1. Control of Eukaryotic Genome
Chapter 19.
Control of Eukaryotic Genome 1

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. 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 3

4. 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. 4

5. 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 5

6. 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
many positively charged amino acids
arginine & lysine
bind tightly to negatively charged DNA
DNA packing movie 6

7. DNA packing Degree of packing of DNA regulates transcription
tightly packed = no transcription
= genes turned off
darker DNA (H) = tightly packed
lighter DNA (E) = loosely packed

8. 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 8

9. Histone acetylation Acetylation of histones unwinds DNA
loosely packed = transcription
= genes turned on
attachment of acetyl groups (–COCH3) to histones
conformational change in histone proteins
transcription factors have easier access to genes 9

10. 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 to RNA polymerase at TATA box of promoter
“enhanced” rate (high level) of transcription

11. Model for Enhancer action
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. 11

12. Post-transcriptional control
Alternative RNA splicing
variable processing of exons creates a family of proteins
One gene can create a variety of polypeptides 12

13. Regulation of mRNA degradation
Life span of mRNA determines pattern of protein synthesis
mRNA can last from hours to weeks 13

14. RNA interference Small RNAs (sRNA) 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”
even though post-transcriptional control, still turns off a gene
siRNA 14

15. mRNA degraded or silenced
RNA interference
sRNAs
mRNA
double-stranded RNA
sRNA + mRNA
mRNA degraded or silenced
functionally turns gene off 15

16. Control of translation
Block initiation stage
regulatory proteins attach to 5’ end of mRNA
prevent attachment of ribosomal subunits & initiator tRNA
block translation of mRNA to protein 16

17. Protein processing Protein processing
folding, cleaving, adding sugar groups, targeting for transport are common steps in the final processing of a protein
Regulation can occur at any of these steps in the modification or transportation of a protein
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. 17

18. Protein degradation “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") 18

19. Proteasome Protein-degrading “machine” cell’s waste disposer
can breakdown all proteins into 7-9 amino acid fragments
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 19

20. 6 4 5 1 3 2 20

21. 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
mRNA transport
in cytoplasm
translation 1
transcription 3 2
mRNA transport out of nucleus
mRNA processing 21

22. Structure of the Eukaryotic Genome22

23. How many genes? Genes only ~3% of human genome
protein-coding sequences
1% of human genome
non-protein coding genes
2% of human genome
tRNA
ribosomal RNAs
siRNAs 23

24. What about the rest of the DNA?
Non-coding DNA sequences
regulatory sequences
promoters, enhancers
terminators
“junk” DNA
introns
repetitive DNA
centromeres
telomeres
tandem & interspersed repeats
transposons & retrotransposons
Alu in humans 24

25. Repetitive DNA
Repetitive DNA & other non-coding sequences account for most of eukaryotic DNA 25

26. Families of genes Multigene family
Collection of identical or very similar genes
Most likely evolved from a single ancestral gene
This evolution occurred long ago. All vertebrates have multiple globin genes & most mammals have the same set of globin genes.
Different versions of each globin subunit are expressed at different times in development, allowing hemoglobin to function effectively in the changing environment of the developing animal. 26

27. Interspersed repetitive DNA
Repetitive DNA is spread throughout genome
interspersed repetitive DNA make up % of mammalian genome
in humans, at least 5% of genome is made of a family of similar sequences called, Alu elements
300 bases long
Alu is an example of a "jumping gene" – a transposon DNA sequence that "reproduces" by copying itself & inserting into new chromosome locations
5% of genome = millions of copies of this sequence!
Alu doesn’t seem to be active anymore. No useful function to organism.
Used to study population genetics = relatedness of groups of people 27

28. Rearrangements in the genome
Transposons
transposable genetic element
piece of DNA that can move from one location to another in cell’s genome
One gene of an insertion sequence codes for transposase, which catalyzes the transposon’s movement. The inverted repeats, about 20 to 40 nucleotide pairs long, are backward, upside-down versions of each other. In transposition, transposase molecules bind to the inverted repeats & catalyze the cutting & resealing of DNA required for insertion of the transposon at a target site. 28

29. Transposons Insertion of transposon sequence in new position in genome
insertion sequences cause mutations when they happen to land within the coding sequence of a gene or within a DNA region that regulates gene expression 29

30. Retrotransposons
Transposons actually make up over 50% of the corn (maize) genome & 10% of the human genome.
Most of these transposons are retrotransposons, transposable elements that move within a genome by means of an RNA intermediate, a transcript of the retrotransposon DNA 30

31. Retrotransposons RNA retrotransposons must be converted back to DNA
The enzyme reverse transcriptase is responsible for this
Reverse transcriptase is encoded in the retrotransposon
It is believed that retroviruses may have evolved from escaped and packaged retrotransposons