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Chromosomes and Gene Regulation
All cells can use their genes
selectively – turning some on and
keeping others off.
In multicellular organisms, gene
expression is under complex
controls. All cells have the same
DNA sequences, they same
chromosomes, and yet they each
look and function very differently.
Cell differentiation is achieved by
changes in gene expression. The
differences between this neuron and
the lymphocyte depend on the
precise control of gene expression.
Same genome
Different genes
expressed
Questions 2-4,6,79,11,12,15
Eucaryotic DNA is packaged into Chromosomes. Human cells
contain two of each chromosome, one maternal and one paternal
– homologous chromosomes Sex chromosomes are nonhomologous chromosomes, X from mom, Y from dad.
Bacteria typically have one circular DNA molecule. It is
also associated with proteins that condense the DNA but
less is know about the structure.
Chromosomes are typically stained by dyes that
distinguish between areas rich in A-T nucleotide
pairs and areas rich in
C-G pairs. This
results in a pattern of
banding that is unique
to each chromosome.
Cytogeneticists use
these to detect major
centromere
chromosomal
abnormalities.
large rRNA
Cells can vary the structure of their chromosomes for DNA
replication and for gene regulation. Chromatin = DNA plus
proteins, the stuff chromosomes are made of.
The state of condensation of chromosomes varies
according to the cell growth cycle. The mitotic
chromosomes are highly condensed, in contrast to that of
the interphase chromosome.
Three types of specialized sequences found in all eucaryotic
chromosomes ensure that chromosomes replicate efficiently.
many, to
ensure speed
kinetochore =
protein
complex that
binds the
spindle and the
centromere
The condensed state is important,
allowing the duplicated chromosomes to
be separated
DNA polymerase requires a primer at the end of the chromatin.
Without telemers, chromosomes would continually shorten.
Also protects ends from attack by the DNA-digesting
enzymes – nucleases.
A. chromatin spilling out of a
lysed interphase nucleus
B. a mitotic chromosome,
which is duplicated already
A. chromatin isolated from an interphase nucleus = 30 nm
B. chromatin “unpacked” after isolation
The nucleosome is the first and
most fundamental packing level of
chromatin.
Histones are small proteins with a
high proportion of positively
charged amino acids (lysine and
arginine). These genes are the
most highly conserved of all know
eucaryotic proteins.
Form a histone octamer
Chromosomes have
several levels of
DNA packing.
Histons H1 pulls the
other histones
together to form the
typical 30-nm
chromatin fiber
1/3 the length
cytoskeleton organizes
the chromatin
In the more compact
state, RNA
polymerase etc can
not bind, and
transcription stops.
A typical mitotic chromosome
• Interphase chromosomes contain both condensed
and more extended forms of chromatin
– highly condensed interphase chromatin, is called
heterochromatin and makes up about 10% of the
chromatin and is transcriptionally inactive.
• normally active genes moved to this region are inactivated
– euchromatin = all of the chromatin except
heterochromatin, varies
– regions that are actively being transcribed into RNA or
easily available for transcription are more extended
(10%) – active chromatin [30-nm fiber or 300nm]
• transcription (RNA polymerase) and replication (DNA
polymerase are not obstructed by histones. It is thought that
the DNA partially detaches from the histone core as a
polymerase moves through, and reassembles immediately.
Happens early in
development
Female becomes a mosaic,
half of the cells in each
organ are maternal, half are
paternal
Position effects
A. ADE2 encodes an enzyme whose
absence leads to the accumulation
of a red pigment.
B. The white gene controls eye
pigment production. When
inactivated the flies have white
eyes.
It is thought the heterochromatin
areas can spread and control the
expression of genes near them
Interphase chromosomes are organized within the nucleus. The
nuclear envelope is supported by two network of protein filaments,
the nuclear lamina inside and intermediate filaments outside the
membrane. Nuclear pores actively and specifically transport.
The interior is not a random jumble of DNA, RNA, etc. It is thought that DNA is organized
by attachment of parts of the chromosomes to sites on the nuclear envelope or the nuclear lamina.
Gene regulation:
As an organism
develops, cells
differentiate into
various types of
cells.
Differentiation
arises because
cells make and
accumulate
different sets of
RNA and protein
molecules – they
express different
genes.
Most of the cells in
an organism
contain the entire
genome. The
difference between
a muscle cell and a
neuron depends on
gene regulation.
Gene (eucaryotic) control.
Transcription is controlled by proteins binding to regulatory
DNA sequences.
Expression depends on
Promotor includes
cell type
its environment
RNA polymerase binding site
its age
initiation site
extracellular signals
Regulatory DNA sequences bound by gene regulatory protiens
some short – simple gene switches
some long and complex (eucaryotic)
molecular microprocessors which
respond to a variety of signals,
integrate them, and determine the
rate of transcription.
Edge of bases
Gene regulatory proteins
Homeodomain – a
insert into the major groove,
structural motif
contacts and binds
(noncovalently) to the edges
of the bases (about 20
interaction), usually without
disrupting the hydrogen
bonds. This binding is very
strong and very specific for
the nucleotide sequence. Zinc finger motif
Frequently DNA-binding
proteins contain alphahelices bind in pairs Leucine
dimerization - which
zipper
doubles the contact area,
motif
increasing the strength and
specificity of the interaction.
