Transcript Lecture 12 – Genetic Circuitry

Lecture - 11
Protein-Protein Interaction
GEB 406
Course Instructor: Sheikh Ahmad Shah
Semester: Summer 2016
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Protein-Protein Interaction
Proteins work together by actually binding to form multicomponent
complexes that carry out specific functions. These functional units
can be as simple as dimeric transcription-factor complexes or as
complex as the 30-plus component systems that form ribosomes.
Biochemists believe that all proteins bind to or interact with at least
one other protein. The discovery that proteins in higher organisms
(e.g., human and mouse) contain higher numbers of functional
domains suggests that many of these proteins have multiple
associations. Understanding how protein complexes work is
essential to understanding how cells work as systems.
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Identifying Protein-Protein Interaction
In the pregenomic era,
immunoprecipitation
was the primary means
of determining proteinprotein interaction.
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Identifying Protein-Protein Interaction
In the post genomic era, few methods have proven especially helpful
for this purpose. Yeast two hybrid (Y2H) system is one of them.
In short, the Y2H method is designed to use a protein of interest as
bait in order to discover proteins that physically interact with the bait
protein, those proteins are termed as preys.
In Y2H method, a single transcription factor is cut into two pieces
called the DNA Binding Domain (DBD) and Activation Domain (AD),
which stimulates the RNA polymerase to begin transcription.
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Identifying Protein-Protein Interaction
Fused to the DBD is the bait protein of interest (B), which cannot
initiate transcription on its own. Fused to the AD the prey ORF, which
can be any known or unknown protein. The prey protein of AD + ORF
fused together cannot initiate transcription either. When the bait and
prey proteins are produced in the same cell, they might interact; and
if they do, transcription of His3 gene is initiated.
Any ORF can be tested with Y2H, which means a proteome-wide
survey can be performed rapidly by transforming a genomic library
into cells that contain bait plasmids. In this way, every protein in a
proteome can be tested individually for its potential interact with
bait.
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Identifying Protein-Protein Interaction
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Lecture - 11
Genetic Circuitry
GEB 406
Course Instructor: Sheikh Ahmad Shah
Semester: Summer 2016
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Introduction
Every cell in an organism contains the exact same DNA. Despite the
genes being all the same, our body can produce different cell types
(like: liver cells, brain cells, skin cells, etc.) by expressing only a
subset of genes.
The subset of genes expressed is tightly controlled during
development, and this control is exerted over:
• Location
• Time
• Amount
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Genetic Circuits
Genetic circuits are functional interaction of proteins and DNA
sequences through inducible transcription factors and cis-regulatory
elements like promoter, enhancers, etc.
Endo16 is one of the most studied genes
which serves as a model for understanding
the genomic control over the expression of
genes. This gene is expressed in the
developing gut of a Sea Urchin embryo.
Sea urchin is transparent in nature and has
been used as a model organism for
developmental biology for over 100 years.
Sea Urchin Embryo
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Genomic Control over Genes
At the upstream of start transcription
site, there are some DNA sequences
which control the transcription process.
These are termed as “cis-regulatory
elements”.
On the other hand, there are some DNA
sequences further away from the coding
sequence, often located on a separate
chromosome, which also regulates
transcription. These are termed as
“trans-regulatory elements”.
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Genomic Control over Genes
Cis-regulatory elements are modular in their organization, which
means that the DNA can be divided into functional units, each of
which performs a particular job.
Each cis-regulatory module is composed of a sequence of DNA to
which one or more DNA binding protein can bind, either to help
initiate transcription (transcription factors) or to repress
transcription (repressors).
The best understood cis-regulatory elements are found in the sea
urchin, at the upstream of Endo16 gene.
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Early Development of Sea Urchin
Sea urchin zygote cell starts to divide mitotically. From one cell to
two cells, from two cells to four cells, and so on. From the mass of
cell, a hollow sphere of embryonic cell called Blastula is formed.
Blastula then undergoes a process called Gastrulation when a subset
of blastula cells begin to invaginate or move into the cavity of the
blastula/gastrula.
As the cells begin to invaginate, they repress some genes and
activate others. The cells that form the elongating tube inside the
gastrula will become the endodermal cells that line the gut of the
future larva. This elongated tube of cells is called the archenteron.
