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Chapter 19
Eukaryotic Genomes:
Organization, Regulation and
Evolution.
Chromatin


The DNA-protein
complex found in
eukaryotes.
It is much more
complex in
eukaryotes than in
prokaryotes.
The DNA Within Cells
undergoes a variety of changes as it
proceeds through the cell cycle.
 in prophase it’s highly diffuse (thin), but as
the cell prepares to divide, it becomes
highly condensed.
 Proteins called histones are responsible
for the first level of DNA packing in
chromatin.

 The
mass of histone is nearly equal to the
mass of DNA.
DNA-Histone Binding

DNA is negatively charged, and histones contain
a high proportion of positively charged aa’s and
enable easy binding of the histones to the DNA.
DNA-Histone Binding
Histones play a very important role in
organizing DNA and they are very good at
it.
 Thus, this is a likely reason why histone
genes have been conserved throughout
the generations in the course of evolution.
 The structure of histones are very similar
among eukaryotes and between
eukaryotes and prokaryotes.

DNA-Histone Binding and DNA
Packing


Electron micrographs show unfolded chromatin and they
look like beads on a string.
These “beads” are referred to as nucleosomes (the basic
unit of DNA packing), and the string is DNA.
The Nucleosome and DNA
Packing
A nucleosome is a piece of DNA wound
around a protein core.
 This DNA-histone association remains in
tact throughout the cell cycle.
 Histones only leave the DNA very briefly
during DNA replication.
 With very few exceptions, histones stay
with the DNA during transcription.

Nucleosome Interaction and
DNA Packing



The next level of DNA packing takes place between the
histone tails of one nucleosome/linker DNA and the
nucleosomes to either side.
The interactions between these cause the DNA to coil
even tighter.
As they continue to coil and fold, eventually the DNA
resembles that of the metaphase chromosome.
DNA Packing

Movie
http://www.travismulthaupt.com/page1/page5/files/19_02DNAPacking_A.swf
Heterochromatin Vs.
Euchromatin



During interphase, some of the DNA remains
condensed as you would normally see it in
metaphase. (centrosomes, and some other
regions of the chromosome).
This is called heterochromatin to distinguish it
from euchromatin which condenses and relaxes
with the cell cycle.
Heterochromatin is rarely transcribed.
The Structural Organization of Chromatin



The structural organization of chromatin is
important in helping regulate gene
expression.
Also, the location of a gene’s promoter
relative to nucleosomes and to sites where
DNA attaches to the chromosome scaffold or
nuclear lamina can also affect whether it is
transcribed or not.
Research indicates that chemical modification
to the histones and DNA of chromatin
influence chromatin structure and gene
expression.
Acetylation



There is a lot of
evidence supporting the
notion that the
regulation of gene
expression is, in part,
dependent upon
chemical modifications
to histones.
When an acetyl group is
added to the histone
tail, the histones
become neutralized and
the chromatin loosens
up.
As a result, transcription
can occur.

The enzymes that interact with histones
are closely associated with, or are
components of transcription factors that
bind to promoters.
Methylation

Addition of a methyl group to a histone tail
leads to condensation of the chromatin.
Histone Code Hypothesis

This hypothesis states that the specific
modifications of histones help determine
chromatin configuration thus influencing
transcription.
DNA Methylation
DNA methylation is completely separate
from histone methylation, but may be a
way in which genes become inactivated.
 Evidence:

 Inactivated
X chromosomes are heavily
methylated.
 In many cells that have inactivated genes, the
genes are more heavily methylated than in
cells where the genes are active.
Control of Eukaryotic Gene
Expression
Recall the idea of the operon and how it
regulated bacterial gene expression.
 The mechanism of gene expression in
eukaryotes is different.
 It involves chromatin modifications, but
they do not involve a change in DNA
sequence. Moreover, they can be passed
on to future generations by what is known
as epigenetic inheritance.

Epigenetic Inheritance
Epigenetic inheritance occurs when traits
are passed on and do not involve the
nucleotide sequences (proteins, enzymes,
organelles).
 It also seems to be very important in the
regulation of gene expression.
 The enzymes that modify chromatin are
integral parts of the cell’s machinery that
regulates transcription.

Chromatin Modifying Enzymes
These provide initial control of gene
expression.
 They make the region of DNA more or less
able to bind DNA machinery.
 Once optimized for expression, the
initiation of transcription is the most
universally used stage at which gene
expression is regulated.

Recall,




Eukaryotic genes have promoters, a DNA sequence
where RNA polymerase II binds and starts
transcription.
There are numerous control elements involved in
regulating the initiation of transcription.
5’ caps.
Poly-A tails.
Also,

RNA modifications
help prevent
enzymatic
degradation of
mRNA, allowing
more protein to be
made.
Movie
Recall,

RNA processing
involves 3 steps:
 1.
Addition of the 5’
cap.
 2. Addition of the polyA tail.
 3. Gene splicing.

