Chapter8

Control of Gene Expression

Molecular Cell Biology 1

BTEC209

May 13, 2024

 An overview of gene expression
• Gene expression
– Complex process by which cells selectively direct the synthesis of
proteins and RNAs
• Cell differentiation
– Cells make and accumulate different sets of RNAs and proteins
• Achieved by changes in gene expression
– Fertilized egg cell gives rise to many cell types that carry out a
range of specialized functions
• b cells of pancreas: insulin
• a cells of pancreas: glucagon
• B lymphocytes: antibody
• Red blood cells: hemoglobin
The different cell types of a multicellular organism contain
the same DNA
• In an organism, cell types differ
– Not b/c they contain different genes
– But b/c they express them differently
– Evidence?
• If chromosomes of differentiated cell
were altered irreversibly during
development,
– Incapable of guiding the development of
the whole organism
 Different cell types produce different sets of proteins
• Differences in gene expression b/t cell types
– By comparing the protein composition of cells
• By 2D gel electrophoresis and mass spectrometry
• Housekeeping proteins: RNA polymerase, DNA repair enzymes,
ribosomal proteins, cytoskeleton proteins etc.
• Cell type specific proteins: hemoglobin
– By cataloging a cell’s RNAs
• By determining the sequence of every RNA
• A typical differentiated human cell expresses perhaps 5,000-15,000
protein-coding genes from a total of about 19,000
• Cause the large variations seen in the size, shape, behavior and
function of differentiated cells
A cell can change the expression of its genes in response to
external signals
• Cells alter pattern of gene expression in
response to extracellular signals
– Steroid hormone cortisol
• Released by adrenal gland during starvation and
intense exercise
• Liver cells boost glucose production from amino
acids and other small molecules
• Liver cells induce enzymes including tyrosine
aminotransferase, which helps convert tyrosine to
glucose
– Different cell types often respond in different
ways to the same signal
• Contribute to the specialization
 Gene expression can be regulated at various steps from
DNA to RNA to protein
• The control of gene expression
1) Controlling when and how often a given gene is transcribed
2) Controlling how an RNA transcript is processed
3) Selecting which mRNAs are exported from nucleus
4) Regulating how quickly certain mRNAs are degraded
5) Selecting which mRNAs are translated into protein
6) Regulating how rapidly specific proteins are destroyed
7) The activity of proteins can be further regulated
 How transcription is regulated
• Until 50 years ago, the idea that genes could be switched on and off
was revolutionary
– Originally came from studies of how E. coli adapt to changes in the
composition of growth medium
• Transcriptional regulators
– Bind to DNA and control gene transcription
 Transcription regulators bind to regulatory DNA
sequences
• Promoters of bacterial and eukaryotic genes
– Bind RNA polymerase and correctly orient enzyme to begin transcription
– Include a transcription initiation site plus nearby sequences that contain
recognition sites for proteins
• Sigma factor in bacteria or general transcription factors in eukaryotes
 Transcription regulators bind to regulatory DNA
sequences
• In addition to promoter, regulatory DNA sequences
– Used to switch gene on or off
– Simple regulatory switches as short as 10 nucleotides predominate in bacteria
and respond to a single signal
– Long (sometimes more than 10,000 nucleotides) sequences in eukaryotes
integrate information from diverse signals
– Must be recognized by transcription regulators
• Bacteria produce several hundred different regulators
• Humans produce about 2,000 different regulators
 Transcription regulators bind to regulatory DNA
sequences
• Transcription regulators in bacteria and eukaryotes
– Surface of protein fits tightly against the major groove of DNA
• Depending on nucleotide sequence
• 10 to 20 intimate molecular contacts at protein-DNA interface are highly specific and
very strong
 Transcription regulators bind to regulatory DNA
sequences
• Transcription regulators in bacteria and eukaryotes
– Bind to DNA as dimers
• Roughly double the area of contact, thereby increasing the strength and specificity of
protein-DNA interaction
Transcription switches allow cells to respond to changes in
their environment
• Gene expression in bacteria
– According to food sources that
available in environment
• Production of tryptophan
– Five genes arranged in operon
• Operon is not transcribed when
tryptophan is abundant
– Operator is within the promoter
• Block access of RNA polymerase to
promoter when repressor binds
• Tryptophan repressor is constitutively
expressed and is an allosteric protein
 Repressors turn genes off and activators turn them on
• Transcriptional regulators
– Transcriptional repressor and activator
• Transcriptional activator
– Able to bind and position RNA
polymerase
– CAP has to bind cAMP
• cAMP rises when glucose is no longer
available
• Drive the production of enzymes that allow
bacteria to digest other sugars
The lac operon is controlled by an activator and a repressor
• Promoter is often controlled by two
different regulators
• Lac operon in E. coli
– To import and digest disaccharide
lactose
• Controlled by Lac repressor and CAP
activator
– In the absence of glucose, cAMP
activates CAP
– If lactose is not present, induction of
lac operon is wasteful
• Lac repressor shuts off operon in the
absence of lactose
 Eukaryotic transcription regulators control gene
expression from a distance
• Enhancers
– Eukaryotic gene activators bind
– Enhance transcription even when they are bound thousands of nucleotides
away from promoter
– Enhance transcription when bound either upstream or downstream from the
gene
 Eukaryotic transcription regulators control gene
expression from a distance
• How do they communicate with
promoter?
