Transcript Chapter 17 Presentation Transcription and Gene Expression

Chapter 18
Eukaryotic Genomes: Organization, Regulation and Evolution.
Gene Regulation
All organisms need to regulate the expression
of genes at any given time.
This regulation is essential for cell
specialization and is done in response to
signals from the external environment.
Differential Gene Expression
The ability to express different genes by
different cells within the same organism is key
to development of that complex, multicellular
organism.
Differential Gene Expression
The regulation of gene expression in
eukaryotes can occur at many different
stages.
Before we discuss these mechanisms of
regulation, we need to learn a little bit about
the organization of the genetic material in a
eukaryotic cell.
Chromatin
The DNA-protein
complex found in
eukaryotes.
It is much more
complex in
eukaryotes than in
prokaryotes.
The DNA Within Cells
The DNA undergoes a variety of changes as it
proceeds through the cell cycle.
Recall, in prophase it’s highly diffuse, 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 the various eukaryotes.
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, and it helps
to supercoil the
DNA.
The Nucleosome and DNA
Packing
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 (supercoil).
Nucleosome Interaction and
DNA Packing
As they continue to coil and fold, eventually
the DNA resembles that of the metaphase
chromosome.
DNA Packing
DNA Packing
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 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.
Epigenetics
The environment of a cell/organism, and the
things a person is exposed to has an effect on
the expression of genes.
The science of epigenetics seeks to
understand these changes and how they
influence the expression of genes.
You may have certain genes, but their level of
methylation often determines if and how they
are expressed.
p://en.wikipedia.org/wiki/Epigenetics#/media/File:Epigenetic_mechanisms.jpg
Histone 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.
Histone Methylation
Addition of a methyl group to a histone tail leads
to condensation of the chromatin and silencing
of the gene.
http://missinglink.ucsf.edu/lm/genes_and_genomes/acetylation.html
Histone Code Hypothesis
The discovery that the many modifications of
the histone tails leads to changes in
chromatin structure and gene expression has
led to the 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
also 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, some of these can be passed on to
future generations by what is known as
epigenetic inheritance.
Chromatin Modifying
Enzymes
These provide initial control of gene
expression.
They make the region of DNA more or less
able to bind DNA machinery--think acetylation
and methylation.
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 and Poly-A tails.
Also,
RNA modifications
help prevent
enzymatic
degradation of
mRNA, allowing
more protein to be
made.
RNA Processing
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.
RNA Splicing
Movie
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.
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.
Movie
Med25-Mediator Subunit, ACID-activator interaction domain, Pol II RNA polymerase II, TFII-transcription factor II,
VWA von Willebrand factor A, TBP-transcription binding protein
Nature Structural & Molecular Biology 18, 404–409 (2011)
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. They are nucleotide
sequences that bind activators and stimulate
gene expression.
Enhancer-Activator
Interaction and Eukaryotic
Gene Expression
When the activators
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.
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.
Control of Eukaryotic Gene
Expression
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 nonsteroid 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.
Control of Eukaryotic Gene
Expression
This process occurs in plants
too.
Movie (with help of a protein channel)
Movie (diffusion through membrane)
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.
Movie
Alternative RNA Splicing
Movie
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