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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
• eukaryotes – polyA signal is transcribed prior to termination
– 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
– it is only the mRNA that is translated
• 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 – only found in eukaryotes
– D. UTRs – only 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
• known as promoter and enhancer regions
– 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
• in bacteria – half of the genes are
clustered into operons
Precursor
Feedback
inhibition
trpE gene
Enzyme 1
trpD gene
Enzyme 2
trpC gene

trpB gene

Enzyme 3
– genes encode the enzymes for a
metabolic pathway
• all the genes are transcribed into
ONE mRNA
Regulation
of gene
expression
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
– usually positioned downstream of the promoter
– 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
• transcription produces a 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
•inactive repressor is incapable of binding the operator
•RNA polymerase can bind the promoter and the downstream genes are expressed AT A
HIGH LEVEL
The trp Operon: Repressible Operons
DNA
No RNA
made
mRNA
Protein
Active
repressor
Tryptophan
(corepressor)
(b) Tryptophan present, repressor active, operon off
-the binding of tryptophan changes the
shape of the active site of the repressor
-allosteric regulation: regulation of an
enzyme (or a protein) via the binding of a
molecule to a site other than the active
site
•when tryptophan is present – the operon does not need to be functional
•tryptophan acts as the co-repressor
•it binds the repressor protein
•changes the shape of the repressor and activates it - allows the repressor to bind and
repress the function of the operator
•MUCH LOWER downstream gene expression
http://www.youtube.com/watch?v=8aAYtMa3GFU
http://bcs.whfreeman.com/thelifewire/content/chp13/1302002.html
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 a 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
• can also convert lactose into allolactose
–
–
–
–
–
2. lacY gene = lactose permease – pumps lactose into the bacterial cell
3. lacA gene = thiogalactase transacetylase – function???
4. lacI gene = codes for a lac repressor
5. operator = binds transcription factors
6. promoter = binds RNA polymerase
• 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
•the 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 the enters 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 concentration of the repressor to
its non-DNA binding conformation
lac operon
lacI
DNA
lacZ
lacY
b-Galactosidase
Permease
lacA
RNA polymerase
3
mRNA
5
mRNA 5
Protein
Allolactose
(inducer)
Inactive
repressor
(b) Lactose present, repressor inactive, operon on
Transacetylase
-the mechanism is shut off upon metabolism
of the allolactose
-b-gal can hydrolyze allolactose
• in nature – the inducer of the lab operon is a
lactose derivative called allolactose
• in the lab – other inducers can be used to turn
the operon on
– e.g. IPTG = isopropyl-b-D-thiogalactoside (allolactose
mimicker)
– IPTG is NOT metabolized by b-Gal
– its concentration will remain constant in the cell and
so will its induction of b-gal expression
• 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 b-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
GO HOME AND STUDY
Next Lecture
EUKARYOTIC GENE
REGULATION
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 chemical
• at the end of these tails are several
positively charged lysine amino acids
– some interact with the negatively charged DNA
in their nucleosome
– others interact with the linker DNA in between
histones
– others interact with neighboring nucleosomes
• some lysines undergo reversible chemical
modification called acetylation
– important for transcription, resistance against
DNA degradation
Histones
Histones
Acetylation and Deacetylation of DNA
• numerous chemical modifications can be done to histone proteins
– e.g. methylation, phosphorylation
– affects how the DNA-histone interacts and ultimately affects the transcription of the
DNA
• some histone lysines undergo reversible chemical modifications called acetylation
and deacetylation
• acetylation = transfer of an acetyl group onto the NH2 terminus of an amino acid
– for histones – performed by a family of enzymes called histone acetyltransferases
(HATs)
– uses acetyl coA as the source of the acetyl group
– principle targets are the H3 and H4 histone subunits
• 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 (e.g. RNA pol II) to the DNA
acetyl
higher rates of transcription result
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 contact 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 by transcription factors
– 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. acetylation of wood proteins increases their hardness
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 -depends on the amino acid
methylated and the number of methyl groups added
• e. g. one methyl group added- increased chromatin condensation
•
two methyl groups added – decreased chromatin condensation
– 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
