1. Overview: How Eukaryotic Genomes Work and Evolve
In eukaryotes, the DNA-protein complex, called chromatin is ordered into higher structural levels than the DNA-protein complex in prokaryotes
Figure 19.1

2. Chromatin in a Developing Salamander Ovum

3. Chromatin, detail

4. Both prokaryotes and eukaryotes
Must alter their patterns of gene expression in response to changes in environmental conditions

5. Levels of Chromatin Packing

6. Concept 19.1: 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

7. Nucleosomes, or “Beads on a String”
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

8. DNA Packing as Gene Control
Degree of packing of DNA regulates transcription
tightly wrapped around histones
no transcription
genes turned off
heterochromatin
darker DNA (H) = tightly packed
euchromatin
lighter DNA (E) = loosely packed H
Form of gene control… DNA is NOT transcribed E

9. (a) Nucleosomes (10-nm fiber)
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
(“bad”)
Histone H1
(a) Nucleosomes (10-nm fiber)
Figure 19.2 a

10. Nucleosomes “Beads on a string” first level of DNA packing
8 histone molecules
“Beads on a string”
first level of DNA packing
histone proteins
8 protein molecules
many positively charged amino acids
bind tightly to negatively charged DNA
Histones = BASIC amino acid (positive) : Lysine and Arginine
DNA is negative… basic amino acids attach

11. Polar, Acidic, and Basic Amino Acids
Generally positive
in charge
Generally negative in charge

12. Concept 19.2: Gene expression can be regulated at any stage, but the key step is transcription
All organisms must regulate which genes are expressed at any given time
During development of a multicellular organism cells undergo a process of specialization in form and function called cellular differentiation

13. Differential Gene Expression
Each cell of a multicellular eukaryote expresses only a fraction of its genes
In each type of differentiated cell a unique subset of genes is expressed to make the 200 different cell types in a human

15. Many key stages of gene expression Can be regulated in eukaryotic cells

16. A Eukaryotic Gene and its Transcript

17. (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
available
for chemical
modification
Histone
tails
Figure 19.4a
(a) Histone tails protrude outward from a nucleosome

18. Seems to loosen chromatin structure and thereby enhance transcription
Histone Acetylation
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

19. Histone Acetylation Acetylation of histones unwinds DNA
loosely wrapped around histones
enables transcription
genes turned on
attachment of acetyl groups (–COCH3) to histones
conformational change in histone proteins
transcription factors have easier access to genes
In histone acetylation, acetyl groups are attached to positively charged lysines in histone tails
This loosens chromatin structure, thereby promoting the initiation of transcription (transcription factors can bind)

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

21. DNA Methylation Methylation of DNA blocks transcription factors
no transcription
 genes turned off
attachment of methyl groups (–CH3) to cytosine
nearly permanent inactivation of genes
ex. inactivated mammalian X chromosome = Barr body
The addition of methyl groups (methylation) can condense chromatin; the addition of phosphate groups (phosphorylation) next to a methylated amino acid can loosen chromatinDNA methylation, the addition of methyl groups to certain bases in DNA, is associated with reduced transcription in some species
DNA methylation can cause long-term inactivation of genes in cellular differentiation
In genomic imprinting, methylation regulates expression of either the maternal or paternal alleles of certain genes at the start of development
DNA is polar… methyl is NON-polar… polar enzymes can’t come in (inhibitor)
- May be important in development: Inactivation of genes in the embryo

22. Epigenetic Inheritance is the inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence

23. (distal control elements)
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
Figure 19.5

24. Proximal control elements are located close to the promoter
Distal control elements, groups of which are called enhancers may be far away from a gene or even in an intron

25. 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
Basal Transcription Factor
Core promoter
and initiation complex
Initiation Complex at Promoter Site binding site of RNA polymerase

26. Model for Enhancer Action
Enhancer DNA sequences
distant control sequences
Activator proteins
bind to enhancer sequence & stimulates transcription
Silencer 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.

27. Activators are proteins that bind to enhancers and stimulate transcription of a gene
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
mRNA
degradation
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
Figure 19.6

28. Some specific transcription factors function as repressors to inhibit expression of a particular gene
Some activators and repressors act indirectly by influencing chromatin structure

29. Post-Transcriptional Control
Alternative RNA splicing
variable processing of exons creates a family of proteins, depending on which RNA segments are treated as exons and which as introns
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
Prokaryotic mRNA degraded after only a few minutes