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Early Development of Sea Urchin
Fate Map of Sea Urchin
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Molecular Dissection of Development
Slightly before gastrulation, cells at the base of the blastula express a
gene called Endo16, and later all cells of the invaginating
archenteron express Endo16. These cells will become endodermal
cells in the larva. During development, the expression of Endo16
gene is tightly controlled for location, time, and amount of RNA.
This expression pattern has made Endo16 an excellent genetic
marker to identify which cells will form the endoderm. Later in
gastrulation, the cells of the foregut and hindgut repress Endo16 so
that only midgut cells still express it. By late gastrulation, the midgut
cells transcribe Endo16 at an even higher rate than before.
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Molecular Dissection of Sea Urchin Development
The modules of Endo16's cis-regulatory elements are located in the
2,300 bp upstream of the coding DNA. Each module (G-A) has a
function when studied individually because DNA-binding proteins
recognize specific DNA sequences within each module, and these
protein-DNA interactions regulate transcription.
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Approach of Davidson’s Group
Several DNA constructs were created to fuse each module
individually onto the most basic promoter (Bp) that allows RNA
polymerase to bind and begin transcription. A reporter gene CAT
(Chloramphenicol Acetyl Transferase) was used in place of the coding
portion of Endo16, because CAT output could easily be monitored.
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Location of Expression
It was found that, constructs 1, 2, 7, and 8 promote the production of
CAT in endodermal cells. These constructs include modules G, B, and
A. Constructs containing modules C, D, E, and F do not promote the
production of CAT in endoderm cells but do permit CAT production in
mesoderm and ectoderm cells.
The roles of modules F-C were unclear and seemingly counterproductive. Therefore, new constructs were created that combined the best
endoderm promoter (GBA+Bp) with each of the remaining modules.
The idea was to test whether modules F-C had any influence on the
transcription promoted by modules G, B, and A.
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Location of Expression
When the new constructs were tested, they exhibited different
capacities to promote the formation of CAT. However, the level of CAT
production was essentially unchanged in endodermal cells, but the
level of "inappropriate" CAT expression in mesoderm and
ectoderm was altered.
Modules DC reduced the capacity of the promoter to function in
mesoderm cells, while modules F and E each reduced the expression
of CAT in ectoderm cells. That suggested that modules F-C appear to
function as cell-type specific repressors of transcription, which helps
explain why Endo16 is not expressed in ectoderm or mesoderm cells.
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Timing of Expression
After establishing which modules promote and repress transcription
and which modules address the location component of Endo16
expression in embryogenesis, scientists then turned to the question
of timing.
They took all the previously made DNA constructs again for this
research. For each DNA construct, CAT enzyme activity was measured
at each time point. First, CAT activity using constructs 1, 2, 7, 8, and
10 were measured, and it was found that the three inducing modules
(G, B, and A) exhibited different temporal profiles.
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Timing of Expression
Module A induced CAT production
during the first 48 hours and then
dropped off. Module B promoted CAT
production primarily at the 60- and
72-hour time points. Module G did
not promote much CAT production by
itself, though there is a marginal
increase around 48 hours. When
modules GBA were combined, the
expression level was almost equal to
the wild-type cis-regulatory element
containing all eight modules.
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The Effect of Module G
To determine the role of module G in
Endo16 transcription, the investigators
built few more DNA constructs. For
these constructs, they removed the
Endo16 Bp (basal promoter) and
replaced it with a weakened viral
promoter (SVp). When each inducing
module was placed individually
upstream of SVp, the output indicated
that modules G, B, and A exerted their
influence on transcription without any
participation by Bp.
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The Effect of Module G
The amplitude of CAT production by module A was increased
approximately fourfold when module G was added onto A+SVp,
though the shape of the curve was essentially unchanged.
Interestingly, module G did not alter the amplitude or shape of
module B's ability to pro-duce CAT.
