Transcript Document

LECTURE PRESENTATIONS
For CAMPBELL BIOLOGY, NINTH EDITION
Jane B. Reece, Lisa A. Urry, Michael L. Cain, Steven A. Wasserman, Peter V. Minorsky, Robert B. Jackson
Chapter 18
Regulation of Gene Expression
Lectures by
Erin Barley
Kathleen Fitzpatrick
© 2011 Pearson Education, Inc.
Gene expression: Bacteria vs. Eukaryotes
• prokaryotes and eukaryotes alter gene expression in response to their changing
environment
– gene expression = refers to the entire process whereby genetic information is decoded
into a protein
• prokaryotes and eukaryotes carry out gene expression in similar ways
– transcription using an RNA polymerase
– translation using ribosomes
• but there are some differences:
–
–
–
–
–
1. RNA polymerases differ – only one in prokaryotes; 3 in eukaryotes
2. transcription factors used by eukaryotes
3. transcription is terminated differently in prokaryotes vs. eukaryotes
4. ribosomes – bacterial ones are smaller
5. lack of compartmentalization in bacteria – transcribe and translate at the same time
So what is a gene?
•
•
•
•
•
unit of inheritance
located on chromosomes
region of specific nucleotide sequence located along the length of DNA
DNA sequence that codes for a specific sequence of amino acids
BUT: some DNA sequences are NEVER translated
– e.g. rRNA and tRNA are transcribed but not translated into anything
• so a gene is a region of DNA that is either
– 1. translated into a sequence of amino acids (polypeptide)  functional protein
– 2. transcribed into a RNA molecule
So what is a gene?
• molecular components of a gene:
–
–
–
–
–
A. coding sequences - eukaryotes have introns within their coding sequence
B. promoter
C. enhancers – found in eukaryotes
D. UTRs – found in eukaryotes
E. poly-adenylation sequence – found within the eukaryotic 3’ UTR
Overview: Conducting the Genetic
Orchestra
• genetic and biochemical work in bacteria identified two things
– 1. protein-binding regulatory sequences associated with genes
– 2. proteins that can bind these regulatory sequences – either activating or repressing
gene expression
• these two components underlie the ability of both prokaryotic and eukaryotic cells
to turn genes on and off
Bacteria often respond to environmental
change by regulating transcription
• natural selection has favored
bacteria that produce only the
products needed by that cell
• bacteria regulate the production
of enzymes by feedback
inhibition or by gene regulation
• gene expression in bacteria is
controlled by the operon model
Precursor
Feedback
inhibition
trpE gene
Enzyme 1
trpD gene
Enzyme 2
Regulation
of gene
expression
trpC gene

trpB gene

Enzyme 3
trpA gene
Tryptophan
(a) Regulation of enzyme
activity
(b) Regulation of enzyme
production
Operons: Definitions & Concepts
• bacteria group functionally related genes so they can be under coordinated
control by a single “on-off regulatory switch”
• the regulatory “switch” is a segment of DNA called an operator
– binding sites for transcription factors that help RNA polymerase II bind the nearby
promoter
• the operator can be controlled by proteins or nutrients
– e.g. can be switched off by a protein called a repressor
– repressor prevents gene transcription - binds to the operator and blocks RNA
polymerase binding to the promoter
– repressor is the product of a separate regulatory gene
– repressor can be in an active or inactive form, depending on the presence of other
molecules
• co-repressor is a molecule that cooperates with a repressor protein to switch
an operon off
– e.g. the amino acid tryptophan
Operons: Definitions & Concepts
• operon = the entire stretch of DNA that includes the
operator, the promoter, and the genes that the
promoter controls
– the transcription of the downstream genes is polycistronic
– produces one long piece of mRNA containing multiple
transcription units
• two kinds – “On” and “Off” operons
trp operon
Promoter
trpE
Operator
Start codon
mRNA 5
Genes of operon
trpD
trpC
trpB
trpA
B
A
Stop codon
E
D
C
Polypeptide subunits that make up
enzymes for tryptophan synthesis
Repressible and Inducible Operons: Two
Types of Negative Gene Regulation
• OFF operon = repressible operon is one that
is usually on but is turned OFF by a repressor
– e.g. the trp operon is a repressible operon
• ON operon = inducible operon is one that is
usually off but is turned ON by an inducer
– e.g. lac operon is an inducible operon
The trp Operon: Repressible Operons
•
•
•
E. coli can synthesize the amino acid tryptophan when it is absent from the
growth media
by default the trp operon is on and the genes for tryptophan synthesis are
transcribed
comprised of the:
– 1. operator – capable of binding a repressor protein
– 2. genes of the operon – for synthesizing tryptophan when it is missing from the
growth media
•
plus a regulatory gene = trpR
– expressed whether tryptophan is absent or present
The trp Operon: Repressible Operons
trp operon
Promoter
