Download Activator Proteins

• 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
Chromatin in a Developing Salamander
Ovum
Chromatin, detail
• Both prokaryotes and eukaryotes
• Must alter their patterns of gene
expression in response to changes in
environmental conditions
Levels of Chromatin Packing
• 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
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
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
E
• 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
DNA double helix
Histones
Histone
tails
Histone H1
Linker DNA
(“string”)
Nucleosome
(“bad”)
(a) Nucleosomes (10-nm fiber)
Figure 19.2 a
10 nm
Nucleosomes
• “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
8 histone
molecules
Polar, Acidic, and Basic Amino Acids
Generally negative in charge
Generally positive
in charge
• 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
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
Many key stages of gene expression
Can be regulated in eukaryotic cells
A Eukaryotic Gene and its Transcript
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
Histone
tails
DNA
double helix
Amino acids
available
for chemical
modification
Figure 19.4a
(a) Histone tails protrude outward from a nucleosome
• Histone Acetylation
• Seems to loosen chromatin structure and
thereby enhance transcription
Unacetylated histones
Figure 19.4 b
Acetylated histones
(b) Acetylation of histone tails promotes loose chromatin structure that permits
transcription
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
DNA Methylation
• Addition of methyl groups to certain bases
in DNA is associated with reduced
transcription in some species
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
• Epigenetic Inheritance is the
inheritance of
traits transmitted by mechanisms not
directly involving the nucleotide sequence
• 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)
Poly-A signal
sequence
Proximal
control elements
Exon
Intron
Intron
Exon
Termination
region
Exon
DNA
Downstream
Upstream
Promoter
Chromatin changes
Transcription
Exon
Primary RNA
5
transcript
(pre-mRNA)
Intron
Exon
Intron
Intron RNA
RNA processing
Coding segment
Translation
Protein processing
and degradation
mRNA
G
P
5
Figure 19.5
Exon
RNA processing:
Cap and tail added;
introns excised and
exons spliced together
Transcription
mRNA
degradation
Poly-A
signal
P
P
Cap
5 UTR
(untranslated
region)
Start
codon
Stop
codon
Poly-A
3 UTR
tail
(untranslated
region)
Cleared 3 end
of primary
transport
• 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
Transcription Complex
Activator Proteins
• regulatory proteins bind to DNA at
Enhancer Sites
distant enhancer sites
• increase the rate of transcription
regulatory sites on DNA
distant from gene
Enhancer
Activator
Activator
Activator
Coactivator
A
Basal Transcription Factor
E RNA polymerase II
F
B
TFIID
H
Core promoter
and initiation complex
Initiation Complex at Promoter Site binding site of RNA polymerase
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
• Activators are proteins that bind to enhancers and
stimulate transcription of a gene
Distal control
element
Promoter
Activators
Enhancer
1
TATA
box
General
transcription
factors
Activator proteins bind
to distal control elements
grouped as an enhancer in
the DNA. This enhancer has
three binding sites.
2
Gene
DNA-bending
protein
Group of
Mediator proteins
A DNA-bending protein
brings the bound activators
closer to the promoter.
Other transcription factors,
mediator proteins, and RNA
polymerase are nearby.
RNA
Polymerase II
Chromatin changes
3
The activators bind to
certain general transcription
factors and mediator
proteins, helping them form
an active transcription
initiation complex on the promoter.
Figure 19.6
Transcription
RNA processing
mRNA
degradation
RNA
Polymerase II
Translation
Protein processing
and degradation
Transcription
Initiation complex
RNA synthesis
• Some specific transcription factors function
as repressors to inhibit expression of a
particular gene
• Some activators and repressors act
indirectly by influencing chromatin
structure
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
Alternative RNA Splicing
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)
• RNA interference by single-stranded
microRNAs (miRNAs)
• Can lead to degradation of an mRNA or
block its translation
The 1
microRNA (miRNA)
precursor folds
back on itself,
held together
by hydrogen
bonds.