Removal of introns and
splicing together of
exons.
Recall,



The transcription initiation
complex assembles on
the promoter sequence.
RNA polymerase II
proceeds to transcribe
the gene making premRNA.
Transcription factors are
proteins that assist RNA
polymerase II to initiate
transcription.
RNA Processing

Movie
Eukaryotic Gene Expression


Most eukaryotic genes are associated with
multiple control elements which are segments of
non-coding DNA that help regulate transcription
by binding certain proteins.
These control elements are crucial to the
regulation of certain genes within different cells.
Eukaryotic Gene Expression

Only after the complete initiation complex
has assembled can the polymerase begin
to move along the DNA template strand,
producing a complementary strand of
DNA.
Eukaryotic Gene Expression
In eukaryotes, high levels of
transcription of a particular gene at the
appropriate time depends on the
interaction of control elements with
other proteins called transcription
factors.
 Enhancers and activators play important
roles in gene expression.

 Enhancers
are nucleotide sequences that
bind activators and stimulate gene
expression.
Enhancer-Activator Interaction
and Eukaryotic Gene
 When
the activators
Expression

bind to the
enhancers, this
causes the DNA to
bend allowing
interaction of the
proteins and the
promoter.
This helps to
position the initiation
complex on the
promoter so RNA
synthesis can occur.
Eukaryotic Gene Expression

Some specific transcription factors
function as repressors to inhibit expression
of a particular gene.
 Certain
repressors can block the binding of
activators either to their control elements or to
parts of their transcriptional machinery.
 Other repressors bind directly to their own
control elements in an enhancer and act to
turn off transcription.
Transcription Initiation

Movie
Blocking Transcription

Movie
Eukaryotic Gene Expression


There are only a dozen or
so short nucleotide
sequences that exist in
control elements for
different genes.
The combinations of
these control elements
are more important than
the presence of single
unique control elements
in regulating the
transcription of a gene.
Recall,

Prokaryotes typically have coordinately
controlled genes clustered in an operon.
The operons are regulated by single
promoters and get transcribed into a single
mRNA molecule. Thus genes are
expressed together, and proteins are
made concurrently.
Control of Eukaryotic Gene
Expression



Recent studies indicate that within genomes of
many eukaryotic species, co-expressed genes
are clustered near one another on the same
chromosome.
However, unlike the genes in the operons of
prokaryotes, each of the eukaryotic genes
have their own promoter and is individually
transcribed.
It is thought that the coordinate regulation of
genes clustered in eukaryotic cells involves
changes in chromatin structure that makes the
entire group of genes available or unavailable.
Control of Eukaryotic Gene
Expression


More commonly, co-expressed eukaryotic
genes are found scattered over different
chromosomes. In these cases, coordinate
gene expression is seemingly dependent on
the association of specific control elements or
combinations of every gene of a dispersed
group.
Copies of activators that recognize these
control elements bind to them, promoting
simultaneous transcription of the genes no
matter where they are in the genome.
Control of Eukaryotic Gene
Expression
The coordinate control of dispersed genes
in a eukaryotic cell often occurs in
response to external signals such as
hormones.
 When the steroid enters the cell, it binds to
a specific intracellular receptor protein
forming a hormone-receptor complex that
serves as a transcription activator.

Control of Eukaryotic Gene
Expression
In an alternative mechanism, a signal
molecule such as a non-steroid hormone
or a growth factor bind to a receptor on a
cell’s surface and never enter a cell.
 Instead, they control gene expression by
inducing a signal transduction pathway.

Post-transcriptional
Regulation and Control of
Gene Expression
The mechanisms we’ve just discussed
involve regulating the expression of the
gene.
 Post-transcriptional regulation involves
regulating the transcript after the mRNA
has been made.
 These modes are unique to eukaryotes.

Alternative RNA Splicing and
Control of Gene Expression


Alternative RNA splicing
is a way in which different
mRNA transcripts are
produced from the same
primary transcript.
This is determined by
which RNA segments are
treated as introns and
which are treated as
exons.
Alternative RNA Splicing and
Control of Gene Expression

Different cells have
different regulatory
proteins that control
intron-exon choices
by binding to
regulatory sequences
within the primary
transcript.
Alternative Mechanisms to
Control Gene Expression
Protein processing is the final spot for
controlling gene expression.
 Often, eukaryotic polypeptides undergo
further processing to yield a functional
protein. Regulation can occur at any of
the sites of protein modification.

Protein Processing

Movie
http://www.travismulthaupt.com/page1/page5/files/19_10_ProteinProcessing_A.swf