When module G was added onto BA+SVp, the amplitude is
substantially increased at the 48-hour time point, which is when
module A is exerting its maximum effect. Furthermore, the addition
of module G (GBA+SVp) has increased the output from module B at
60 and 72 hours, when module B becomes active. In short, module G
acts as an amplifier for module A and B* (*while combined with A).22
Circuit Diagram of Endo16 Cis-Regulatory Elements
A circuit diagram can be drawn which can explain the cis-regulatory
elements function of Endo16 in early and late development.
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Integrating Single-Gene Circuits
the complexity of wholegenome regulation is too
overwhelming to diagram
as simple circuits.
Genomic information is
accumulating faster than
ever, and new tools are
needed to visualize all of it
simultaneously. So, many
gene circuits are being
integrated.
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Integrated Genomic Circuit
Our genes are regulated to be activated in some cells and repressed
in others. Genetic expression changes dynamically in response to
environmental influences and aging.
Cells need a mechanism to switch genes from on to off and vice
versa. Genes need to sense their intracellular environment and
respond accordingly. But cells should also be tolerant of some
cellular variations. Furthermore, cells need to have alternative
means for accomplishing vital functions. Our genomes must be
prepared for circumstances that might block one circuit from
performing its cellular role.
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Bistable Toggle Switches
Bistable Toggle Switch is the switch that is used to turn any
instrument or device on or off in a stable way. A biological, bistable
toggle switch will remain in one position (on or off ) until the circuit
determines the switch should be toggled to the other position.
A biological toggle switch typically consists of three factors:
Promoters, Repressors, and Inducers.
• (Constitutive) Promoters encourage expression of a gene.
• Repressors bind to promoters, inhibiting expression of genes.
• Inducers bind to repressors, preventing repressor binding to
promoters. Thus inducers encourage expression of genes.
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Bistable Toggle Switches
The bistability of the toggle arises from the mutually inhibitory
arrangement of the repressor genes. In the absence of inducers, two
stable states are possible: one in which promoter 1 transcribes
repressor 2, and vice versa. Switching is accomplished by transiently
introducing an inducer of the currently active repressor. The inducer
permits the opposing repressor to be maximally transcribed until it
stably represses the originally active promoter.
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How Do Toggle Switches Work?
Genetic switches have to deal with a degree of uncertainty, which is
termed as “Noise”. Gene activation occurs when transcription factors
bind to cis-regulatory elements. When a cell undergoes mitosis and
cytokinesis (eukaryotes) or cell division (bacteria), the first source of
noise is introduced as transcription factors may not split in 50:50
ratio. For this kind of uncertainty, the process is called “Stochastic”.
For example, if a cell had 50 copies of the Otx transcription factor, 6%
of the time a particular daughter cell might get 19 or fewer copies
(instead of 25 copies), while 6% of the time it might get at least 31.
That could have a profound effect on the subsequent regulation of
Endo16 expression.
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How Do Toggle Switches Work?
Another component of genetic noise is the random binding of
proteins (transcription factors) to its target DNA. As each cisregulatory element must be found by a small number of DNA-binding
proteins, it results in an increased range of times when all the
transcription factors are in the right places for any given gene.
Again, once the cis-regulatory element is fully occupied and ready to
initiate transcription, the first RNA will be produced after a variable
amount of time due to noise in the initiation of the transcription
machinery. For these kind of stochastic behaviors, the time of the
transcription of a particular gene can not be predicted very precisely.
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Effect of Noise and Stochastic Behavior
In prokaryotes and eukaryotes, proteins are produced in bursts of
translation of varying durations and with varying outputs. Therefore,
the total number of proteins produced from any gene is not the same
each time, but rather an average with a normal distribution (a bell
shaped curve, with “average” being the highest point of that curve).
By producing proteins in bursts rather than at a constant rate, the cell
provides proteins a higher probability of forming a quaternary
structure (e.g., a dimer) that may be required for full function. So,
there exists a chaotic and mildly disorganized environment for
protein expression inside the cell.
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Effect of Noise and Stochastic Behavior
Protein A can bind to the cisregulatory elements of genes b
and c to initiate transcription for
both genes. Protein B has three
possible fates: it can be
degraded by the cell; it can diffuse away and perform other functions;
and, most importantly for us, it can repress the expression of gene c.