Promoter
Genes of operon
DNA
trpE
trpR
Regulatory
gene
mRNA
3
RNA
polymerase
Operator
Start codon
trpD
trpC
trpB
trpA
C
B
A
Stop codon
mRNA 5
5
E
Protein
Inactive
repressor
(a) Tryptophan absent, repressor inactive, operon on
D
Polypeptide subunits that make up
enzymes for tryptophan synthesis
•when tryptophan is absent – the operon needs to function to make tryptophan
•the repressor protein is made but it is inactive & is incapable of binding the operator
•RNA polymerase can bind the promoter and the downstream genes are expressed
The trp Operon: Repressible Operons
DNA
No RNA
made
mRNA
Protein
Active
repressor
Tryptophan
(corepressor)
(b) Tryptophan present, repressor active, operon off
•when tryptophan is present – the operon does not need to be functional
•tryptophan acts as a co-repressor & binds the repressor protein
•this allows the repressor to bind and repress the function of the operator
•MUCH lower downstream gene expression vs. when the operon is ON
The lac Operon: Inducible Operons
• proposed by Francois Jacob and Jacques Monod - 1960s
• E.coli can use glucose and other sugars (such as lactose) as their
sole source of carbon and energy
• the normal situation is for the bacteria to use glucose
– levels of a bacterial enzyme called beta-galactosidase (lactose
breakdown) are very low
• when lactose is given to the bacteria – b-Gal levels increase
– said to be induced
• the lac operon is an inducible operon
– contains genes that code for enzymes used in the hydrolysis and metabolism
of lactose
• when E. coli are grown with glucose – no need for the enzymes of
the lac operon since there is no lactose in the medium
– so the operon is turned OFF
• but with media containing lactose – need to turn the operon ON to
make the enzymes for metabolizing and using lactose
The lac Operon: Inducible Operons
• genes of the lac-operon:
– 1. lacZ gene = beta-galactosidase – splits the lactose into glucose and
galactose
– 2. lacY gene
– 3. lacA gene
– 4. lacI gene = codes for a lac repressor
– 5. operator = binds transcription factors
– 6. promoter = binds RNA polymerase II
• THIS IS IMPORTANT!!! - without any outside control - the lac
repressor gene lacI is constitutively active and acts to eventually
switch the lac operon OFF
– through the constitutive production of a lac repressor protein
• a molecule called an inducer is needed to inactivate the repressor
to turn the lac operon ON
Regulatory
gene
Promoter
Operator
lacZ
lacI
DNA
No
RNA
made
3
mRNA
5
Protein
RNA
polymerase
Active
repressor
(a) Lactose absent, repressor active, operon off
lac repressor protein
•when lactose is absent – an active repressor is made
•the genes metabolizing lactose are NOT needed
•repressor gene lacI is constitutively active – makes a lactose repressor
•repressor binds the operator and hinders the binding of the RNA polymerase
to the promoter
•downstream genes are transcribed AT A VERY LOW LEVEL
•when lactose is present – an inducer is required to turn the operon ON
•metabolizing and using lactose is now needed
•ALLOLACTOSE ACTS AS AN INDUCER
•allolactose – form of lactose that can enter bacterial cells
•the inducer binds the repressor and prevents it from binding to the operator
•the downstream genes are expressed AT A HIGH LEVEL
•lactose binding to the repressor shifts the repressor to its non-DNA binding
conformation
lac operon
lacI
DNA
lacZ
lacY
-Galactosidase
Permease
lacA
RNA polymerase
3
mRNA
5
mRNA 5
Protein
Allolactose
(inducer)
Inactive
repressor
(b) Lactose present, repressor inactive, operon on
Transacetylase
• in nature – the inducer of the lab operon is a
lactose derivative
• in the lab – other inducers can be used to turn
the operon on
– e.g. IPTG = isopropyl-b-D-thiogalactoside
– IPTG is NOT a substrate of -Gal
• we can also give the bacteria a specific b-Gal
substrate that will turn colors
– X-Gal – turns blue with broken down by b-gal enzyme
– used to identify bacteria containing cloned genes
– can insert additional genes into plasmids containing
the b-gal gene
• insertion of your desired gene INTO the plasmid
disrupts -gal expression
• inability to breakdown X-Gal – colonies are white
• inducible enzymes usually function in catabolic
pathways
– their synthesis is induced by a chemical signal
• repressible enzymes usually function in anabolic
pathways
– their synthesis is repressed by high levels of the end
product
• regulation of the trp and lac operons involves
negative control of genes because operons are
switched off by the active form of the
repressor
Positive Gene Regulation: CAP proteins
• some operons are also subject to positive control
• when bacteria are given both lactose AND glucose - the
bacteria will use glucose
– the enzymes for glycolysis are continually present in bacteria
• when lactose is present and glucose is short supply – it
makes the enzymes for lactose metabolism
• how does the bacteria sense the low levels of
glucose??
