molecular biology lecture 7
TRANSCRIPT
8/12/2019 Molecular Biology Lecture 7
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BIOL321 - Prokaryote gene regulation
Madigan et al. 2003. Biology of Microorganisms. Prentice Hall
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Objectives
- Overview of Prokaryotic gene transcription
- Introduction of gene regulation
- Promoters
- Operators
- Introduction to lac regulation
- History
- Lactose breakdown
- Induction of lac operon
- Mutations of lac
- The hunger signal and cAMP signaling- Introduction to t rp regulation
- Roles and functions of trp
- Repression of trp by TrpR
- Translational control of trp through attenuation
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A Quick Review of Transcription
• The step where a molecule of mRNA is
assembled, based on DNA
• RNA polymerase is responsible for
reading the DNA and assembling the
growing mRNA strand
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Prokaryotic vs. Eukaryotic
Transcription
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Getting Started
• RNA polymerase must find a promoter
sequence in DNA before it can start
transcribing
• The polymerase can bind the DNA
directly (in bacteria) or seek outtranscription factors that bind to the
promoter (in eukaryotes)
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In bacteria…
DNA
Promoter
RNA polymerase
Transcription Start
Site
RNA Polymerase (RNAP) ‘scans’ along the DNA,
looking for a promoter
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In bacteria…
DNA
Promoter
Transcription Start
Site
When a promoter sequence is recognized, RNAP ‘melts’ the D
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In bacteria…
DNA
Promoter
Transcription Start
Site
RNAP starts synthesizing the mRNA at the transcription start si
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Transcription: Always On?
• Some genes (constitutive genes) are always
expressed within a cell
• However, other genes only need to beexpressed at certain times – inducible genes
• Therefore, to reduce wasted effort, many
genes are regulated , and only expressed
under certain conditions
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Regulating Your Genes
• This regulation can occur at many
different steps of gene expression,
including: – Transcription
– mRNA processing (several kinds)
– Translation
• However, the majority of regulation
takes place at the transcriptional level
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Regulating Your Genes• In bacteria, regulated genes have an
downstream region adjacent to the promotercalled the operator
• The operator is a binding site for proteins thathelp to regulate gene expression
DNA
Promoter Operator
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Regulatory Proteins
• There are two main types of regulatory proteins in
bacteria:
– Repressors bind to the operator and prevent RNA
polymerase from initiating transcription – Activators bind to sequences near the promoter
and allow RNA polymerase to initiate transcription
• A given gene (or group of genes) may use either or
both types of protein for regulation
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Regulatory Proteins
DNA
Promoter Operator
Repressor – Transcription Blocked
Activator – Transcription EnabledDNA
Promoter Operator
RNAP
repressor
activator
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The lac Operon
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The lac operon
• Jacob and Monod studied the regulation ofgenes required for the metabolism of lactose
in bacteria (coordinate regulation)
François Jacob
(1920 - )
Jacques Monod
(1910 – 1976)
Won the Nobel Prize in Physiology or Medicine, 1965
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Lactose Catabolism
• A transcriptionally regulated system
• When the bacterium is in an environment that contains lactose,
the cell would want to turn on genes for enzymes that are
required for lactose catabolism• When lactose is absent, the cell would want to turn these genes
OFF
• Actual regulation is more complicated
• Glucose = prime source of food
• So, if both glucose and lactose are available, the bacterium willturn off lactose metabolism in favour of glucose
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Lactose Breakdown in E. co li
by the enzyme beta-galactosidase
+
GLUCOSE GALACTOSE
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The lac operon
• Studied extensively by François Jacob and Jacques Monod• If lactose is present then three protein products are made:
– Lac Z = -galactosidase:
(lactose glucose +galactose)(lactose allolactose)
– Lac Y = Permase
(active transport of lactose across cell membrane)
– Lac A = Transacetylase
(galactose acetylgalactose)
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The lac operon
• All three genes share the same promoter (lacP ), the same
operator (lacO), and are transcribed as a single mRNA
( polycistronic mRNA)
• The gene for a regulatory protein (LacI), encoding a
repressor, is found near the operon
lacI lacP lacO lacZ lacY lacA
lac operon
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lac Operon Regulation
• Since the role of proteins encoded by lac isto break down the sugar lactose, the
structural (protein-coding) genes lacZ, lacYand lacA should only be expressed whenlactose is present in the cell
•
The lacI gene encodes a repressor proteinthat shuts the system down when lactose isnot present
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Induction of the lac operon
• If lactose is present in the environment (growth medium), the lac
operon is ‘induced’
• Lactose enters the cell and binds to the lac operon
• This induces a conformational change that allows the repressor to
fall off the DNA
– Now RNA polymerase is free to move along the DNA
– RNA can be made from the three genes
– Lactose can be metabolized
• When the inducer (lactose) is removed, the repressor returns to its
original conformation and binds to the DNA
– RNA polymerase can no longer get past the promoter
– No RNA or protein is made
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Induction of the lac operon -1
Repressor (LacI) bound to operator, RNA
polymerase cannot initiate transcription
at the promoter
No lactose - system turned OFF
lacP lacO lacZ lacY lacA
LacI
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Induction of the lac operon -2
If lactose is added, an isomer (allolactose) will bind
to the repressor
lacP lacO lacZ lacY lacA
Allolactose
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Induction of the lac operon -3
The allolactose-bound repressor undergoes a conformational
change and dissociates from the operator sequence.