30. Alternative RNA Splicing

31. small single-stranded RNA molecules that can bind to mRNA
MicroRNAs (miRNAs)
small single-stranded RNA molecules that can bind to mRNA
These can degrade mRNA or block its translation
Inhibition of gene expression by RNA molecules = RNA INTERFERENCE (RNAi)
Made from longer RNA precursors that fold back on themselves, forming one or more short double stranded hairpin structures, each held together by hydrogen bonds.
After each hairpin is cut away from the precursor, it is trimmed by an enzyme (called a dicer) inot a short double stranded fragment
One of the two strands is degraded, while the other strand, which is the miRNA, forms a comples with one or more proteins, the miRNA allows the complex to bind to any mRNA molecule with 7-8 nucleotides of complementary sequence
The miRNA protein complex then either degrades the target mRNA or blocks its translation
It has been estimated that expression of at least one half of all human genes may be regulated by miRNAs.
RNAi = researchers had found that injecting double stranded RNA molecules into a cell somehow turned off expression of a gene with the same sequence as the RNA

32. 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

33. 5 3 Figure 18.15 Hairpin Hydrogen bond miRNA Dicer
(a) Primary miRNA transcript
miRNA
miRNA- protein complex
Figure Regulation of gene expression by miRNAs.
mRNA degraded
Translation blocked
(b) Generation and function of miRNAs

34. Small Interfering RNAs (siRNAs)
RNA interference (RNAi) is caused by siRNAs
Ex: Yeast: siRNA’s play a role in heterochromatin formation and can block large regions of the chromosome
siRNAs are similar in size and function to mirNAs.
Difference? miRNA is usually formed from a single hairpin in a precursor RNA. siRNAs are formed from a much longer, linear, double stranded RNA molecule

35. The initiation of translation of selected mRNAs can be blocked by regulatory proteins that bind to specific sequences or structures of the mRNA
Alternatively, translation of all the mRNAs in a cell may be regulated simultaneously

36. After translation various types of protein processing, including cleavage and the addition of chemical groups, are subject to control

37. Ubiquitin “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

38. Proteasomes
Are giant protein complexes that bind protein molecules and degrade them
Enzymatic components of the
proteasome cut the protein into
small peptides, which can be
further degraded by other
enzymes in the cytosol. 3
The ubiquitin-tagged protein
is recognized by a proteasome,
which unfolds the protein and
sequesters it within a central cavity. 2
Multiple ubiquitin mol-
ecules are attached to a protein
by enzymes in the cytosol. 1
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
Figure 19.10

39. 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

40. Degradation of a Protein by a Proteasome

41. Concept 19.4 Eukaryotic genomes can have many noncoding DNA sequences in addition to genes
The bulk of most eukaryotic genomes consists of noncoding DNA sequences, often described in the past as “junk DNA”
However, much evidence is accumulating that noncoding DNA plays important roles in the cell

42. The Relationship Between Genomic Composition and Organismal Complexity
Compared with prokaryotic genomes, the genomes of eukaryotes
Generally are larger
Have longer genes
Contain a much greater amount of noncoding DNA both associated with genes and between genes

43. Now that the complete sequence of the human genome is available
We know what makes up most of the % that does not code for proteins, rRNAs, or tRNAs
Exons (regions of genes coding
for protein, rRNA, tRNA) (1.5%)
Repetitive
DNA that
includes
transposable
elements
and related
sequences
(44%)
Introns and
regulatory
(24%)
Unique
noncoding
DNA (15%)
DNA
unrelated to
(about 15%)
Alu elements
(10%)
Simple sequence
DNA (3%)
Large-segment
duplications (5-6%)
Figure 19.14

44. The first evidence for wandering DNA segments
Came from geneticist Barbara McClintock’s breeding experiments with Indian corn
Figure 19.15

45. Eukaryotic transposable elements are of two types:
Transposons, which move within a genome by means of a DNA intermediate
Retrotransposons, which move by means of an RNA intermediate
Transposon
New copy of
transposon
is copied
DNA of genome
Insertion
Mobile transposon
(a) Transposon movement (“copy-and-paste” mechanism)
Retrotransposon
retrotransposon
RNA
Reverse
transcriptase
(b) Retrotransposon movement
Figure 19.16a, b

46. Retrotransposon Movement

47. Transposons in Corn

48. Rearrangements of Parts of Genes: Exon Duplication and Exon Shuffling
A particular exon within a gene could be duplicated on one chromosome and deleted from the homologous chromosome

49. 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
Figure 19.20

50. 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
Some mechanisms can alter the functions of genes or their patterns of expression and regulation

52. Part of a family of identical genes for ribosomal RNA

53. The evolution of human -globin and -globin gene families

54. DNA rearrangement in the maturation of an immunoglobulin (antibody) gene

55. Genetic changes that can turn proto-oncogenes into oncogenes

56. Signaling pathways that regulate cell growth

57. A multi-step model for the development of colorectal cancer