Regulation of Gene Expression

1
Regulation of Gene Expression
constitutive genes genes expressed at all times
and not regulated (ie. RNA polymerase, tRNAs,
glycolysis enzymes, etc) aka housekeeping
genes regulated genes genes whose expression
varies based on the needs of the cell-- often
controlled at the level of transcription cataboli
c pathways group of enzymes to degrade something
(often for generating energy) anabolic
pathways group of enzymes that make something
(ie. tryptophan, vitamins, etc) in bacteria (and
to a lesser extent eukaryotes), small organic
molecules known as effectors act as allosteric
transcriptional regulators
2
Regulation of Lactose Catabolism
lactose disaccharide composed of glucose and
galactose connected by a b glycosidic bond--
commonly found in milk often hard for adults
to utilize (ie. lactose intolerance) and an
inducible pathway in bacteria Jacob and Monod--
two french scientists who developed the
regulation of lactose genes as the general
model for how gene regulation occurs control of
lactose utilization involve 2 types of genes
1) structural-- enzymes involved in lactose
uptake and utilization 2) regulatory--
proteins that regulate the activity of structural
genes 3 structural genes involved in lactose
metabolism 1) lacZ- codes for b-galactosidase,
breaks b glycosidic bonds 2) lacY- galactoside
permease, membrane protien transporting lactose
3) lacA- transacetylase which adds an acetyl
group to lactose all 3 lie near each other in
the genome and each protein is only made
when lactose is present
3
Regulation of Lactose Catabolism
operon a single regulatory unit that controls
the expression of a set of genes with related
functions and which are transcribed
together operons are the most common gene
regulatory system in bacteria not typically
used in eukaryotes (which does use the same
principles) lacI- gene which controls inducible
expression of lacZ, lacY and lacA if lacI is
deleted, the lac operon is always expressed lacI
is a repressor (inhibitor) of lac opteron
transcription lac operon is organized as a
promoter and the 3 structural lac genes
overlapping the promoter is a DNA sequence
called the operator- regulatory regions of DNA
often have several overlapping protein sites
4
Regulation of Lactose Catabolism
note that the 3 lac genes have only 1 regulatory
region all 3 genes are expressed from the same
RNA molecule polygenic RNA single RNA that
codes for more than 1 protein chain found in
most if not all prokaryotes and some viruses
allows several structural genes to be regulated
at once the lac repressor protein binds to the
operator sequence in the promoter binding
prevents the RNA polymerase from binding since
RNA polymerase can't bind, transcription can't
occur lacI is located elsewhere in the genome
under a different promoter lacI protein is
produced regardless of whether the lac operon
is active
5
Regulation of Lactose Catabolism
the lac repressor normally binds the operator
element in the lac promoter lactose
allosterically regulates the lac repressor so
that it cannot bind to the operator when bound
to lactose-- inhibits the inhibitor lactose
causes the repressor protein to come off the DNA,
which allows RNA polymerase to bind and the
polygenic lac operon to be transcribed note
that the lacI protein is still present and being
transcribed if lactose goes away, repressor
protein can bind to DNA again inducible operon
operon that gets turned on only in specific
conditions
6
Regulation of Lactose Catabolism
initial evidence for the regulation of the
lactose operon was genetic mutations were
either in the structural genes (lacZ, lacY,
lacA) mutations also found in regulatory
elements (lacI, operator, promoter) mutations in
structural genes affected only 1 of the
proteins mutations in regulatory gene/regions
affected all 3 structural proteins damage the
promoter, RNA polymerase can't bind lower
expression damage the operator and RNA
polymerase always transcribes the operon
damage lacI DNA binding, repressor cannot bind so
operon transcribed damage lacI lactose
binding, repressor always binds, no
transcription suppressor mutation change which
prevents operon transcription whether or not
the inducer is present
7
Regulation of Lactose Catabolism
trans protein factor
cis local DNA
cis acting mutation (or sequence) acts near the
site of that DNA ie. promoters or operator
elements that regulate local operon
expression trans acting mutation gene that acts
at a distance from what it regulates ie.