• the inheritance of traits transmitted by mechanisms not directly involving the
nucleotide sequence is called epigenetic inheritance
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 – known as enhancers
– proximal – associated with promoters
• 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 in eukaryotes
• 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
• activators have two domains
Activation
domain
DNA-binding
domain
DNA
1. DNA binding domain
2. activation domain - site
that activates transcription by
helping to form the
transcription initiation
complex
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
Promoters
•
•
•
•
sequences 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
in eukaryotes the promoter also binds transcription factors
Promoters
• 1. Core promoter – found in most eukaryotic genes
– gives a low level of transcription
• 2. proximal promoter elements/promoter
proximal elements (PPEs) – found in addition to the
core in many genes
– gives regulated transcription
– e.g. in certain cells at certain times
Core promoter
• in bacteria: binds RNA polymerase and an associated sigma factor (part of the RNA
polymerase complex)
– 10 nucleotides upstream from the start site of transcription is a key region =
TATA box (consensus sequence TATAAT)
– is another site – called the -35 site – TTGACA
– the core promoter binds a sigma factor called s70 – recognizes the TATA box
Core Promoter
• in eukaryotes:
– TATA box – conserved from the bacterial TATA box
•
•
•
•
A/T rich sequence - ~ 30 base pairs upstream of start site (-30 position)
in most genes but not all – not found in housekeeping genes
together with the transcription start site – considered to be the core promoter
like the bacterial promoter – TATA box accurately positions the RNA polymerase
at the start site
• it also binds general transcription factors
• BUT needs proximal promoter elements (PPEs) to increase transcriptional control
The Core Promoter binds General Transcription
Factors
• the core promoter binds general TFs
• there is a specific order to binding
– 1. binding of first three general transcription factors at
the TATA box– TFIIA, TFIID & TFIIB
– 2. binding of the RNA polymerase II at the TF/TATA box
complex
– 3. additional general TFs join
– 4. binding of gene-specific TFs + interaction with
activators bound to the enhancer
Additional Control Elements for Transcription
• core promoter only provides basic control over
transcription
• eukaryotic genes have additional control elements to
better control
• 1. proximal promoter elements/promoter proximal
elements (PPEs)
• 2. enhancer sequence elements or enhancers
Proximal Promoter Elements (PPEs)
• located further upstream (or downstream) of the core promoter – couple
of hundred base pairs upstream
• increase the efficiency of transcription by binding additional
transcription factors
• numerous types – most common:
• 1. octamer (Oct) consensus
• 2. GC-rich regions
• 3. CCAAT box
most eukaryotic genes use a combination
Proximal Promoter Elements (PPEs)
Histone 2A gene promoter
Proximal Promoter Elements (PPEs)
• PPEs are bound by cell-type specific transcription factors
• work with the TATA box (with its general transcription factors and
RNA polymerase) in the initiation of transcription
Enhancers
• distal control elements of a gene
• are DNA sequences called sequence elements that act to enhance
eukaryotic transcription
• bind transcription factors called activators
• the enhancer interacts with the promoter to enhance transcription
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
Eukaryotic gene elements: a summary
• so the typical eukaryotic gene consists of up to 3 distinct
control elements
– 1. core promoter– upstream of the transcription start site
• for basic transcriptional control
– 2. promoter proximal elements (PPEs) located close to the
promoter
• required for efficient transcription in any cell – increase transcription
rate
– 3. 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 core promoter, PPEs, enhancers and a multitude of
transcription factors together with the RNA polymerase II
• these components come together to form a transcription
initiation complex
Transcription Initiation
Promoter
Activators
Gene
DNA
TATA box
Enhancer
•
General
transcription
factors
activators bind to the distal
enhancer
DNAbending
protein
•
a DNA bending protein “bends” the
distal part of the DNA – bringing it
close to the promoter
Group of mediator proteins
RNA
polymerase II
•
•
general TFs, PPE-specific transcription factors,
mediator proteins and RNA polymerase II are
added
form a transcription initiation complex with
the enhancer and its activator/transcription
factors
RNA
polymerase II
Transcription
initiation complex
RNA synthesis
Transcription Initiation – Pretty Picture, eh?
activator-enhancer
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
specific for
liver genes
• 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
• the lens cell has a different set of
TFs that activates transcription in
these cells
• 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
environment cue
Available
activators
specific for lens genes
Albumin gene
not expressed
Crystallin gene
expressed
(b) Lens cell
Go have lunch!
