Transcript Gene Regulation

Gene Regulation
Intro
• What causes gene products to be synthesized in some
cells under some conditions, but not in others? A large
part of research in molecular biology in aimed at trying to
determine this. We are going to talk about a few basic
systems, but a whole course could easily be devoted to
the subject.
• For most genes, the essential regulation point is
transcription: whether the gene is transcribed or not.
Regulation also occurs at other points: availability of the
DNA to be transcribed at all, whether the mRNA is
translated, stability of the mRNA, how quickly the protein
is degraded, etc.
Gene Regulation in Prokaryotes
• The first system of gene regulation that was
understood was the lac operon in E. coli, worked
out by Francois Jacob and Jacques Monod in
1962. Many other prokaryotic genes are
regulated in a similar fashion, and the basic
principles carry over into eukaryotes.
• The lac operon codes for enzymes involved in
the degradation of lactose. Lactose is a
disaccharide that can be used as food in the
absence of glucose. A lac- mutant is a
chemoauxotroph that can’t use lactose.
Structure of the lac Operon
• The lac operon consists of 3 protein-coding genes plus
associated control regions.
• The 3 genes are called z, y, and a. lacZ codes for the
enzyme beta-galactosidase, which splits lactose into
glucose plus galactose. lacY codes for a “permease”
protein that allows lactose to enter the cell, and lacA
codes for an enzyme that acetylates lactose. Together
these three genes are called the “structural genes”. We
will mainly focus on lacZ.
• All 3 genes of the lac operon are transcribed on the
same messenger RNA. Ribosomes translate the 3
proteins independently. This is a feature of prokaryotes
that is only very rarely seen in eukaryotes, where 1 gene
per mRNA is the rule.
Control Regions
• Near the lac operon is another gene, called lacI, or just
“i”. It codes for the lac repressor protein, which plays an
essential role in lac operon control. The lac repressor
gene is expressed “constitutively”, meaning that it is
always on (but at a low level). It is a completely separate
gene, producing a different mRNA than the lac operon.
• Just upstream from the transcription start point in the lac
operon are two regions called the operator (o) and the
promoter (p). Neither region codes for protein: they act
as binding sites on the DNA for important proteins.
• The promoter is the site where RNA polymerase binds to
start transcription. Promoters are found upstream from
all protein-coding genes.
• The operator is where the actual control occurs.
Visual
Control
• The lac repressor protein (made by lacI) has 2 states: it can either
bind to lactose (technically, to a lactose derivative called allolactose)
or it can bind to the operator region of the lac operon.
• In the presence of lactose, the repressor binds to it, and the
repressor-lactose complexes float freely in the cytoplasm away from
the DNA. In this situation, RNA polymerase can bind to the
promoter, and the gene is transcribed. It makes beta-galactosidase
which digests the lactose.
• In the absence of lactose, the repressor binds to the operator DNA.
The repressor is a large molecule, and when it is bound to the
operator, RNA polymerase is blocked from reaching the promoter.
The lac operon is not transcribed, and no beta-galactosidase is
made.
• If lactose appears, the operon is said to be “induced”. The lactose
binds to the repressor, which then falls off the operator and allows
transcription to occur.
Picture
Genetic Analysis with Mutants
• Jacob and Monod developed their model of lac
regulation through the use of mutants. Later,
biochemical techniques showed that their model was
correct.
• They isolated two main types of mutant: lac- mutants,
which can’t use lactose as a food source, and
“constitutive” mutants, in which the lac operon is always
on regardless of external conditions.
• They then tested these mutants alone and in
combination with each other, with extensive use of
merodiploids (partial diploids) to test for dominance. In
particular, they created and used several F’ strains.
Constitutive Mutants
• There are two ways of making a mutant strain
where the lac operon is always on, regardless of
whether lactose is present or not.
• One way is an i- mutation: the lacI gene does not
produce a functional repressor protein. Since
there is no repressor to bind to the operator,
RNA polymerase is never inhibited, and the lac
operon is always transcribed.
• i- mutants are recessive: an i+ / i- heterozygote
has normal gene regulation, because the wild
type allele produces a normal repressor.
Operator Mutants
• The operator is a region of DNA upstream from the structural genes
that binds the promoter protein. It doesn’t make a protein product.
• Mutations in the operator can only affect the gene to which it is
attached. Such mutants are said to act in cis, or to be “cisdominant”.
• In contrast, repressor mutants make a protein which can move freely
through the cell to any copy of the gene. Repressor mutants are
“trans-acting”. (This terminology comes from cis and trans in organic
chemistry.)
• Many operator mutants are constitutive, oc. The operator is mutated
so that the repressor can no longer bind to it. Transcription occurs
and the lac operon is on when no repressor is bound to the operator.
