MOLECULAR BIOLOGY TEST #3

Gene Regulation in Prokaryotes

Regulation of transcription initiation by transcription factors (transcriptional
activators or repressors)


-When a gene is turned OFF, it is not literally turned OFF completely. The mechanism is more like a tap; it can
be opened to increase gene expression or closed to decrease gene expression
* Expression of housekeeping genes is different in different tissues. This up or down regulation is
performed by the cell
-Most gene regulation occurs at the Initiation Stage: where RNA polymerase binds the promoter and activates
transcription (95% of regulation occurs at this time)
* This happens b/c the cell doesnt want to be wasteful = less waste if regulated before expression.
*Also because it is easier to regulate one or two copies of a gene instead of having to modify
hundreds or thousands of RNA molecules.
-Outside chemicals can enter bacteria and affect transcription
-When bacteria is grown at a higher temperature than the ideal (37C), a group of genes (and therefore proteins),
called heat-shock proteins, are activated.
* The function of these proteins is to protect the bacteria against the heat




-Promoters in Prokaryotic cells: since the promoter is not 100% promoter for the consensus sequence, the
RNA polymerase has a relatively weak attraction and can only perform small amount of transcription = basal
level transcription (Transcription of a gene in the absence of any activator)
-Repressor molecules can recognize and bind the promoter region, thus inhibiting the binding of RNA
polymerase and transcription.
-Activator molecules help the RNA polymerase to bind the promoter region. The activator molecule itself will
NOT bind the promoter, but a site adjacent to it. This allows the RNA polymerase to bind the promoter. The
activator will bind both the DNA and the RNA pol.



-Open or Closed Complex: Refers to the DNA. When the RNAP is attached, it is in the open complex. When it
is not attached, it is called the closed complex.
* For DNA to be transcribed it must be in the Open Complex
* The Activator cause the change in complex: It can either change the 3D conformation of the RNAP
or it can act directly on the DNA (separating the two strands of the double helix.)


-Sometimes, DNA Binding Proteins work together synergistically. In the above example:
Protein A cannot bind its site unless Protein B is already bound to its site; the presence of Protein B is
aiding in the binding of Protein A.
*This provides a biological switch to turn on transcription. Sometimes, some genes may only be turned
on when all 4 (example) specific proteins are present.
-Binding sites of proteins may be very far apart. DNA can bend to allow for proteins to interact.




-Bacteria prefer glucose as an energy source b/c it is a monomer, unlike lactose which must be broken down

-Lactose DOES NOT diffuse into the bacteria; it must use a channel formed by the Lac Permease.
*Once inside, lactose is broken down by Beta-galactosidase into Galactose & Glucose

-Lac operon consists of 3 main genes: lacZ, lacY, and lacA Each encodes something different (MEMORIZE)

-How do activator and repressor know when to bind to the DNA? They are regulated.

-cAMP (in the absence of glucose) binds CAP and causes a conformational change in the molecule (BELOW)

-The conformational change allows CAP to bind the DNA (Major Groove)
-Activator is in INVERSE CORRELATION with Glucose


-DNA Binding Proteins usually sit on the DNA as homodimers or heterodimers.
* Homodimer: Two of the same molecule
* Heterodimer: Two different molecules

-Restriction enzymes are HOMODIMERS = have symmetry (bind DNA as homodimers)


X-gal: B-galactosidase activation will cause cleavage of X-gal, producing a blue color


-Footprinting utilizes a DNase which cuts the DNA at random sites, except those areas bound by a DBP
(Footprint will correspond to area that is protected)


-The core RNA polymerase in bacteria is the same, what is different is the promoters.
* Many different sigma factors bound in bacteria (protein bring RNAP to a promoter; it recognizes the
DNA sequence)
-To hijack bacteria, bacteriophages take over the host cell with the use of certain operons which produce
proteins that correspond to each sigma factor. These new sigma factors produced by the bacteriophage infecting
the host will give the RNAP a different specificity for a different type of promoter, causing it to bind a different
promoter and transcribing it.
END PCB 1.9