An operon = a cluster of bacterial or viral genes transcribed
from a single promoter.
A sequence of DNA within the promotor is
recognized by regulatory proteins - the operator.
Repressor protein switch genes on or off.
These regulators allow fast
response to the environment
because they are always present in
the cell - constitutively expressed
Is recognized well by RNA
polymerase.
Bacteria in your gut after you
eat a steak
Regulatory proteins like these are allosteric.
Activator proteins act on promoters that do not bind RNA
polymerase on their own. These promoters are made fully
functional by the addition of a bound activator protein
which is also allosteric, activated by binding another
molecule at a site different from the DNA binding site.
Example: CAP has to bind cyclic AMP (an intracellular
signaling molecule) to bind DNA. Genes regulated by CAP are
switched on in response to increases in cAMP, which is
triggered by signals received by cell membrane receptors.
Initiation of transcription in eucaryotic cells is
more complex as compared to the simple,
economical procaryotic genetic switches.
1. Three RNA polymerases (Table 1)
2. Eucaryotic RNA polymerases require
general transcription factors to bind the
promoter.
3. Gene regulatory proteins (repressors and
activators) can influence initiation of
transcription from regulatory sequences found
thousands of nucleotide pairs away from the
promoter - enhancers.
4. Transcription is impacted by the chromatin
structure.
General transcription factors assemble on
all promoters transcribed by RNA
polymerase II
Start with binding of TFIID which binds a
short DNA sequence with many A-Ts TATA box - typically 25 nucleotides
upstream from the transcription start site.
This binding causes a dramatic distortion in
the DNA. Figure 24.
Other factors assemble along with RNA
polymerase = transcription initiation
complex. TFIIH contains a protein kinase
subunit which adds phosphates to RNA
polymerase, releasing it from the complex
to start transcription
Start with binding of TFIID
which binds a short DNA
sequence with many A-Ts TATA box
This binding causes a
dramatic distortion in the
DNA.
TBP is a subunit of TFIID
which binds the TATA box.
Partially
unwound
areas
A model for gene activation
from an enhancer
Enhancers can be
thousands of base pairs
away, either upstream or
downstream and can either
increase or decrease
transcription.
“Action at a distance”
General
transcription
factors usually can
not efficiently
initiate
transcription
alone.
Chromatin structure effects gene
transcription.
The presence of nucleosomes
does not generally block
elongation but they may inhibit
initiation if they are positioned
over a promoter
The effect of chromatin
structure on transcription
initiation is not well understood
yet. More compact chromatin
does block transcription.
The regulatory mechanisms
involved in packaging
interphase chromatin
(heterochromatin, inactive X)
are also still a mystery.
Summary of gene activation and regulation in
procaryotes and eucaryotes.
Gene Regulatory sequences of a typical eucaryotic cell
Regulated by combinations of proteins over up to 50,000 nucleotide pairs
Combinatorial control can be positive or negative
Model for the control of the human beta-globin gene
Some gene regulators like CP1 are present in many types of cells
Others like GATA-1 are only found in a few types of cells.
These are thought to contribute to the cell-type specificity of gene expression
The expression of different genes can be coordinated by a single
protein in eucaryotic cells. This can result in rapid switching on or
off of whole groups of genes.
Glucocorticoid steroid hormone
is released in response to
starvation or intensive physical
activity. This hormone
stimulates many different cells
to do many different things,
including liver cells to increase
expression of many different
genes, some to stimulate the
production of glucose from
amino acids. All of these genes
responding to glucocorticoid
steroid are regulated by the
binding of the hormoneglucocorticoid receptor complex
to a regulatory site.
Regulatory proteins specific
for this gene
Regulatory proteins
(combination control)
specific for this gene
Combinatorial control
many control one
Single protein control
one controls many
This system allows the dramatic
differences between cell types to be
controlled during differentiation and
produced by differences in gene
expression
Myoblasts (mucle precursor cells) fuse
and produce actin and myosin, ion
channel proteins, etc. One regulatory
protein involved is MyoD.
Fiberblasts from the skin, transfected with
MyoD behave like myoblasts. Other cells
do not.
Stained
green with
an antibody
that binds a
muscle cellspecific
protein.
Combinatorial gene
control generates
different cell types.
A limited set of gene
regulatory proteins
can control the
expression of a much
larger number of
genes.
Different
combinations
will produce
different
phenotypes
Notice that each
daughter cell
remembers the
previous gene is
activated.
When differentiate cells divide, they produce the same type
of cells. Muscle and Neurons do not divide, but others like
fibroblasts, smooth muscle cells hepatocytes do. Two
possible mechanisms include positive feedback loops and
propagation of chromatin structure.
Positive feedback loops
include proteins that
regulate their own genes.
Model for propagation of a condensed chromatin structure.
As in X chromosome inactivation.
The formation of an entire organ can be triggered by a single
gene regulatory protein. Example, eye development in
Drosophila, mice, and humans.