Promoter
DNA
lacI
lacZ
CAP-binding site
cAMP
Operator
RNA
polymerase
Active binds and
transcribes
CAP
Inactive
CAP
Allolactose
Inactive lac
repressor
(a) Lactose present, glucose scarce (cAMP level high):
abundant lac mRNA synthesized
-when glucose is scarce  accumulation of a small molecule called cyclic AMP
(cAMP)
-cAMP functions as a “2nd messanger” to signal that glucose levels are low in the
growth medium
- high levels of cAMP activate a regulatory protein called catabolite activator protein
(CAP)
-cAMP binds CAP and activates it
- activated CAP attaches to the lac operon promoter and accelerates transcription
(functions as a transcription factor)
- enhances the affinity of RNA polymerase for the promoter
• CAP helps regulate other operons that encode
enzymes used in catabolic pathways
• when glucose levels are low and lactose levels are
high
– 1. lactose binds the lactose repressor and prevents it
from binding the operator and inhibiting gene
transcription = genes for lactose metabolism are made
– 2. cAMP activation of CAP and its binding to the lac
promoter increases transcription = lactose genes are
made at a higher rate
• when glucose levels increase and lactose levels
decrease
– 1. CAP activation will eventually decrease and so will its
enhancement of transcription
– 2. the lactose repressor is now able to bind the
operator and inhibit transcription
• so the lac operon is actually under dual control as lactose increases
and glucose decreases:
– positive – as levels of cAMP rise – so does CAP activation and the activity of
the lac operon
– negative – as repressor activity decreases & the activity of the lac operon
increases
– THEREFORE: it is the allosteric state of the lac repressor that determines if
transcription happens
– it is the presence of CAP that controls the rate at which transcription will
happen
Eukaryotic gene expression is regulated at
many stages
Signal
NUCLEUS
Chromatin
•
•
•
•
•
all organisms must regulate which genes are
expressed at any given time
in the same organism – the genomes are identical
from cell to cell
so why do different cells express different
genes/proteins??
differences result from differential gene
expression = the expression of different genes by
cells with the same genome
several steps along the
replication/transcription/translation path are
control points for differential gene expression
– control of DNA transcription – modification
of DNA-histone interaction
– post-transcriptional control
– post-translational control
DNA
Chromatin modification:
DNA unpacking involving
histone acetylation and
DNA demethylation
Gene available
for transcription
Gene
Transcription
RNA
Cap
Exon
Primary transcript
Intron
RNA processing
Tail
mRNA in nucleus
Transport to cytoplasm
CYTOPLASM
mRNA in cytoplasm
Degradation
of mRNA
Translation
Polypeptide
Protein processing, such
as cleavage and
chemical modification
Degradation
of protein
Active protein
Transport to cellular
destination
Cellular function (such
as enzymatic activity,
structural support)
Control of DNA Transcription: Histone Acetylation
• each of the histone proteins (H2A, H2B, H3,
H4) contain flexible extensions of 20 to 40
amino acids called “tails”
• these histones can be modified posttranslationally by the addition of functional
groups
• at the end of these tails are several
positively charged lysine amino acids
• some of these lysines undergo reversible
chemical modification called acetylation
– important for transcription, resistance against
DNA degradation
Histones
Histones
Acetylation and Deacetylation of DNA
• numerous post-translational modifications can be done to histone proteins
– affects how the DNA-histone interacts and ultimately affects the transcription of the
DNA
• some histone lysines undergo reversible chemical modifications called acetylation
and deacteylation
• acetylation = transfer of an acetyl group onto the NH2 terminus of an amino acid
– for histones – performed by a family of enzymes called histone acteyltransferases
(HATs)
• acetylation neutralizes the +ve charge of these lysines
– its interaction with the DNA is eliminated
– the DNA becomes less tightly associated with the histone
– results in better access for the transcriptional machinery to the DNA
acetyl
coA “donor”
lysine
R-group
Acetylation and Deacetylation of DNA
• deacetylation = removal of this acetyl group from the
histone by a family of enzymes called histone deacetylases
(HDACs)
– increases the interaction between DNA and the histone by
removing the acetyl group and increasing the “positivity” of the
lysine residues
– 11 eukaryotic HDACs !!!
