19. RNA interference Small interfering RNAs (siRNA)
short segments of RNA (21-28 bases)
bind to mRNA
create sections of double-stranded mRNA
“death” tag for mRNA
triggers degradation of mRNA
cause gene “silencing”
post-transcriptional control
turns off gene = no protein produced
siRNA

20. Blockage of translation
RNA interference by single-stranded microRNAs (miRNAs)
Can lead to degradation of an mRNA or block its translation
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. 5 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
Figure 19.9

21. 1990s | 2006
RNA interference
“for their discovery of RNA interference — gene silencing by double-stranded RNA”
Andrew Fire
Stanford
Craig Mello
U Mass

22. 5. Control of translation
Block initiation of translation stage
regulatory proteins attach to 5' end of UTR of mRNA
prevent attachment of ribosomal subunits & initiator tRNA
block translation of mRNA to protein
Control of translation movie

23. 6-7. Protein processing & degradation
folding, cleaving, adding sugar groups, targeting for transport
Protein degradation
ubiquitin tagging
proteasome degradation
The cell limits the lifetimes of normal proteins by selective degradation. Many proteins, such as the cyclins involved in regulating the cell cycle, must be relatively short-lived.
Protein processing movie

24. Ubiquitin 1980s | 2004 “Death tag” mark unwanted proteins with a label
76 amino acid polypeptide, ubiquitin
labeled proteins are broken down rapidly in "waste disposers"
proteasomes
Since the molecule was subsequently found in numerous different tissues and organisms – but not in bacteria – it was given the name ubiquitin (from Latin ubique, "everywhere")
Aaron Ciechanover
Israel
Avram Hershko
Israel
Irwin Rose
UC Riverside

25. Proteasome Protein-degrading “machine” cell’s waste disposer
breaks down any proteins into 7-9 amino acid fragments
cellular recycling
A human cell contains about 30,000 proteasomes: these barrel-formed structures can break down practically all proteins to 7-9-amino-acid-long peptides. The active surface of the proteasome is within the barrel where it is shielded from the rest of the cell. The only way in to the active surface is via the "lock", which recognises polyubiquitinated proteins, denatures them with ATP energy and admits them to the barrel for disassembly once the ubiquitin label has been removed. The peptides formed are released from the other end of the proteasome. Thus the proteasome itself cannot choose proteins; it is chiefly the E3 enzyme that does this by ubiquitin-labelling the right protein for breakdown
play Nobel animation

26. Cancer results from genetic changes
Growth factors can create cancers
proto-oncogenes
normally activates cell division
growth factor genes
become oncogenes (cancer-causing) when mutated
if switched “ON” can cause cancer
example: RAS (activates cyclins) 30% cancers
tumor-suppressor genes
normally inhibits cell division
if switched “OFF” can cause cancer
example: p53 - more than 50% cancers

27. Cancer & Cell Growth
Cancer is essentially a failure of cell division control
unrestrained, uncontrolled cell growth
What control is lost?
lose checkpoint stops
gene p53 plays a key role in G1/S restriction point
p53 protein halts cell division if it detects damaged DNA
options:
stimulates repair enzymes to fix DNA
forces cell into G0 resting stage
causes apoptosis of damaged cell
p53 discovered at Stony Brook by Dr. Arnold Levine

28. p53 — master regulator gene
NORMAL p53
p53 allows cells
with repaired
DNA to divide.
p53
protein
DNA repair enzyme
p53
protein
Step 1
Step 2
Step 3
DNA damage is caused
by heat, radiation, or
chemicals.
Cell division stops, and p53 triggers enzymes to repair damaged region.
p53 triggers the destruction of cells damaged beyond repair.
ABNORMAL p53
abnormal
p53 protein
cancer
cell
Step 1
Step 2
DNA damage is
caused by heat,
radiation, or
chemicals.
The p53 protein fails to stop
cell division and repair DNA.
Cell divides without repair to
damaged DNA.
Step 3
Damaged cells continue to divide.
If other damage accumulates, the
cell can turn cancerous.

29. How Transposable Elements Contribute to Genome Evolution
Movement of transposable elements or recombination between copies of the same element
Occasionally generates new sequence combinations that are beneficial to the organism
“copy and paste” mechanism
Typically noncoding sequences make up these transposable elements

30. Duplication and Divergence of DNA Segments
Repetitive DNA probably arose by mistakes in DNA replication or recombination
The basis of change at the genomic level is mutation
Accidents in cell division
Can lead to extra copies of all or part of a genome, which may then diverge if one set accumulates sequence changes
Ex: errors in meiosis can result in extra sets of chromosomes
Ex: duplications of genes on one chromosome

31. Duplication and Divergence of DNA Segments
Unequal crossing over during prophase I of meiosis
Can result in one chromosome with a deletion and another with a duplication of a particular gene
Nonsister
chromatids
Transposable
element
Gene
Incorrect pairing
of two homologues
during meiosis
Crossover
and

32. Evolution of Genes with Related Functions: The Human Globin Genes
The genes encoding the various globin proteins
Evolved from one common ancestral globin gene, which duplicated and diverged
Ancestral globin gene      2 1   G A  
-Globin gene family
on chromosome 16
 -Globin gene family
on chromosome 11 
Evolutionary time
Duplication of
ancestral gene
Mutation in
both copies
Transposition to
different chromosomes
Further duplications
and mutations

33. Evolution of Genes with Novel Functions
The copies of some duplicated genes
Have diverged so much during evolutionary time that the functions of their encoded proteins are now substantially different
Ex: similar amino acid sequence in lactalbumin and lysozyme enzyme
Lysozyme – enzyme that helps prevent infection
Lactalbumin – protein in milk production in mammals

34. Portions of ancestral genes TPA gene as it exists today
In exon shuffling
Errors in meiotic recombination lead to the occasional mixing and matching of different exons either within a gene or between two nonallelic genes
EGF
Epidermal growth
factor gene with multiple
EGF exons (green) F
Fibronectin gene with multiple
“finger” exons (orange)
Exon
shuffling
duplication K
Plasminogen gene with a
“kfingle” exon (blue)
Portions of ancestral genes
TPA gene as it exists today
Plasminogen – this protein is involved in limiting blood clotting, it has four domains of three types each encoded by an exon. This gene is believed to have arien by several instances of exon shuffling and duplication.
Epidermal growth factor and plasminogen –untangles blood cells from fibrinogen. Prevents clotting as well.
Figure 19.20