• Demonstrating cis-dominance: oc z+ / o+ z- is always on: the normal
lacZ allele is attached to the constitutive operator. In contrast, oc z- /
o+ z+ has normal regulation. The constitutive operator is attached to
a defective lacZ gene, so expression of this gene is not detected,
while the normal operator is attached to a normal lacZ gene, giving
normal gene regulation.
Lac- Mutants
• Most mutations in the 3 structural genes of the lac operon, lacZ,
lacY, and lacA, affect only that particular gene. Thus, a lacZ- mutant
is usually lacY+ and lacA+.
• However, mutations in the control regions usually affect all 3 genes
simultaneously.
• One control mutation that affects all 3 genes is the super-repressed
mutation, iS. The repressor protein made by this mutant binds very
tightly to the operator and does not bind to lactose. The effect is
that the lac operon is always repressed, even when lactose is
present.
• iS mutants are dominant: an iS / i+ merodiploid shows the superrepressed phenotype: it is always off. A mixture of normal
repressor proteins and super-repressor proteins will end up with the
super-repressor sticking permanently to all copies of the operator
that are present in the cell. In contrast, i- mutants are recessive.
Cis-Acting oS Mutants
• The operator can also be mutated to a
super-repressed state, in which the
operator binds so tightly to the repressor
that it never gets released.
• oS mutants act in cis. For example, a oS z+
/ o+ z- strain is always off, because the
only functional lacZ gene is attached to the
super-repressed operator. A oS z- / o+ z+
strain shows normal regulation.
Summary of Lac Control Mutants
• lacI = repressor gene, makes repressor protein. Mutants
act in trans.
– i+ = normal regulation: ON in presence of lactose, OFF in
absence of lactose.
– i- = no repressor made, gene always ON, recessive.
– is = super-repressor permanently bound to operator. Gene
always OFF, dominant.
• lacO = operator region, controls expression of the
attached lacZ, Y and A genes. Mutants act in cis only.
– o+ = normal expression of attached structural genes.
– oc = constitutive, represor unable to bind to operator. Lac genes
in cis are always ON.
– os = super-repressed operator. Normal repressor protein stays
permanently bound. The attached lac genes are always OFF.
A Few Examples
• What is the lacZ (beta-galactosidase) phenotype
of these genotypes: normal expression, always
on, or always off?
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i+ oc z+
i+ oc zis oc z+
i+ os z+ / i+ oc zi- o+ z+ / i- oc zi- os z- / is oc z-
Negative and Positive Regulation
• As described above, the lac operon is negatively
regulated: the regulatory protein (repressor)
causes transcription to stop.
• Positive regulation, where the regulatory protein
causes transcription to start, is more common.
• The lac operon also contains an example of
positive regulation, called “catabolite
repression”. E. coli would prefer to use glucose
as its food source. In the presence of glucose,
the lac operon (and other similar genes) are
turned off, even if lactose is present in the
medium.
Catabolite Repression
• Catabolite repression uses a regulatory protein called CAP
(catabolite activator protein). It also uses the small molecule cyclic
AMP (cAMP).
• cAMP is made from ATP. When the glucose level in the cell is high,
the cAMP level is low, because glucose inhibits synthesis of cAMP.
When the glucose level is low, the cAMP level is high.
• cAMP combines with the CAP protein to form a complex that binds
to part of the lac operon promoter. This complex bends the DNA in
a way that makes it much easier for RNA polymerase to bind to the
promoter. This allows transcription to occur, but only if the lac
repressor isn’t present.
• Thus, low glucose levels cause high cAMP levels. When cAMP is
high, it combines with CAP The CAP-cAMP complex then binds to
the promoter to allow transcription to occur.
• This is positive regulation because the binding of CAP to the DNA
causes transcription to occur.
Eukaryotic Gene Regulation
• Things are a bit more complex in higher eukaryotes (such as
humans).
• Major factors affecting gene expression:
– Proteins and DNA sequences that affect binding of RNA polymerase to
the promoter
– Changes in chromatin structure
– Alternate RNA splicing patterns
– Regulation by small RNA molecules after transcription
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Other forms of regulation also exist.
Eukaryotic Transcription Initiation
• In eukaryotes, there are 3 RNA
polymerases. However, all proteincoding genes are transcribed by RNA
polymerase 2 (pol2).
– Pol1 transcribes the ribosomal RNA
genes
– Pol3 transcribes transfer RNA genes
• Pol2 binds to the “TATA” box, a region
of about 8 bases which are mostly A
and T. This is the equivalent of the
promoter in prokaryotes.
• Pol2 binding can only occur if several
other proteins, the general transcription
factors, have already bound to the
DNA.
Activator Proteins, Response Elements
and Enhancers
• Just upstream from the TATA box there are a set of short DNA sequences
(response elements) that regulate the time, tissue, and amount of
transcription that occurs.