acetyl
coA “donor”
lysine
R-group
Control of DNA Transcription: Acetylation and
Deactylation of DNA
• acetylation/deacetylation is a transient histone modification that
affects transcription
– euchromatin – higher HAT activity  more transcriptionally active form of
chromatin
– heterochromatin – higher HDAC activity  less transcriptionally active form
of chromatin
heterochromatin
euchromatin
Increased binding
of transcription factors
and RNA Pol II
to “opened” acetylated
chromatin
Protein
Control of DNA Transcription: Acetylation and
Deacetylation of DNA
• the HAT/HDAC enzymes are part of a large complex of proteins that binds the
DNA
– includes transcription factors, other regulatory proteins, RNA polymerase II
• it is now thought that HAT and HDAC enzymes are recruited into this complex
– once there – they modify the DNA and give the rest of the transcription machine
better “access” to the DNA helix
• non-histone proteins can also be acetylated!!
– e.g. transcription factors are also acetylated/deacetylated – changes their activity
level and therefore transcription
Histone Methylation
• histone methylation = the addition of methyl groups (CH3) to
certain amino acids on histone tails
– lysines or arginines – usually lysines
– is associated with reduced transcription in cases, increased transcription in
others
– usually results in increased association between the histone and the DNA
and a decrease in transcription in that area
– histone methylation is considered an epigenetic modification
• alteration of gene expression by mechanisms outside of DNA structure
• performed by a family of enzymes called histone methyltransferases
DNA Methylation
• in addition to histones – methyl groups can be attached to
certain DNA bases = DNA methylation
–
–
–
–
usually cytosine
done by a different set of enzymes than those that methylate histones
is associated with reduced transcription in some species
i.e. the more methylated, the more inactive the gene
• DNA methylation essential for long-term inactivation of genes
during cellular differentiation
– DNA methylation can last through several rounds of replication
– when a methylated DNA sequence is replicated – the daughter strand is
methylated too
– can affect transcription rates over several rounds of replication
Regulation of Transcription Initiation
• chromatin-modifying enzymes provide initial control of
gene expression by making a region of DNA either more
or less able to bind the transcription machinery
• additional transcriptional levels are also found
– enhancers
– promoters
Organization of a Typical Eukaryotic Gene
Enhancer
(distal control
elements)
DNA
Upstream
Proximal
control
elements
Transcription
start site
Exon
Promoter
Intron
Exon
Intron
Poly-A
signal
sequence
Exon
Transcription
termination
region
Downstream
• most eukaryotic genes are associated with multiple control
elements
– segments of noncoding DNA that serve as binding sites for transcription
factors that help regulate transcription
– distal elements– known as enhancers
– proximal elements – associated with promoters
• many of these control elements and the transcription factors
they bind are responsible for the differential gene expression
seen in different cell types
Transcription Factors
• proteins that bind sequences of DNA to control transcription
• can act as activators or repressors to transcription
– activating TFs - proteins that recruit the RNA polymerase to a promoter region
– repressing TFs – proteins that prevent transcription in many ways
• must contain a DNA binding domain to be a transcription factor
• not always one protein – can be multiple subunits together in a complex
• two broad categories:
– 1. general transcription factors
– 2. specific transcription factors
Transcription Factors
•
two broad categories:
– 1. general transcription factors are essential for the transcription of all protein-coding
genes
• assist the RNA polymerase in binding the promoter region – only give a low level of
transcription!!