Ch17 Eukaryotic Gene Regulation
Initiation of Translation is the MAIN CONTROL STEP
**Most Eukaryotic regulation occurs at the INITIATION STAGE
As in prokaryotes, Transcription is regulated by ACTIVATORS and REPRESSORS
**Mechanisms of Translation have been relatively conserved from yeast to mammals
Information we have learned about how Translation works has been learned by studying yeast.
Easier to use yeast in experiments rather than human cells
Eukaryotic cells have additional complexities UNIQUE to them:
1. Nucleosomes (The name of the structural unit made up of a HISTONE) and their modifiers
influence access to genes (unique problem but also an opportunity for regulation).
The function of histones is to package DNA in an orderly way. Aids when going back and
accessing different components of the DNA.
Gene regulation in Eukaryotes also works by modifying the histones.

2. Splicing (and its regulation) is unique to eukaryotes Remove introns, etc

3. Many eukaryotic genes have more regulatory binding sites and are controlled by more
regulatory proteins than are typical bacterial genes.
In eukaryotes binding sites are more numerous and positioned further from the start site of
transcription.
** Coupled Transcription/Translation exists in prokaryotes but NOT in eukaryotes. After it is transcribed, pre-
mRNA (Still has introns that must be excised) leaves the nucleus to be translated by ribosomes in the
cytoplasm.

Above Diagram:
- Bacterial gene has regulatory sequence right next to promoter
- Yest is between bacterial and human gene. Promoter is present, as are regulatory sequences which
are slightly upstream of the promoter, but not very far.
- The human gene has tolls for regulatory elements, of which there are many more present (They can
be both positive and negative) than in prokaryotes
Regulatory elements can be very far away from initiation site, which may create many problems as
well. (Action from a distance requires insulators or boundary elements).
Eukaryotes can also have regulatory elements downstream of the initiation site; the reason for this
is that many eukayotic genes have introns, which are not transcribed/translated. Regulatory elements
can be present within these introns.

-----BE ABLE TO NAME 5 DIFFERENCES/SIMILARITIES BETWEEN EUKS & PROKS-----


ACTIVATORS
**Activators have separate DNA binding and activating functions/DOMAINS

**BOTH domains are necessary & essential for activation of transcription
If activation domain is removed, the DNA-binding domain will be able to bind the DNA by itself,
but it WILL NOT be able to activate transcription.
If activation domain is present in the absence of the DNA-binding domain, nothing will happen
either.
Example of a Yeast Gene

** GAL1 gene has Upstream Activating Sequence consisting of 4 DNA motifs/elements where GAL4 is going
to bind.
Activating sequences often shown as one element, but most times they have various binding sites
Having more binding sites allows for more activators to bind and for the upregulatory effect to be
multiplied

Figure Above:
Shows that an activating domain from Yeast can be used in a mammalian cell.
Shows that these proteins are modular, have different domains, and can function in the context of
other domains.
What is unique about a DNA-Binding Domain?
There are 4 families of DNA-Binding Domains.
1. Homeodomain Proteins - Involved in Embryonic development. It has 3 alpha-helixes. The #3 alpha
helix is the one that interacts with the major groove of the DNA.

2. Zinc containing DNA-binding domains (Zinc Finger Proteins) - Require zinc atoms to function.
To inactivate them, you add the ZFP to a solution with EDTA or EGTA which will chelate the zinc out
of the protein, protein will collapse. Biggest family of DNA-binding proteins

3. Leucine Zipper Motif Forms a HOMODIMER that interacts with the DNA. Is a major class of
transcriptional factors.


4. Helix-Loop-Helix Proteins Two alpha-helixes. Can form HOMODIMERS as well as
HETERODIMERS. This gives the protein more flexibility. Example of H-L-H is the Nik (sp?) family of
genes

Activation Domain Part of the protein that interacts with the RNA Polymerase (In Prokaryotic Cells) or the
Mediator (In Eukaryotic Cells). It was discovered by analyzing many different ones that there is no particular
motif or structure to activating domains.
They have a higher concentration of acidic amino acids.
Some are glutamine rich, others are proline rich.
Activating domains can be synthesized to fit your specific needs (Genetic Engineering)
In Bacteria, the activators interact directly with the RNA Polymerase. In Eukaryotics, they interact with the
MEDIATOR. (**Major Difference**)
Activators in Eukaryotes can recruit nucleosome modifiers.