RNA polymerase is then free to initiate transcription
Lactose present – system turned ON
lacP lacO lacZ lacY lacA
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Mutations affecting lac
• lacZ -, lacY -, lacA- Structural gene mutations lead to
non-functional proteins
• lacP - Non-functional promoter, RNAP cannot bind so
genes will not be expressed• lacOC Non-functional operator so repressor cannot
bind. Since the system cannot be shut off, this is a
constitutive mutation
lacI lacP lacO lacZ lacY lacA
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Mutations affecting the
lac operon
• lacI - Non-functional repressor, unable to
bind the operator to shut off transcription
• lacI S Super-repressor, unable to dissociatefrom operator. System always off.
lacI lacP lacO lacZ lacY lacA
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Summary of the E.coli lactose operon
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Regulation of lac
• When a repressor is used to turn the system off, that
system is under negative control . The LacI repressor
is an example of this.
• The lac operon is also under positive control , where
an activator protein is used to increase the efficiency
of transcription
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Glucose is the Favourite
Carbon Source of E. co li • Whereas E. coli can metabolize lactose using the
products of the lac operon, it actually ‘prefers’ glucose
as its carbon source• Remember that glucose can be directly introduced into
glycolysis
• Therefore, when glucose is present, there is no need to
express the genes in the lac operon
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No glucose?
• If a cell runs out of glucose, a small molecule – cyclic3’5’adenosine monophosphate (cAMP) – is producedfrom ATP by the enzyme adenylate cyclase
• cAMP is a ‘hunger signal’ that stimulates the
expression of genes that produce enzymes to breakdown alternate sugars, such as lactose
• cAMP binds the activator protein CRP (cAMPreceptor protein) or CAP (catabolite activatorprotein), which can then bind to lacP to help activatetranscription
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The Hunger Signal
lacP lacO lacZ lacY lacA
CRP binding site
Inactive CRP
When glucose is present (NOT hungry), no
cAMP is produced so CRP is inactive. RNAP
cannot initiate transcription of the lac operon.
RNAP
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The Hunger Signal
If glucose is depleted, cAMP is produced, which
binds to and activates CRP. Activated CRP can
then bind to the promoter.
lacP lacO lacZ lacY lacA
CRP binding site
Active CRPcAMP
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The Hunger Signal
2 Actions combine:• Lactose present – then allolactose will bind to repressor to cause its
dissociation.
• Activated CRP binds the promoter, so RNAP can enter and initiate
transcription
• Genes of the lac operon are transcribed
lacP lacO lacZ lacY lacA
allolactose bound to repressor
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On vs. Off
Lactose
present
Lactose
absent
Glucose present Glucose absent
No repressor bound
No activator bound
OFF
Repressor bound
No activator bound
OFF
No repressor bound
Activator bound
ON
Repressor bound
Activator bound
OFF
Note: this option would occur
when a third kind of sugar was present
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The t rp Operon
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• In addition to sugars, like glucose and lactose, E. coli cells
also require amino acids
• One essential amino acid is tryptophan
• When E. coli is in tryptophan (milk and poultry) it will absorb
the amino acids from the media
• When tryptophan is not present in the media then the cell
must manufacture its own amino acids
The t rp Operon
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The t rp Operon
• Responsible for biosynthesis of the essential amino acidtryptophan
• If tryptophan levels are low, turn on expression toproduce more
• If tryptophan levels are high, turn off the system
• All 5 genes are transcribed together as a unit
trpP trpO trpE trpD trpC trpB trpA
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The t rp Repressor (TrpR)
• Encoded by the trpR gene, located far from the trp
operon
• TrpR can only bind to the trp operator if activated by
a tryptophan molecule (co-repressor)
trpP trpO trpE trpD trpC trpB trpA
TrpR bound to tryptophan
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Translational Control of t rp
gene expression
• Another method of control is through attenuation • The first protein-coding gene in the trp operon is the‘leader peptide’ trpL, which contains adjacent codonsfor tryptophan
• Therefore, if tryptophan - tRNA is abundant in the
cell, translation of this peptide will be quick
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Attenuation
• mRNA secondary structure is essential in thisregulation
• If tryptophan is abundant, the ribosome willspeed quickly through the trp codons
BUT
The mRNA BEHIND the codons will form astem-loop terminator structure, and RNAP willfall off before it can transcribe trpEDCBA(slide to come)
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Attenuation
• If tryptophan is rare in the cell, the ribosome
will pause at the Trp codons
• This pausing will prevent the formation of theterminator signal, and RNAP will continue
transcribing the trpEDCBA genes
Stem loops form by complimentary base pairing
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Stem loops form by complimentary base pairing
in the Trp leader RNA
Movement of the ribosome through the Trp codons produces alternate stem
loops:
Fast movement (Trp present) = termination structure (UUUUU) (no
transcription)
2 3
Tryptophan
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trpE trpD trpC trpB trpATrp
Codons Ribo
4
Stem loop
Termination
structure
RNA
pol.
RNA polymerase
falls offRibosome
proceeds quickly
through Trp
codons
trpE trpD trpC trpB trpATrp
Codons
2 3
4
AttenuationRibosome stalls
at Trp codons
Transcription
proceeds through
Trp Operon
yp p
high
Tryptophan
low
RNA
pol.
Ribo