• activity is enhanced by specific transcription factors
– 2. specific transcription factors control the high-level, differential expression of specific
genes within a specific cell type
•
•
•
•
bind the promoter and enhancer regions of a gene
can function to activate or repress transcription
e.g. Runx-2 – transcription factor that is found in osteoblasts
directs the expression of several osteogenic genes involved in making bone
Transcription Factors bind DNA
• binding of a TF to DNA is easy to see experimentally using a DNA mobility shift
assay
• Step #1 – radioactively label your DNA
• Step #2 – mix your labelled DNA with your possible TFs
• Step #3 – Southern blot - run the DNA on a gel and transfer it to a filter paper
-control – DNA not mixed with the possible TF
• Step #4 – expose the filter to film to develop the radioactive signal
DNA footprinting assay
Promoters
•
•
•
•
sequence of DNA located immediately upstream of the transcription start site
promotes transcription of DNA into RNA
site of RNA polymerase binding in both prokaryotes and eukaryotes
contain sequences for the binding of RNA polymerase and sequences for the
binding of transcription factors
Promoters
• initial work done in bacteria
– found two kinds of DNA sequences in the promoter
• 1. those that are found in the promoters of all bacterial genes
• 2. those that are found in a more limited number of genes that respond to a
specific signal
• core bacterial promoter: binds RNA polymerase and an associated sigma factor
(part of the RNA polymerase complex)
– two key DNA sequences located 10 and 35 bp upstream from the transcription start site
(TSS) – for the binding of the RNA polymerase
– -10 site = TATA box (consensus sequence TATAAT)
– -35 site - TTGACA
Promoters
• eukaryotic promoters: 10 classes of promoters known
– DEFINITION: DNA sequence that binds the RNA polymerase II (plus 7
additional factors)
– BUT – more complicated than prokaryotic promoters
– are additional upstream DNA sequences that regulate transcription
– three well-known regions studied
• -30, -75 and -90 sites
– two kinds of DNA sequences associated with transcription of a eukaryotic gene:
– 1. part of the promoter (i.e. control elements) involved in the basic process of
transcription
– 2. control elements active in a particular tissue type or in response to a specific
signal – regulated transcription
Promoters
• promoter sequences involved in basic transcription
– TATA box – conserved from the bacterial TATA box
•
•
•
•
•
•
•
A/T rich sequence - 30 base pairs upstream of start site
in most genes but not all – not found in housekeeping genes
together with the transcription start site – considered to be the core promoter
accurately positions the RNA polymerase at the start site
also binds general transcription factors
actually provides a very low level of transcription
needs upstream promoter elements (UPEs) to increase transcriptional control
– UPEs – also essential for transcription
•
•
•
•
increase the efficiency of transcription
e.g. SP1 sequence &/or CCAAT box
some genes can have an SP1 and CCAAT box – e.g. hsp70 gene
others may only have one – e.g. metallothionein gene
• promoters sequences involved in regulated transcription
– promoters also have control elements that are shared with a more limited
number of genes
– interspersed among the UPEs
– provide cell-type specific or signal-specific transcription
– some well-known sequences are known as Response Elements
– e.g. metallothionein gene – metal response elements (MREs)
• gene binds metals such as zinc – acts to decrease oxidative stress
– many hormones act through these response elements
• e.g. estrogen
Histone 2A gene promoter
Metallothionein 1 gene promoter
Promoters
– functions of promoter were discovered through
• mutation or deletion of specific regions
• putting these regions in front of a reporter gene  is the reporter gene
transcribed?
• e.g. insertion of the metallothionein gene promoter into a reporter plasmid
containing the firefly luciferase gene (reporter gene)
• transcription of the luciferase gene will be driven by the inserted promoter
• put in the entire promoter and measure luciferase activity
• put in “pieces” of the promoter with specific regions deleted – measure luciferase
activity
Activation of Metallothionein
Gene Expression by Hypoxia
Involves Metal Response
Elements and Metal Transcription
Factor-1
Cancer Res March 15, 1999 vol. 59
no. 6
pp1315-1322
Enhancers
• distal control elements of a gene
• DNA sequences that act to enhance eukaryotic transcription
• can be found either:
–
–
–
–
upstream of the gene
downstream of the gene
within the gene
even on a different chromosome!!!