22
An enzyme
called Dicer moves
along the doublestranded RNA,
cutting it into
shorter segments.
One strand of
each short doublestranded RNA is
degraded; the other
strand (miRNA) then
associates with a
complex of proteins.
3
4
The bound
miRNA can base-pair
with any target
mRNA that contains
the complementary
sequence.
55 The miRNA-protein
complex prevents gene
expression either by
degrading the target
mRNA or by blocking
its translation.
Chromatin changes
Transcription
RNA processing
mRNA
degradation
Translation
Protein processing
and degradation
Protein
complex
Dicer
Degradation of mRNA
OR
miRNA
Target mRNA
Figure 19.9
Hydrogen
bond
Blockage of translation
Figure 18.15
Hairpin
Hydrogen
bond
miRNA
Dicer
5 3
(a) Primary miRNA transcript
miRNA
mRNA degraded
miRNAprotein
complex
Translation blocked
(b) Generation and function of miRNAs
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
• 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
• After translation various types of protein
processing, including cleavage and the
addition of chemical groups, are subject to
control
Ubiquitin
• “Death tag”
• mark unwanted proteins with a label
• 76 amino acid polypeptide, ubiquitin
• labeled proteins are broken down rapidly in "waste
disposers"
• proteasomes
Aaron Ciechanover
Israel
Avram Hershko
Israel
Irwin Rose
UC Riverside
• Proteasomes
• Are giant protein complexes that bind
protein molecules and degrade them
3 Enzymatic components of the
Chromatin changes
The ubiquitin-tagged protein
2
1
is recognized by a proteasome,
Multiple ubiquitin molwhich unfolds the protein and
ecules are attached to a protein
by enzymes in the cytosol. sequesters it within a central cavity.
proteasome cut the protein into
small peptides, which can be
further degraded by other
enzymes in the cytosol.
Transcription
RNA processing
mRNA
degradation
Proteasome
and ubiquitin
to be recycled
Ubiquitin
Translation
Proteasome
Protein processing
and degradation
Protein to
be degraded
Ubiquinated
protein
Protein entering a
proteasome
Figure 19.10
Protein
fragments
(peptides)
Proteasome
• Protein-degrading “machine”
• cell’s waste disposer
• breaks down any proteins
into 7-9 amino acid fragments
• cellular recycling
Degradation of a Protein by a
Proteasome
• 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
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
• Now that the complete sequence of the human
genome is available
• We know what makes up most of the 9798% 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%)
Unique
noncoding
DNA (15%)
Repetitive
DNA
unrelated to
transposable
elements
(about 15%)
Alu elements
(10%)
Figure 19.14
Introns and
regulatory
sequences
(24%)
Simple sequence
DNA (3%)
Large-segment
duplications (5-6%)
• The first evidence for wandering DNA segments
• Came from geneticist Barbara McClintock’s
breeding experiments with Indian corn
Figure 19.15
• 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
DNA of genome
Transposon
is copied
Insertion
Mobile transposon
(a) Transposon movement (“copy-and-paste” mechanism)
Retrotransposon
New copy of
retrotransposon
DNA of genome
RNA
Reverse
transcriptase
Figure 19.16a, b
(b) Retrotransposon movement
Insertion
Retrotransposon Movement
Transposons in Corn
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
• 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
EGF
EGF
EGF
Epidermal growth
factor gene with multiple
EGF exons (green)
Exon
shuffling
F
F
F
Fibronectin gene with multiple
“finger” exons (orange)
Exon
duplication
F
F
EGF
K
K
Plasminogen gene with a
“kfingle” exon (blue)
Figure 19.20
Portions of ancestral genes
Exon
shuffling
TPA gene as it exists today
K
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
Part of a family of identical genes for
ribosomal RNA
The evolution of human -globin and globin gene families
DNA rearrangement in the maturation
of an immunoglobulin (antibody) gene
Genetic changes that can turn protooncogenes into oncogenes
Signaling pathways that regulate cell
growth
A multi-step model for the development
of colorectal cancer