• Simplest model for “action at a
distance”
– DNA between enhancer and
promoter loops out
• Allow eukaryotic activators to directly
influence
– Mediator serves to link transcription
regulators to proteins at promoter
– Form a large “transcription
initiation complex” at promoter
 Eukaryotic transcription regulators control gene
expression from a distance
• Eukaryotic transcription regulators
– Attract proteins that modify chromatin structure
• Affect the accessibility of promoter to general
transcription factors and RNA polymerase
– Control the assembly of transcription initiation
complex
 Eukaryotic transcription regulators help initiate
transcription by recruiting chromatin-modifying proteins
• Problem of DNA packaging
– DNA is wound around clusters of
histones to form nucleosomes
– If nucleosomes are positioned over a
promoter,
• Inhibit the initiation of transcription by
physically blocking the assembly on promoter
• Chromatin structure can be altered
– By chromatin-remodeling complexes
– By histone-modifying enzymes
 Eukaryotic transcription regulators help initiate
transcription by recruiting chromatin-modifying proteins
• Transcription activators
– Attract chromatin-modifying proteins to
promoters
• Recruitment of histone acetyltransferases (HAT)
• Recruitment of chromatin-remodeling
complexes
– HAT
• Promote acetyl group attachment to selected
lysines in histone tail
• Acetyl groups attract proteins that promote
transcription
– Chromatin-remodeling complexes
• Make nearby DNA more accessible
 Eukaryotic transcription regulators help initiate
transcription by recruiting chromatin-modifying proteins
• Transcription repressors
– Reduce the efficiency of transcription initiation
– Attract histone deacetylase (HDAC)
• Remove acetyl groups from histone tails
• Reverse the positive effects that acetylation has on transcription initiation
– Some work on a gene-by-gene basis
– Others orchestrate the formation of large swathes of
transcriptionally inactive chromatin
• Heterochromatin
• Inactive X chromosome
 The arrangement of chromosomes into looped domains
keeps enhancers in check
• Eukaryotic transcription regulators
– Act across very long stretches of DNA
• If a transcription regulator forms a loop in the wrong direction,
– Inappropriately influence the transcription of a neighboring gene
 The arrangement of chromosomes into looped domains
keeps enhancers in check
• If a transcription regulator forms a loop in the wrong direction,
– To avoid such cross-talk, DNA is arranged in a series of loops
• Formed by specialized proteins
• The localization restricts the action of enhancers
• Mutations preventing loops from properly forming lead gene expressions at the wrong
time and place, causing cancers and inherited diseases
 Generating specialized cell types
• Some changes in gene expression are often only transient
– Tryptophan repressor
• Cell memory
– Once a cell becomes committed to differentiate into a specific
cell type
– The choice is generally maintained through subsequent cell
divisions
– Changes in gene expression are remembered
 Eukaryotic genes are controlled by combinations of
transcription regulators
• Most eukaryotic transcription regulators
– Work as part of a large “committee” of regulatory proteins
– Cooperate to express the gene in the right cell type, in response to the right
conditions, at the right time and in the required amount
• Combinatorial control
– Groups of regulators work together to determine the expression of gene
• Lac operon is a simple example
 Eukaryotic genes are controlled by combinations of
transcription regulators
• Combinatorial control
– In eukaryotes,
• A typical gene is controlled by dozens of
regulators
• Direct assembly of mediator, chromatin-
remodeling complexes, histone-modifying
enzymes, general transcription factors and
RNA polymerase
• In many cases, multiple repressors and
activators are present
 The expression of different genes can be coordinated by a
single protein
• To coordinate the expression of
different genes
– In bacteria,
• Coordinate the expression of operon under
the control of a promoter
• Such clustering is not seen in eukaryotic
cells
– In eukaryotic cells,
• Control of gene expression is combinatorial
• How do eukaryotic cells rapidly and
decisively switch groups of genes on or off?