• act to increase the activity of the promoter
– DO NOT have promoter activity themselves
• some enhancers are active in all tissues and increase promoter activity
constitutively
• others are only expressed in specific cells
• made up of several DNA sequences (sequence elements) that bind transcription
factors which interact together
• many of these sequence elements are also found in the promoter
Enhancer
(distal control
elements)
DNA
Upstream
Proximal
control
elements
Transcription
start site
Exon
Promoter
Intron
Exon
Intron
Poly-A
signal
sequence
Exon
Transcription
termination
region
Downstream
Enhancers & their Transcription Factors
•
transcription factors than bind enhancers are called activators
– positively acting transcription factors
•
•
activators = proteins that bind to DNA sequences and stimulate/activate transcription of a
gene
activators have two domains
– 1. DNA binding domain
– 2. activation domain - site that activates transcription by helping to form the transcription
initiation complex
Activation
domain
DNA-binding
domain
DNA
Eukaryotic gene elements: a summary
• so the typical eukaryotic gene consists of up to 4
distinct control elements
– 1. core promoter itself – upstream of the transcription start
site
– 2. upstream promoter elements (UPEs) located close to the
promoter – required for efficient transcription in any cell
– 3. elements that intersperse among the UPEs and activate
transcription of genes in specific tissues or in response to
specific stimuli – regulated transcription elements
– 4. distant elements called enhancers
Transcription Initiation
•
transcription can happen as long as the core promoter is present
– but transcription rates will be very low
•
•
•
so efficient transcription of eukaryotic genes requires the activity of
the promoter, enhancers and a multitude of transcription factors
together with the RNA polymerase II
these components come together to form a transcription initiation
complex
stepwise assembly
– 1. binding of three general transcription factors at the TATA box– TFIIA,
TFIID TFIIB
– 2. recruitment and binding of the RNA polymerase II at the TF/TATA box
complex
• in some organisms – polymerase is bound to these 3 TFs before
binding DNA first = RNA polymerase holoenzyme
– 3. additional general TFs join
– 4. binding of gene-specific TFs + interaction with enhancer/activators
Promoter
Activators
Gene
DNA
Distal control
Enhancer element
-activators bind to the DNA of the
enhancer via their DNA-binding domains
-the activators bind to regions called
distal control elements
TATA box
General
transcription
factors
DNAbending
protein
Group of mediator proteins
a DNA bending protein “bends” the
distal enhancer region – bringing it close
to the the promoter
RNA
polymerase II
general transcription factors, promoterspecific TFs, mediator proteins and RNA
polymerase II form a transcription
initiation complex with the enhancer
and its activators
RNA
polymerase II
Transcription
initiation complex
RNA synthesis
Transcription Initiation
activator-enhancer
Mediator protein = co-activator
-multi-subunit protein complex
found in the transcription initiation
complex of all eukaryotes
-31 proteins!!!!
-functions as a bridge between
transcription factors
promoter-specific
transcription factors
Repressors
• some transcription factors can also function as
repressors or silencers
– inhibiting expression of a particular gene by a variety of
methods
– some repressors bind activators and prevent their binding to
enhancers
– some bind the distal control elements in the enhancer directly
– others bind proximal control elements or the promoter
Cell-Type Specific Transcription
Enhancer Promoter
Control
elements
LIVER CELL
NUCLEUS
Albumin gene
Crystallin
gene
Available
activators
• both liver and lens cells have the same
genome
• so why does a liver cell make albumin and a
lens cell make crystallin?????
• it’s the transcription factors and control
elements
• liver cell has a unique complement of
transcription factors that activate albumin
transcription
Albumin gene
expressed
Crystallin gene
not expressed
(a) Liver cell
Enhancer
Control
elements
Promoter
LENS CELL
NUCLEUS
Albumin gene
Crystallin
gene
Available
activators
• the lens cell has a different set of TFs that
activates crystallin transcription
• these transcription factors may only be
made within a lens or liver cell at a precise
time in development or in response to an
extracellular signal (e.g. growth factor or
hormone) or even an environmental cue
Albumin gene
not expressed
Crystallin gene
expressed
(b) Lens cell
Coordinately Controlled Genes in Eukaryotes
• unlike the genes of a prokaryotic operon, each coexpressed eukaryotic gene has a core promoter and
several other control elements
• these genes can be scattered over different
chromosomes, but each has the same combination of
control elements as one another
• multiple copies of activators recognize these control
elements on each gene and promote their