Mediator Complex is needed because since there is so many activators and repressors regulating one
single gene, Mediators weigh out the many positive and negative interactions and expresses the
final outcome on the RNA Polymerase.
Chromatin Immunoprecipitation (ChIP) Not Tested On This?

ChIP: You have an activator (or repressor); want to find out what is the target of the activator-- and how many
genes it activates (since most times one activator will regulate multiple genes.)
Proteins that sit on the DNA will be cross-linked and frozen in that particular location.
Next, use a DNase/other enzyme/sonication to cut DNA into smaller pieces.
Fragments of DNA with proteins attached to them. Use an antibody to precipitate out the protein that
you are interested in. By precipitating the protein, you will also precipitate the piece of DNA that is
attached to it.
Use a chemical method to remove the protein from the DNA
Perform a PCR to extend the DNA, then sequence them and see that those specific pieces of DNA
interact with your activator.
You get multiple sequences as a result; a computer will tell you where each specific sequence is
located. You hope to find that this sequence is located in the promoter region of a gene, letting you
know that this specific activator (protein) regulates this specific gene.
10/30
Activators also recruit nucleosome modifiers that help the transcription machinery bind at the
promoter
**Two types of nucleosome modifiers:
1. Histone acetyl transferases (HATs) [A] - Transfer acetyl groups
2. Those that remodel the nucleosomes (ATP-dependent activity of SWI/SNF) [B]

A: Activator will recruit the histone acetylase to interact with it. The conformation of the DNA
wrapped around the nucleus is changed, making previously inaccessible promoters once again
accessible and allowing for transcription to take place.
B: Activator once again recruits the remodeling complex which changes the structure of the
nucleosome, allowing transcription to take place.
**It is believed that these types of modifications precede others because they make the DNA more accessible**

Some of the enhancer sequences that are recognized by activators (or repressors) can be many thousands of BP
away. Other genes may be in the vicinity of an activator. To make sure that the right promoter is activated, the
cell utilizes insulators.
Insulators: proteins sit on the DNA between an enhancer and the promoter; by doing so, they block
the effects of the enhancer on an undesired promoter. This will allow for the enhancer to interact with
the desired promoter.

Examples of Cooperativity

Signal Integration
The HO gene is controlled by two regulators; one recruits nucleosome. Modifiers and the other recruits
mediator.
SWI5 is the activator; when it recruits the CRC and HA to the DNA promote,r they change structure
of the DNA next to the promoter and allow the SBF factor to bind and initiate transcription

Signal Integration: Cooperative binding of activators at the human -interferon gene. 3
regulators come together to form enhancer sequence + HMG that form brackets (Need all)

Combinatorial control lies at the heart of the complexity and diversity of eukaryotes.
Since there is only about 2000 regulatory proteins, and over 25000 genes to regulate, different
combinations of the same proteins must be used to regulate each gene.


Transcriptional Repressors: **(ways in which Transcriptional Repressors work)**
1. Competition [a] Overlapping binding sites of activator & repressor
2. Inhibiton [b] when activator & repressor bind to adjacent sites, the repressor will push of the activator
3. Direct repression [c] A repressor will interact with the mediator, sending a negative signal to the RNA
Polymerase
4. Indirect repression [d] A repressor will recruit a histone deacetylase (vs an Activator Histone
Acetylase) that removes the acetyl groups, decreasing transcription.


Signal Transduction and the control of transcriptional regulators.
- Signals are often communicated to transcriptional regulators through signal transduction pathways.