Conversely, protein C has three fates, one of which is to repress gene
b. Here, A can bind with either B or C, resulting in the repression of C
and B respectively. Stochastic factors like the amount of A and its
ability to find a limited number of binding sites upstream of b and c
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determine which protein will be expressed by the cell.
Toggle Switch in λ Phage
In bacteriophage λ,
there is a naturally
evolved toggle switch
which controls whether
the phage will go into
lytic phase or lysogenic
phase. Here, the
deciding factor is a
single protein called CII
or C two. (This CII is
equivalent of protein A
of the previous slide).
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Toggle Switch in λ Phage
If CII finds the promoter PRE, transcription will proceed toward the left
of PRE and lead to the transcription of CI (C one) further downstream.
Dimer of CI can bind to the promoter PL upstream of CIII and lead to
the production of CIII. CIII prevents the destruction of CII; thus, CIII
indirectly reinforces its own production in a positive feedback loop.
Transcription of CI
Transcription of CIII by CI
Prevention of CII degradation by CIII
Toggle Switch in λ Phage
Dimerized CI (or CI2) reinforces its own production indirectly by
binding to sites labeled OR1 and OR2 to repress the production of Cro
protein (CI2 acting as a repressor of cro). CI2 binding to OR1 and OR2
also promotes its own production in a positive feedback loop by
acting as a transcription factor for its own gene, CI.
CI2 repressing Cro
Positive feedback loop of CI
Toggle Switch in λ Phage
Once CII initiates this bistable toggle switch, λ is locked into peaceful
lysogenic coexistence with its host E. coli unless new environmental
forces disturb the system (e.g., UV light, change in nutrient
availability).
However, the toggle switch could have flipped the other way,
depending on the noise and stochastic protein behaviors. CII protein
could have been degraded if it took too long to find PRE, because E.
coli makes a protease that can destroy CII. If Brownian motion
(random motion driven by kinetic energy) causes the protease to find
CII before CII finds PRE, the lytic lifestyle is chosen.
Toggle Switch in λ Phage
In the absence of CII, the promoter labeled
PR is weakly active and begins transcribing to
the right, resulting in the production of Cro
protein. Cro2 binds to OR3 and OR2, which
leads to repression of CI and increased
transcription of cro. The positive feedback
loop keeps the bistable toggle switch flipped
toward cro transcription and a lytic lifestyle
that eventually leads to the production of
hundreds of fully mature viruses that swell
and lyse the E. coli host cell.
Toggle Switch in λ Phage
There are several noisy factors in the choice made by λ phage, such
as the limited number of proteins and binding sites, variable
amount of time for transcription, burst of protein production for
efficient dimerization. Environmental influences can also skew this
decision. For example, if the bacterium host happens to be growing
in a nutrient-rich environment, the bacterium produces more
protease, resulting in faster destruction of CII and the production of
many new λ phage (lytic lifestyle). Conversely, if the bacterium
happened to be in a nutrient-poor environment, there are fewer
protease molecules, so CII has a higher probability of finding its
binding site on PRE before being destroyed. A longer half-life for CII
leads to peaceful coexistence (lysogenic lifestyle).
Genomic Control of Different Genes
In a study, investigators from Tufts University had examined the
amount of noisy factors generated by different aspects of a genomic
circuit. They placed two different promoters recA and lacZ upstream of
the reporter gene GFP and then measured the expression in 200
individual cells when under control conditions or under induction.
It was found that the recA promoter is constitutively on (always
activated) at a low level with varying amount of noise. When induced,
recA promoter stimulates large amounts of mRNA. In contrast, lacZ
exhibits a very low background level of transcription with little noise
under control conditions, and induction does not produce as much
increase over basal rate.
Genomic Control of Different Genes
The behavior of recA and lacZ promoters makes sense considering
their roles. RecAp is an essential promoter used to repair DNA damage
which is a vital process. Thus, when the cell senses DNA damage, the
promoter requires only one step to switch to a higher expression rate
with relatively less noise.
In contrast, lacZp metabolizes lactose, and the gene is induced in the
absence of glucose and the presence of lactose. Basal expression of
lacZ is normally low because alternative sugars would be available.
The toggle switch for lacZ induction requires several other proteins,
and each of those proteins has its own level of noise. Therefore, lacZ
induction is a much noisier system than RecA.