simultaneous transcription
Mechanisms of Post-Transcriptional
Regulation
• transcription alone does not account for gene expression
• regulatory mechanisms can operate at various stages after transcription
• allow a cell to fine-tune gene expression rapidly in response to
environmental changes
• post-transcriptional processing:
– 1. mRNA structure – cap and tail; UTRs
– 2. mRNA splicing
– 3. mRNA half life and degradation
Post-Transcriptional Regulation: mRNA structure
• pre-RNA processing to mRNA involves the addition of the 5’ methylated cap and
3’ poly-A tail
• cap is added shortly after transcription initiation – by a capping enzyme which is
associated with the RNA polymerase II
– cap - 7-methylguanosine
– function of the cap
• 1. protection against mRNA degradation
• 2. export out into the cytoplasm
• 3. binding of the small subunit for translation
Post-Transcriptional Regulation: mRNA structure
• cap is followed by the 5’UTR region (untranslated region)
– found between the transcription start site and ends one nucleotide before
the ATG/start codon of the coding sequence
– ontains elements for controlling gene expression and mRNA export
– contains sequences that are involved in translation initiation
– in bacteria – contains a sequence for docking of the ribosome = Shine
Delgarno Sequence
TSS
Post-Transcriptional Regulation: mRNA structure
• poly A tail – in animal cells, all mRNAs (except histone mRNAs) have
polyA tails
– prevent degradation of the mRNA and induces export from the nucleus
– two special mRNA sequences are needed – located in the 3’UTR
• 1. Poly Adenylation signal – AAUAAA
• 2. Poly A site – downstream from the signal – area rich in Gs ands Us
– area where the mRNA is cut and the poly-A tail is added
-complex of proteins binds the poly-Adenylation signal
-a Poly(A) polymerase is recruited – cleaves the mRNA at the poly A site and adds
hundreds of “A” nucleotides
Post-Transcriptional Regulation: mRNA structure
• 3’UTR – second of the two UTRs that flank a transcription unit’s coding sequence
– length is important – longer the UTR, the lower level of gene expression
– can be an area of variability – controls expression levels and localization of a protein within a cell
– contains numerous regulatory regions for
• 1. poly –adenylation – contains the polyA signal and polyA site
• 2. mRNA creation – silencer regions to repress transcription of mRNA
• 3. mRNA stability – contain AU-rich elements (AREs) that increase the stability of the mRNA
• 4. mRNA export – contains sequences that attract nuclear export proteins
• 5. translation efficiency – also affected by AREs
Post-transcriptional control: mRNA degradation
• the life span of mRNA molecules in the cytoplasm is a
key to determining protein synthesis
• eukaryotic mRNA is more long lived than prokaryotic
mRNA
• numerous enzymes (RNases) can breakdown mRNA
• the binding of small RNAs called microRNAs (miRNAs)
to the mRNA can target it for degredation
Post-transcriptional Regulation: Splicing
•
•
removal of introns and the joining of exons
performed in the nucleus by the spliceosome
– small nuclear RNAs – U1, U2, U4, U5 and U6
– associated with protein subunits = snRNPs
– snRNPs form the core of the spliceosome
•
•
spliceosome recognizes conserved sequences at the
start and end of an intron = splice sites
introns are removed as a lariat structure – a 5’ G at
the end of the intron is joined in an unusual
phosphodiester bond (2’ to 5’) to the A at the end of
the intron
–
“A” nucleotide is called a branch point
Post-transcriptional Regulation: Splicing
•
•
•
•
•
•
•
the snRNPs are numbered based on their
“entrance” into the splicing reaction
1. first is U1 – base pairs with the 5’ G at the
start of the intron
2. next is U2 which binds the branch point A
3. U4/U6 and U5 enter next – formation of the
completed spliceosome
4. rearrangements among these snRNAs “loops
out” the intron and cuts the intron at the 5’
end
5. departure of U1 and U4 + joining of the 5’
end of the intron to the A (completes the
lariat)
6. U6 snRNA cuts at the 3’ end of the intron
and joins the two exon sequences
Splicing
• In alternative RNA splicing, different mRNA molecules
are produced from the same primary transcript,
depending on which RNA segments are treated as
exons and which as introns
Exons
DNA
1
4
3
2
5
Troponin T gene
Primary
RNA
transcript
3
2
1
5
4
RNA splicing
mRNA
1
2
3
5
or
1
2
4
5
Initiation of Translation
• initiation of translation of selected mRNAs can be blocked by regulatory
proteins that bind to sequences or structures of the mRNA
• alternatively, translation of all mRNAs in a cell may be regulated
simultaneously
• for example, translation initiation factors are simultaneously activated in an
egg following fertilization
Protein Processing
• after translation - various types of protein processing, including
folding, cleavage and the addition of chemical groups take place
• known as post-translational processing
• numerous kinds of chemical additions