STATs: Transcriptional regulators that are present in the cytoplasm when they are not activated.
When phosphorylated, stats dimerize and travel to the nucleus and affect transcription.
Ras Pathway: most common pathway. A receptor is triggered by a signal molecule (usually growth
factors found in medium).
Importance in Human Cancers: A single mutation makes Ras permanently active, even when
growth factors are not present in the plasma membrane, causing uncontrolled growth.
Why so many steps in a cascade? Different receptors can feed onto pathway at different points.
More steps = finer regulation = increased amplification

Imprinting
Some genes are only expressed if they are on the paternal chromosome. The maternal chromosome
will translate the H19 gene because the insulator prevents the translation of other genes.
In the paternal chromosome, the insulator is not present and therefore the enhancer will activate the
Igf2 gene.
This demonstrates that some proteins are only coded for by parent-specific chromosomes.
(Mitochondria = Mother)


RNAs in gene regulation
Double-stranded RNA inhibits expression of genes homologous to that RNA

Short interfering RNAs (siRNAs) are produced from dsRNA and direct machinery that switches off
genes in various ways. After integration into the RISC, siRNAs base-pair to their target mRNA and
induce cleavage of the mRNA, thereby preventing it from being used as a translation template.


Possible end results:
Degradation (dsRNA is 100% homologous to cell DNA)
Translation Inhibition (dsRNA partially homologous to cell DNA)
Amplification believed to occur b/c very few molecules of siRNA in the cell are able to inhibit
many hundreds of
Translocation to nucleus shut down gene translation
Signals Control the activities of eukaryotic transcriptional regulators in a variety of ways:
(ppt 2.0 not reviewed)

Gene Silencing by modification of histones and DNA. (ppt 2.0, not reviewed)
Silencing is a position effect; a gene is silenced because of where it is located, NOT in response to
environmental signals.
Silencing can spread over large stretches of DNA, switching off multiple genes, even ones quite a
distance from the initiating event.


END PCB2.0

11/4
Bioinformatics
DNA Chip Microarrays
Put a large number (~100K) of cDNA sequences or synthetic DNA oligomers onto a glass slide (or
other subtrate) in known locations on a grid.
Label an RNA sample and hybridize
Measure amounts of RNA bound to each square in the grid
Make comparisons
- Cancerous vs. normal tissue
- Treated vs. untreated
- Time course
* Many applications in both basic and clinical research

What genes are expressed determines what each cell is and what it does. With DNA microarrays,
you can compare, for example, cells from the liver and cells from a cancer.
Scientists use DNA microarrays to measure the expression levels of large numbers of genes
simultaneously or to genotype multiple regions of a genome.
From lecture: Compare the expression of every single RNA that is present in the cell. Can compare
cancer vs. normal cells. Treated vs. untreated (by a specific drug).
Each dot on grid is a particular gene. Any RNA that has come from that specific gene during
translation can hybridize the gene (binding of the DNA by the complementary RNA). You label the
cDNA (YOU MUST USE DNA b/c it is more stable than RNA) population with fluorescent dyes. A
gene expressed only in normal cells will be one color; a gene expressed in cancer cells will be
another color. You can have colors in between the two that give proportions of gene expression.
Use this method to find a protein that is made in cancer cells but not in normal cells so that they can
be targeted
Single-nucleotide polymorphisms (SNPs) - the most common known variation in the human
genome sequence.

Any two unrelated individuals differ by one base pair every 1000

These are referred to a single nucleotide polymorphisms (SNPs)

It is believed that SNPs are the cause of most common genetic disorders

Many SNPs have no effect on cell function

Some could predispose people to disease or influence drug response

SNPs must happen in at least 10% of the population (MUTATIONS = much less common)


Why do we care about SNPs?
93% of all annotated genes contain at least one SNP.

59% of human genes have five or more SNPs

39% have ten or more SNPs

Discover how SNPs can cause diseases

Examine how SNPs can affect medical therapies
Common Diseases
If we carry a particular set of SNPs, we can be predisposed to one common disorders with a genetic
component such as diabetes, asthma or cancer

There are a number of classic genetic diseases caused by mutations of a single gene.

There are also many diseases that are the result of the interactions of many genes:
Asthma, Heart Disease, Cancer

Each of these genes may be considered to be a risk factor for the disease.