Next lecture: Post-transcriptional
and translational control
Mechanisms of Post-Transcriptional
Regulation
• 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
Post-Transcriptional Regulation: mRNA structure
– function of the cap
• 1. protection of mRNA against degradation
• 2. export of mRNA out into the cytoplasm
• 3. binding of the small subunit to mRNA for translation
Post-Transcriptional Regulation: mRNA structure
• cap is followed by the 5’UTR region (untranslated region)
– part of the transcription unit
– found between the transcription start site and ends one nucleotide before
the ATG/start codon of the coding sequence
– contains sequences for controlling gene expression, mRNA export,
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
– “A” nucleotides are added following enzymatic cleavage of the mRNA
Post-Transcriptional Regulation: mRNA structure
• poly A tail
– two special mRNA sequences are needed – located in the 3’UTR
• 1. Poly A signal – AAUAAA
• 2. Poly A site – downstream from the signal – area rich in Gs ands Us
-a Poly(A) polymerase binds the poly A signal – 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
– much longer than the 5’UTR – average is 800 NTs long
– length is important – longer the UTR, the lower level of gene expression (due
to miRNA action)
– numerous regions for a variety of functions
Post-Transcriptional Regulation: mRNA structure
• 3’UTR - contains numerous regulatory regions for
• 1. poly–adenylation – contains the polyA signal and polyA site
• 2. mRNA stability – contain AU-rich elements (AREs) that increase the
stability of the mRNA; also interacts with miRNAs (repress translation and
degrade mRNA)
• 3. mRNA export – contains sequences that attract nuclear export
proteins + attract proteins that will associate the mRNA with the
cytoskeleton
• 4. improves translation efficiency
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 degradation
Post-transcriptional Regulation: Splicing
• removal of introns and the joining of exons
– for short mRNAs – happens after poly-adenylation at the 3’ end
– for longer mRNAs with multiple exons – happens during transcription
• 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
Post-transcriptional Regulation: Splicing
• 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 start 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
Differential Splicing
• In differential 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
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
• numerous chemical groups can be added
–
–
–
–
–
a. phosphorylation
b. methylation
c. acetylation
d. glycosylation
e. iodination
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 proteins don’t fold the quickly
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
– work by interacting with exposed hydrophobic AAs – hydrophobic residues are
dangerous
– 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
Proteasome
and ubiquitin
to be recycled
Ubiquitin
Proteasome
Protein to
be degraded
Ubiquitinated
protein
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 (20S
core)
• top and bottom are additional protein complexes that feed the
abnormal protein into the core (19S cap)
– 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
Proteasome
and ubiquitin
to be recycled
Ubiquitin
Proteasome
Protein to
be degraded
Ubiquitinated
protein
Protein entering
a proteasome
• signal to enter the proteosome is the
chemical attachment of a poly-ubiquitin
chain
Protein
fragments
(peptides)
Noncoding RNAs play multiple roles in
controlling gene expression
• only a small fraction of DNA codes for proteins
– 30,000 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
– e.g. snRNA
MicroRNAs
• MicroRNAs (miRNAs) are small singlestranded RNA molecules that can bind to
mRNA
• described between 2000 and 2005
• can degrade mRNA or block its translation
• over 600 miRNAs in humans
• regulate at least 1/3 of all protein coding
genes in humans
• miRNAs are made by RNA polymerase II &
are capped and polyadenylated
– large and have complex secondary structure that
needs to be processed
• pre-miRNA molecules are exported out to
the cytoplasm
Hairpin
miRNA
5 3
(a) Primary miRNA transcript
MicroRNAs
• large pre-miRNA molecule is exported
out to the cytoplasm
• requires processing
• a protein called Dicer cleaves the premiRNA into the mature miRNA
• one strand of miRNA associates with
proteins  RNA-induced silencing
complex (RISC)
Hairpin
Hydrogen
bond
miRNA
Dicer
5 3
(a) Primary miRNA transcript
miRNA
RISC
mRNA degradedTranslation blocked
(b) Generation and function of miRNAs
MicroRNAs
• the RISC base pairs with its complementary
mRNA nucleotides – usually in the 3’UTR
• if base pairing is extensive – cleavage of
mRNA
– happens in plant cells
• if base pairing is limited – repression of
translation
– happens in animal cells