repressors or activators change transcription at
a different operon IPTG is an alternative
inducer of the lactose operon-- binds like
lactose but is not metabolized for energy so
lower amounts are needed to induce the protein
expression lac promoter/operon is very often
used to express a protein in bacteria
8
Tryptophan Synthesis Regulation
many catabolic processes are regulated by
inducible operators (repressors) many anabolic
processes are regulated by repressible elements
(activators) trp operon structural genes and
regulatory elements needed to produce the
amino acid tryptophan when there is enough
tryptophan, turns off trp operon
transcription trpR is an allosteric repressor
that inhibits transcription with
tryptophan corepressor requires both tryptophan
and trpR protein to repress operon both lac and
trp operons are examples of negative regulation-
inhibitors of transcription
9
Positive Regulation
positive regulation key regulator turns on gene
transcription instead of turning it
off catabolite repression ability of glucose
(the preferred energy source of most cells)
to inhibit the synthesis of other catabolic
operons glucose is the sugar directly
utilized to make ATP-- other sugars get
converted into glucose or intermediate before
they can be used instead of glucose, catabolite
repression uses a secondary signal that is the
inverse of the level of glucose in a cell--
cyclic AMP (cAMP) binds to cAMP receptor
protein cAMPreceptor protein binds within
operons to CRP recognition sites to enhance
transcription-- activator-- positive effector
10
Dual Control Regulation
CRP recognition sites allow operons to be turned
on at high levels when glucose is not
present lac operon is under dual control-- very
common for inducible genes one regulatory
element activates operon (CAP site binding CRP
protein) one regulatory element represses
operon (Operator binding lacI) balance of
activation and repression controls overall rate
of transcription
activator
11
Sigma factor regulation of transcription
multiple different sigma factors regulate
transcription initiation s72 initiates
transcription at most bacterial promoters s32
binds at higher temperature-- changes some
proteins that get made s54 allows transcription
of genes for nitrogen utilization bacteriophage
often make a unique sigma factor-- by making
their own sigma factor, they can take over the
RNA polymerase of the cell to preferentially
transcribe their own genes viral sigma factors
allow virus to make more of themselves at the
expense of cellular functions
12
Gene Regulation in Eukaryotes vs. Prokaryotes
general similarities between eukaryotic and
prokaryotic regulation cis DNA elements
control transcription of a nearby gene trans
acting proteins (transcription factors) regulate
RNA transcription however eukaryotes have
much more complicated and elaborate cis
elements multiple transcription factors at
several locations regulate transcription
additional transcriptional regulators do not
recognize DNA directly but interact with
proteins bound to DNA several levels of
post-transcriptional control to further regulate
when a protein is made from the mRNA
13
Gene Regulation in Eukaryotes vs. Prokaryotes
Eukaryotic genomes are 2-3 orders of magnitude
larger than prokaryotes larger regulatory
regions (can be over a million bases long!)