Post-translational modifications of Proteins
• usually involves enzymes adding
–
–
–
–
–
–
1. hydrophobic groups (fatty acids) for membrane localization of protein
2. cofactors to enhance enzymatic activity
3. additional peptides or proteins – e.g. ubiquitin
4. structural changes – disulfide bridges between cysteines
5. proteolytic cleavage
6. chemical groups
• numerous chemical groups can be added
–
–
–
–
–
a. phosphorylation
b. methylation
c. acetylation
d. glycosylation
e. iodination
• non enzymatic modifications – changes the chemistry of an amino
acid without the use of an enzyme
– e.g. chemical addition of biotin (vitamin B7) as a cofactor
Protein Folding: Chaperones
• protein function is completely dependent upon 3D structure
• the information for folding is contained within the amino acid sequence of the
polypeptide chain
– the hydrophobic residues are “buried” within the center of the folding protein –
spontaneous process
– large numbers of interactions between the R groups of the AAs form
• ionic
• van der waals – between hydrophobic groups
• disulfide bridges
• folding can begin the minute the PP chain emerges from the ribosome
• but most protein don’t
• these proteins are met at the ribosome by a class of proteins called molecular
chaperones
Protein Folding: Chaperones
• molecular chaperones
– best described class of chaperones – heat shock proteins or HSPs
– numerous families – e.g. HSP40, HSP60, HSP70
– work by interacting with exposed hydrophobic AAs – hydrophobic residues are
dangerous
– distinct mechanisms for each chaperone
– e.g. HSP60 forms a “barrel-like” structure that “isolates” folding proteins AFTER they
are made = known as chaperonin
– bind to the hydrophobic residues and ensures correct folding
• some chaperones bind to misfolded proteins and sequester them until they are
destroyed
Proteasome
and ubiquitin
to be recycled
Ubiquitin
Proteasome
Protein to
be degraded
•
•
•
•
Protein entering
a proteasome
Protein
fragments
(peptides)
if the protein is not folded properly – will have to be degraded
Proteasomes are giant protein complexes that bind protein molecules and degrade them
consists of a protein complex that forms a hollow cylinder
top and bottom are additional protein complexes that feed the abnormal protein into the core
–
–
–
•
Ubiquitinated
protein
protein is unfolded as it is fed in
exposed to proteases within the core
keeps the protein in the core until the entire protein is cleaved into peptides
signal to enter the proteosome is the chemical attachment of a poly-ubiquitin chain
– ubiquitin is prepared by an enzyme (Ubiquitin activating enzyme)
– attached to lysines by enzymes (Ubiquitin ligase)
– done over and over  poly-ubiquitin chain
Noncoding RNAs play multiple roles in
controlling gene expression
• only a small fraction of DNA codes for proteins
– 30,000 to 100,000 genes
• a fraction of the non-protein-coding DNA pieces are genes for
RNAs such as rRNA and tRNA
• but most are transcribed into noncoding RNAs (ncRNAs)
– e.g. miRNA
– e.g. siRNA
• noncoding RNAs regulate gene expression at two points: mRNA
translation and chromatin configuration
MicroRNAs
•
•
•
•
•
•
•
•
MicroRNAs (miRNAs) are small single-stranded RNA
molecules that can bind to mRNA
Hairpin
Hydrogen
miRNA
can degrade mRNA or block its translation
bond
miRNAs are made by RNA polymerase II & are capped
Dicer
and poly-adenylated
exported out to the cytoplasm
(a) Primary miRNA transcript
miRNA
miRNAprotein
a protein called Dicer cleaves the primary miRNA into the
complex
mature miRNA
one strand of miRNA associates with proteins  RNAinduced silencing complex (RISC)
then the RISC base pairs with it complementary mRNA
nucleotides – usually in the 3’UTR
mRNA degraded Translation blocked
(b) Generation and function of miRNAs
if base pairing is extensive  cleavage of mRNA
5 3
–
•
happens in plant cells
if base pairing is limited  repression of translation
–
happens in animal cells
Small Interfering RNAs
•
•
•
through RNA interference (RNAi) viruses can
target and destroy host mRNAs
RNAi is done through the production of small
interfering RNAs (siRNAs) – target and destroy
mRNA just like miRNA
siRNAs and miRNAs are similar but form from
Dicer cutting up different RNA precursors
– miRNA made from single stranded RNA made by
the cell
– siRNA made from double stranded RNA (shRNA)
made by viruses
•
siRNA is now used in the lab to target specific
mRNAs
–
introduce artificial shRNA into cells  siRNA that will
bind and degrade your desired piece of mRNA
1. viral production
of dsRNA (shRNA)
2. complexing with
DICER  siRNA
3. integration of
siRNA with RISC
4. binding to target
mRNA
5. DESTRUCTION of
mRNA
The Evolutionary Significance of Small
ncRNAs
• Small ncRNAs can regulate gene expression at
multiple steps
• An increase in the number of miRNAs in a species
may have allowed morphological complexity to
increase over evolutionary time
• siRNAs may have evolved first, followed by
miRNAs