– Response elements work in cis only
– The order of response elements near a gene isn’t important
• The proteins that bind to response elements, the activator proteins, influence
the ability of RNA polymerase to start transcription. They are directly
responsible for controlling the pattern of gene expression in higher
organisms.
– Activator proteins are trans-acting factors
– There are also repressor proteins that reduce transcription.
• Most response elements are directly upstream (say, within 200 bp) of the
transcription start.
– However, enhancers are groups of response elements found further away, either
upstream or downstream from the gene.
– Enhancers work the same way as regular response elements: they bind activator proteins
that affect RNA polymerase binding.
– Silencers are enhancers that repress transcription instead of increasing it.
Activator Proteins Interact with
RNA Polymerase
Chromatin Structure
• In eukaryotes, the DNA is organized into
nucleosomes: about 200 bp of DNA
wrapped around a protein core.
• The protein core consists of 8 histone
proteins
• Histones are basic (i.e. alkaline): they
contain positively charged amino acids
that bind to the negative charges on the
DNA (backbone phosphate groups).
• DNA tightly wrapped around histones is
inaccessible to RNA polymerase.
• Thus, one important event in preparing a
gene for transcription is “chromatin
remodelling”: sliding the nucleosomes
along the DNA to expose the promoter
region.
Histone Acetylation
• A second event needed for transcription
affects large regions of the chromosome
instead of individual genes.
• DNA is normally tightly wrapped around
the histones and is inaccessible to
transcription factors. The structure can
be loosened by acetylating the histones.
• Acetyl groups are added to lysines,
which removes their positive charge.
The binding of the DNA to the histones is
lessened, and the DNA structure opens
up, allowing access to transcription
factors.
• Conversely, deacetylation tightens the
chromatin structure, preventing
transcription throughout that region of the
chromosome.
Alternative RNA Transcription and
Splicing
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Many genes in eukaryotes contain
introns.
– This is especially true of large
multicellular organisms like humans.
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Introns are spliced out of the primary
RNA transcript of the gene before it gets
translated into protein.
Variant mRNAs (and resulting proteins)
can be generated by skipping some
introns, or by using a sequence as an
intron in one cell type and as an exon in
another cell type.
Alternative promoters or polyadenylation
sites. Are also used to generated
variants at the beginning and end of the
mRNA (and protein).
Variant proteins are called isoforms.
Micro RNAs
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Micro RNAs (miRNA) are the products of a new type of
RNA-only (i.e. not translated into protein) genes. Their
discovery and significance has only been known since
about 2000.
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miRNAs regulate gene activity in the cytoplasm, by
binding to messenger RNA molecules.
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An example of “junk” DNA that was later found to have a
function.
This causes the mRNA to be untranslatable, or to be
degraded.
Different sets of miRNAs are expressed in different
tissues.
miRNA genes are transcribed into an RNA molecule that
spontaneously forms a hairpin.
After some RNA processing, the miRNA is joined to a
protein complex called RISC.
When the miRNA in RISC binds to a cellular messenger
RNA, RISC acts as an RNAase to destroy the mRNA so
it can’t be translated into protein.
Epigenetics
•
Epigenetics is the study of inherited changes caused by mechanisms other
than changes in the DNA sequence.
– This can between parent and offspring, or between cells within a single
organism.
– Within an organism, epigenetic changes are the main reason why it isn’t easy to
take the nucleus from any random cell and use it to grow a whole new organism
(i.e. reproductive cloning).
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The molecular basis for (most) epigenetic mechanisms seems to be the
methylation of cytosines in the DNA. The C must be followed by a G (CpG)
for this to happen.
Methylation of C’s near the promoter region of a gene prevents
transcription. This means a heavily methylated gene is permanently
inactivated.
Each cell type and tissue has its own methylation pattern, keeping some
genes functional and others permanently inactivated. This provides cells
with "memory": after cell division, the daughter cells know what their type is.
More Epigenetics
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How is the methylated state preserved? All of the cytosine
methylations are renewed with every DNA replication: an
enzyme called hemimethylase recognizes a 5-methyl C on
the old strand, then methylates the corresponding C in the
new strand.
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Recalling that the methylation sequence CpG is also CpG on
the opposite strand.
The methylation pattern is (mostly) reset in the early
embryo, allowing embryonic cells to develop into any cell
type.
An example of epigenetic effects: a small deletion of part of
the long arm of chromosome 15 results in Prader-Willi
syndrome if the single copy of chr 15 is inherited from the
mother, and Angelman syndrome if the chromosome is
inherited from the father.
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This chromosomal region has different methylation patterns
depending on whether it come from the sperm or the egg.
Prader-Willi is characterized by an uncontrollable appetite for
food (among other things).
Angelman has been called the Happy Puppet syndrome: jerky
movements and happy, laughing demeanor.