Groups of SNP markers may be associated with a disease without determining mechanism


END PCB2.3.1

11/6
Ch18 - Gene Regulation during Development
Humans have more than 200 different cell types, each of which has distinct programs of gene
expression.
Simpler organisms, including some bacteria, can generate distinct cell types that contribute to
developmental events
What differentiates them is the identity and amount of gene expression
How do genetically identical cells establish different programs of gene expression?
Stem Cell: cell that is able to differentiate and make different kinds of cells
Adult stem cells are limited to how many different cells they can differentiate into. Compared to
embryonic stem cells, from which any of the 200 cell types can be made.

Strategies for controlling developmental gene expression - Each of these events establishes
asymmetry in gene expression which, in turn, leads to morphological asymmetry

mRNA Localization mRNA is localized to one part of the cell. When the cell divides, most of this mRNA
will go with only one of the daughter cells. Use actin/myosin filaments to localize. An adapter protein binds
UTR and is bound by filament to be localized. Ex. Yeast Mating Type Switching

Cell-cell Contact - Cell-cell contact and secreted signaling molecules often act by promoting changes in the
activity and/or localization of transcription factor. Signal tells cell WHAT PHENOTYPE TO BECOME

Secretion of a Signaling Molecule - Since many transcription factors exhibit cooperative binding to their DNA
sites, a very small change in the active concentration of such transcription factors can lead to a situation in
which a target gene goes from being not expressed to being fully expressed. CLOSER TO SOURCE,
CONCENTRATION FO CHEMICALS/SIGNALS IS HIGHER, MORE RECEPTORS ARE ACTIVATED
AND A STRONG SIGNAL IS TRASNMITTED = TRANSCRIPTION OF A LOT OF GENES WHICH
DETERMINE THE FATE OF THE CELL.
Ex. Sonic Hedgehog

HO Gene: Changes mother cell from a cell to alpha
END PCB2.1

Ch19 - Comparative Genomics and Evolution of animal Diversity
- Most Animals have essentially the same genes.
- Three ways gene expression has changed during evolution.
- Genome evolution and human origins.
**Flattish round worm believed to be the common ancestor**
How does GENE DUPLICATION give rise to biological diversity? (Forms of evolution?)
How does increasing the number of copies of certain genes lead to increased morphological diversity?
1. Genes are created that encode related proteins with slightly different activities.
-If mutations occur in coding regions, the function of the new gene will be slightly different
2. Duplicated genes acquire new regulatory DNA sequences = different pattern of expression
within the developing organism.
**Gene duplication events offer the opportunity to expand the repertoire of protein
Both forms of evolution are seen in the globin genes in mammals; e and g are used by the fetus
which lacks functional lungs.
Three ways gene expression is changed during evolution:
1. A given pattern determining gene can itself be expressed in a new pattern. (Changes in
expression pattern)
- This in turn will cause those genes whose expression it controls (target genes) to acquire new
patterns of expression.


2. The regulatory protein encoded by a pattern determining gene can acquire new functions
(Changes in function of encoded regulatory protein) e.g. an activator domain can be converted into a
repression domain. [b]
3. Target genes of a given pattern determining gene can now acquire new regulatory DNA
sequences and thus come under the control of a different regulatory gene (Changes in the enhancers that
are recognized and regulated by pattern determining proteins.) Thus their pattern of expression is
altered. [c] Can have mutations occur in site of activator binding, leading to inability of activator to
bind to DNA, turning the gene OFF

Experimental manipulations that alter animal morphology.
Fruit flies expressing the squid Pax6 gene; eyes obtained in tissues where pax6 is expressed. 30%
overall similarity between Drosophila and squid Pax6.

Genome Evolution and Human Origins
Humans contain surprisingly few genes.
25,000-30,000 protein coding genes
Organismal complexity is not correlated with gene number, but instead depends on THE NUMBER
OF GENE EXPRESSION PATTERNS.
The human genome is very similar to that of the mouse and virtually identical to the chimp; There
are few if any new genes in the humans that are completely absent in mice.

**FOXP2 fosters speech in humans
END PCB2.2