 The expression of different genes can be coordinated by a
single protein
• Effect of single transcription regulator can still be decisive in switching
any particular gene on or off
– Simply by completing the combination needed to activate or repress the gene
• Just as same number can complete the combination for different locks
• Same protein can complete the combination for several different genes
 The expression of different genes can be coordinated by a
single protein
• Coordinated regulation in response to
cortisol
– Liver cells produce glucose from small
organic molecules by increasing the
expression of many genes
– To switch on such cortisol-responsive genes,
cortisol-receptor complex binds to a
regulatory sequence
– A single transcription regulator can
coordinate the expression of many different
genes
Combinatorial control can also generate different cell types
• Key transcription regulators
– Useful in the day-to-day regulation of
cell function
– Cells diversify into particular types of
cells during embryonic development
Combinatorial control can also generate different cell types
• Development of skeletal muscle cells
– Distinguished structures from other cells
• Production of muscle-specific forms of actin and myosin
• Receptor proteins and ion channels to allow cells to contract in response to stimulation
by nerve
– A small number of key transcription regulators
• Expressed only in potential muscle cells
• Coordinate muscle-specific gene expression
• Crucial for muscle-cell differentiation
Combinatorial control can also generate different cell types
• Other sets of transcription regulators
– Activate expression of genes that are specific
for other cell types
• How different combinations of
transcription regulators can tailor the
development of different cell types is
illustrated
Combinatorial control can also generate different cell types
• Differentiation of a particular cell type
– Small number of regulators acting in
different combinations can form complex
regulatory networks
• Given transcription regulator often controls
the expression of hundreds or thousands of
genes
– Estimated that about 1,000 transcription
regulators are sufficient to control 24,000
genes that give rise to an individual human
 The formation of an entire organ can be triggered by a
single transcription regulator
• Single master transcription regulator
called Ey
– Trigger the formation of a whole organ
– Ey gene can be artificially expressed in
embryos in cells that would normally
give rise to a leg
– Control additional transcription
regulators
• Can produce a cascade of regulators
• Lead to the formation of many different
types of cells
 Transcription regulators can be used to experimentally
direct the formation of specific cell types in culture
• Some transcription regulators can
convert one specialized cell type to
another
– Reprogramming
– Transcription regulator MyoD introduced
into fibroblast forms musclelike cells
• Fibroblasts have already accumulated other
necessary transcription regulators
• MyoD completes the unique combination
– A set of nerve-specific transcription
regulators can convert liver cells to
functional neurons
 Transcription regulators can be used to experimentally direct the
formation of specific cell types in culture
• Other transcription regulators
– Can maintain cells in an undifferentiated state
• Embryonic stem cell are capable of giving rise to all the specialized cell types
• Induced pluripotent stem (iPS) cells
– De-differentiate specialized cell types into iPS cells
• Generate a variety of specialized differentiated cells
• Differentiated cells produced from iPS cells are currently used in the study or treatment
of disease
 Differentiated cells maintain their identity
• Specialized cell types give rise only to cells like themselves
– Differentiated cells such as fibroblasts, smooth muscle cells and liver cells
divide many times
• Cell memory
– Proliferating cell maintains its identity
– Patterns of gene expression must be remembered and passed on to daughter
cells
• Cells have several ways for cell memory
– Positive feedback loop
– DNA methylation
– Modification of histones
 Differentiated cells maintain their identity
• Positive feedback loop
– One of the simplest and most important
– A master transcription regulator activates transcription of its own gene and
other cell-type specific genes
– Ey protein
 Differentiated cells maintain their identity
• DNA methylation
– Occur on certain cytosine bases
– Generally turn off genes by attracting proteins
that bind and block gene expression
– DNA methylation patterns are passed on to
progeny cells
• By the action of enzyme that copies methylation
pattern
 Differentiated cells maintain their identity
• Modification of histones
– Each daughter DNA receives half of parent’s histone proteins, which contain