introns within genes (transcribed DNA that does
not code for proteins) alternative splicing
generates several mRNAs from the same
DNA Eukaryotic genomes are compartmentalized--
stuck in the nucleus transcription and
translation are separated in both time and space
barrier allows mRNA processing but prevents
attenuation control Eukaryotic chromosomes are
more complex than bacterial DNA eukaryotic DNA
sometimes has to be 'uncoiled' before it is
transcribed each gene is made as a separate
mRNA but promoter regions are alike gives
flexibility at the expense of added complexity
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Gene Regulation in Eukaryotes vs. Prokaryotes
Eukaryotic mRNAs can (not always!) persist longer
than in prokaryotes eukaryotes have a more
predictable environment-- slower response
time eukaryotes modify proteins more than
bacteria-- signal sequences, glycosylation,
etc. bacteria modify proteins, but not to the
same extent eukaryotic cells need to
specifically degrade unnecessary proteins
bacteria constantly divide so can 'remove'
proteins by dilution eukaryotic cells often
stop dividing and survive for a long time
15
Stem Cells and Differentiation
most eukaryotic cells are no longer dividing--
multicellular organsisms usually stop growing
at some point eukaryotic cells are also
differentiated-- cell types specialize for
certain functions and manufacture different
proteins stem cells immature, nonspecialized
cells that can divide an unlimited number of
times and which can give rise to several (or all)
types of cells found in the adult stem cells
are found in embryos, but many are found in
adults as well hematopoietic stem cells are
found in the bone marrow-- make blood neural
stem cells are found in the brain, make neurons
and glia, etc. 5 different levels of regulating
protein function in eukaryotes genome,
transcription, RNA processing/export,
translation, post-translational
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Stem Cells and Differentiation
17
Transcriptional Control
different genes are transcribed in different cell
types with some overlap general transcription
factors (discussed in Chapter 19) are essential
for the transcription of all genes RNA pol II
(for making mRNA) has the core promoter with BRE
element, TATA box, initiator segment and
DPE core promoter will give a low minimal level
of transcription-- basal level proximal
promoter DNA elements within 200 bp of the core
promoter that can change the level of
transcription regulatory transcription factors
transcription factors outside the core
promoter which can change (either increase or
decrease) transcription
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Transcriptional Control
other control elements lie even further away from
the proximal promoter enhancers control region
that increases transcription silencer control
region that decreases transcription both
enhancers and silencers can regulate
transcription from nearby or far away-- distance
is not a critical feature for them for
enhancers (or silencers) to function, they must
be bound by protein transcription factors
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Transcriptional Control
activator transcription factor which binds
enhancers and increase the level of
transcription for a gene repressor protein that
binds silencers and reduces transcription from a
gene both activators and repressors have
several of the same characteristics both need
to loop DNA to bring them close to the proximal
promoter both interact with other proteins
(coactivators or corepressors) which
recognize both the regulated and general
transcription factors many coactivators have
histone acetyltransferase activity-- catalyze
the acetylation of histones which can increase
transcription combinatorial model of gene
regulation 'relatively' small number of
regulatory proteins work in various combinations
to generate many different patterns of gene
expression using activators and repressors
20
Transcriptional Control
enhancers bound by activators work with
coactivators to increase transcription Silencer
elements would be bound by repressors and work
with corepressors to block TFIID or other
general transcription factor
21
Transcriptional Control
if the brain would have a repressor that binds in
the albumin promoter even less transcription
only a few common families of activators and
repressors
22
Transcriptional Control
Regulatory transcription factors need 2 key
features 1) DNA binding domain-- part of the
transcription factor that physically
interacts with DNA 2) activation domain--
protein region that interacts with the
transcription apparatus to enhance
transcription (or for repressors, reduce it) DNA
binding and transcription activation are
generally separable features an activation
domain from one factor can work with a DNA
binding domain from another many activation
domains have an a helix with many acidic amino
acids giving it a fairly strong negative
charge other structures can also form an
activation domain
23
DNA binding motifs
motif relatively small region of protein that
forms a consistent 3D structure helix turn
helix 2 a helices separated by a loop (or turn)