covalent modifications
– Enzymes responsible for modifications may bind to parental histones
 Differentiated cells maintain their identity
• Epigenetic inheritance
– These cell-memory mechanisms transmit patterns of gene expression to
daughter cells
– Epigenetic changes are important in controlling patterns of gene expression
w/o altering DNA sequence
 Post-transcriptional controls
• Post-transcriptional controls
– Play a crucial part in regulating the expression
– Alternative RNA splicing allows different forms of protein
• Post-translational modifications
– Regulate protein’s concentration and activity
 mRNAs contain sequences that control their translation
• mRNA’s lifetime
– By specific nucleotide sequences
• Lie upstream and downstream of protein-coding sequence
• Binding sites for proteins that are involved in RNA degradation
– Most bacterial mRNAs last only a few minutes
• Allow a bacterium to adapt quickly to environmental changes
– Most eukaryotic mRNAs last less than 30 minutes
• Most short-lived are transcription regulators
 mRNAs contain sequences that control their translation
• Bacterial mRNAs contain a ribosome-binding sequence
– Form base pairs w/ RNA in small ribosomal subunit
– Ideal target for translational control by blocking
• Eukaryotic mRNAs possess a 5’ cap
– Help guide ribosome to the first AUG
– Repressor can inhibit translation initiation by binding the 5’ region of mRNA
 Regulatory RNAs control the expression of thousands of
genes
• Coding RNAs
• Noncoding RNAs
– Structural and catalytic roles in
ribosomes
– Regulatory RNAs: roles in regulating
gene expression
• microRNAs, small interfering RNAs and
long noncoding RNAs
 MicroRNAs direct the destruction of target mRNAs
• MicroRNAs (miRNAs)
– Control gene expression by base-
pairing w/ specific mRNAs
• Reduce their stability and translation
into protein
– Undergo processing to produce
mature miRNA
• About 22 nucleotides in length
• Packaged w/ proteins to form RISC
(RNA-induced silencing complex)
• Patrol the cytosol in search of mRNAs
 MicroRNAs direct the destruction of target mRNAs
• MicroRNAs (miRNAs)
– Once base-pairs w/ RISC,
• Destroyed by a nuclease that is part of
RISC
• Translation is blocked and other
nucleases eventually degrade
• Seek out additional mRNA target
• Efficiently block production of encoded
protein
 MicroRNAs direct the destruction of target mRNAs
• Roughly 500 different miRNAs in human genome
– May regulate as many as one-third of protein-coding genes
– Play a critical part in regulating gene expression
– Thereby influence many cell functions
 Small interfering RNAs protect cells from infections
• RNA interference (RNAi)
– Serve as a cell defense mechanism
• By eliminating dsRNAs produced by viruses and
transposable genetic elements
– dsRNAs are cut into short fragments (about 22
nucleotides) by Dicer
• Called small interfering RNAs (siRNAs)
– ds siRNAs are taken up by RISC
• RISC discards one strand of the siRNA duplex
• Seek and destroy complementary RNA molecules
 Small interfering RNAs protect cells from infections
• RNAi
– Selectively shut off the synthesis of foreign RNAs by
host’s RNA Pol
• siRNAs are packaged into a RITS (RNA-induced
transcriptional silencing)
• RITS complex attaches to complementary RNA sequences
transcribed from RNA polymerase
– RNAi-directed heterochromatin formation
• RITS complex attracts proteins that modify nearby
histones
• Promote the localized formation of heterochromatin
• Limit the spread of transposable genetic elements
throughout the host genome
 Small interfering RNAs protect cells from infections
• RNAi
– Operate in diverse organisms, including fungi, plants and worms
• Indicate that it is an evolutionarily ancient defense mechanism
• Particularly against viral infection
– Invading pathogen elicits the production of siRNAs or antibodies
• Custom-made to inactivate the specific invader and thereby protect the host
 Thousands of long noncoding RNAs may also regulate
mammalian gene activity
• Long noncoding RNAs
– More than 200 nucleotides
– Upwards of 5,000 of these lengthy RNAs in human and mouse
– Roles in the biology of organism are not entirely clear
• May promote silencing of specific genes
• Xist
– Best understood of long noncoding RNAs
– Key player in X inactivation
– Produced by only one of X chromosomes
• Transcript sticks around, coating chromosome and attracting enzymes and chromatin
remodeling complexes
• Promote the formation of heterochromatin
 Thousands of long noncoding RNAs may also regulate
mammalian gene activity
• Some long noncoding RNAs
– Fold into specific, 3D structures via complementary base pairing
• Serve as scaffolds, which bring together proteins
• One of roles of RNA molecule in telomerase is to hold its different protein subunits
together