one a helix (recognition helix) binds DNA in
the major groove second a helix stabilizes the
recognition helix
24
DNA binding motifs
zinc finger motif 3D shape composed of 1 a helix
and a 2 strand b sheet held together by a zinc
atom bound by 4 cysteines and/or
histidines several are usually found together in
one transcription factor individual 'fingers'
stick into the major groove of DNA for
recognizing particular sequences of DNA
25
DNA binding motifs
leucine zipper motif an a helix which binds the
major groove of DNA and a second a helix that
has regularly spaced leucines the leucines form
a hydrophobic binding surface for a second
leucine zipper protein to wrap around it into
a coiled coil, forcing these proteins to
function as a dimer some zippers form homodimers
(2 identical subunits) others form heterodimers
(2 different subunits)
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DNA binding motifs
helix loop helix similar to both
helix-turn-helix leucine zipper proteins,
has 1 a helix binding the DNA in the major groove
and a second helix these proteins dimerize and
form a 4 a helix bundle instead of a coiled
coil forms heterodimers or homodimers like
leucine zippers
27
Coordinating Gene Expression
in eukaryotes, genes turned on or off at the same
time are usually scattered through the
genome response elements DNA control sequences
that turn on transcription in response to some
environmental or developmental signal response
elements can be either proximal or a component of
enhancers genes that respond to the same signal
have the same response element many different
types of response elements which act together
with all the other control circuits to give
the final expression pattern ie. different
tissues (ie. brain or liver or muscle) can turn
on different genes using the same response
element
28
Steroid Hormone Receptors
steroids are hormones related to cholesterol that
are released into the bloodstream because
steroids are lipids, they are able to cross the
cell membrane bind to nuclear receptors in the
cytoplasm of a cell where they cannot bind to
DNA and are usually bound to an inhibitory
protein subunit steroid binding induces an
allosteric change and causing its binding
partner to dissociate and revealing a nuclear
localization sequence-- allows the protein to
enter the nucleus and bind DNA binds to hormone
response elements (15 bp total) using zinc
fingers usually need 2 nuclear receptors to turn
on transcription-- generally have inverted
repeat sequences, each recognized by one of the 2
proteins very common theme among regulatory
transcription factors
29
Steroid Hormone Receptors
5'-TCCAGTACTGGA-3' 3'-AGGTCATGACCT-5'
thyroid hormone response element inverted
repeat sequence nuclear receptors bind to all of
the correct response elements regardless of
their location on the chromosome some hormone
receptors bind hormone binding sites that are
either excitatory or inhibitory-- one protein
can stimulate some genes and repress others
30
Homeotic Genes
homeotic gene transcription factor that
determines body organization mutations cause
one part of the body to be replaced by
another homeotic genes control how other genes
get expressed so that they form the
appropriate type of structure -- critical
regulator has an amazingly highly conserved
helix turn helix that binds DNA
31
Translational Eukaryotic Gene Regulation
RNA interference (RNAi) targeted degradation of
particular mRNAs using a 22-23 bp piece of
double stranded RNA which is complimentary to
the sequence of the mRNA if the double stranded
RNA has some mismatches with the mRNA, it can
simply block translation of that mRNA believed
to have evolved as an anti-viral response to
protect against RNA viruses mRNA naturally
occurring RNAs which can inhibit the translation
of different normal mRNAs mRNAs are regularly
found within introns, so that the transcription
of one gene results in the negative
translational regulation of a different gene
32
Posttranslational Gene Regulation
several features regulate the control of protein
degradation, including the protein
sequence rate of protein degradation is also
under cellular control-- some proteins can be
targeted for destruction while others are left
alone ubiquitination mechanism by which
eukaryotes mark proteins for death
discoverers won the Nobel Prize in Chemistry in
2004 uses a common 76 aa protein chain
(ubiquitin) attached to lysines requires 3
enzymes 1) a ubiquitin-activating enzyme
(E1) activates ubiquitin using ATP 2) a
ubiquitin-conjugating enzyme (E2) intermediate
holding activated ubiquitin 3) a substrate
recognition protein (E3) targets addition of
ubiquitin to a particular protein or few proteins
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Posttranslational Gene Regulation
additional ubiquitins get added, forming a
short chain proteasome large, protein
degradation structure which recognizes
ubiquitinated protein and breaks it down via
several proteases selectivity is based on the
substrate recognition enzymes, or E3 amino
acid at the very N-terminus is an important
position for determining how quickly a
protein is ubiquitinated and degraded