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The dogma
o Stimulus leads to biological change (aka protein synthesis)
How we respond to the environment
DNA structure
o 5’ end has the phosphate while 3’ end doesn’t have it
o DNA strand is made up of many bases
ATCG
Know where the protein is interacting on the strand of DNA
Transcription
o Process that requires sequence specific regulators = transcription factor (interacting with
DNA sequences)
Specific DNA sequences are called motifs
Interact with DNA sequences in response to cellular signals (APs that initiate
the transcription)
o Diagram
Chromatin = unwinding the chromosome to expose strands of DNA for transcription
The unwinding process is needed to read the DNA strand sequences
RNA polymerase II
What is needed for initiation of transcription
o Can be blocked, or be more efficient by various interactions
Around RNA polymerase II, we need to build a transcription complex assembly
To initiate transcription
Made up of many different transcription factors (TF)
o Molecules that regulate gene transcription
TFII
Specific group related to polymerase II (for functioning properly)
Main job
o Proteins that help position RNA polymerase II at the correct
promoter region
Find polymerase, then put it into the right region and make
sure the unwinding of the DNA is done
Activator molecules (co-activators) also work alongside transcription factor to
facilitate polymerase II
o Second diagram (initiation)
(1) Starts with assembly of TFIID
Recognizes an area of DNA strand that has TATA (called TATA box =area of
strand rich in Ts and As)
o Specifically, the TATA binding protein (portion of TFIID) recognizes
the TATA box
(2) Causes the distortion of the DNA strand; signals all the other TF to come together
(and bind)
Attracts TFIIA and TFIIB as well as the RNA polymerase (etc.)
o Each have unique functions
TFIIH has the helicase to unwind the DNA
(3) Together make the transcription initiation complex
(4) Once ready to transcript, all of the TF are not required and disassembly
o Disassembly signifies transcription
3’ to 5’
Table
o Binding in a specific order
Transcription
o Step 2: Elongation
o Step 3: Termination
Capping
o A cap that stops anything from happening to it
A m7G cap
Splicing
o While introns are there, the mRNA is called pre-mRNA
o Diagram
During splicing, there is removing of introns
Now is a mature mRNA strand (what’s actually translated)
o Step 1
U1 snRNP helps cleaves the 5’ end of the intron
o Step 2
U2, U4 and U6 snRNP
Brings 5’ close to the branch point
o There is a bend in the branch point as a result
U5 brings the 3’ end close
3’ end is cut and joined to 5’ end
Left with a loop
Called an RNA lariat
o What is removed from the mRNA
Exons are now joined together
Splicing diagram
o Lariat is removed later on
Splicing video
o All processes happen all the same time (in multiple places)
Not like one process happens in one time and in one position (at one period of times)
Spliceosomes at different areas and RNA pol II transcribing
Transcription factors
o Binds to parts of DNA called specific consensus sequences (DNA binding motifs)
Important for supporting specific behavioural processes
o Distinct DNA sequences areas offer TFs patterns of hydrogen bonds (donor)
TF can accept the hydrogen bonds and that’s how they are linked to the DNA strand
o TF bind to DNA binding motifs via structural motifs
Actual part of the TF that binds DNA
CREB (most important TF)
o CREB recognizes the CRE DNA sequence
o Diagram
Linked to all forms of behaviour
CREB is involved somehow to basically all forms of behaviour
o Diagram (structure)
CREB has 3 main structural features
(1) bZIP domain (at C terminus) facilitates dimerization (joining of 2 things)
between different family members
o Called the leucine zipper = bZIP side of the CREB molecule
o bZIP is the part of CREB that recognizes the CRE sites
(2) KID
o A site for phosphorylation by PKA and others
(3) Q2 domain
o Recruits the transcriptional machinery to the promoter
o Diagram
A
All pathways that lead to CREB activation
o Commonly activated
B
Leucine zipper or zipper region
D
Placed on a strand
CREB
o Diagram (bottom)
Idea that when CREB is activated, there is 2 of them
They are phosphorylated which allows the attraction of CBP molecule
o That’s how RNA pol II is brought in (attracted RNA pol II so it
facilitates transcription)
o Diagram
Actually, the HAT within the CBP molecule attracts the RNA pol II
The domain that enables chromatin remodeling to allow RNA pol II to come
in
Co-factors
o Other molecules that interact with TF
Example
HAT interacts with CREB process
o All co-factors
Serve to enhance capabilities of the TF and its efficiency
Example
o HAT
Enhances unwinding of the chromatin so transcriptional
process takes place
Does it by weakening the DNA histone binding
o Without HAT, not as effective at
transcription
o Compressors (opposite of co-factors)
Example
HDAC (enzyme)
o Makes the transcription less efficient
By increasing DNA histone binding
Week 3:
Chromatin
o Chromatin regulation is outside of the genetic code
Controls genetic expression outside of being genetic code
o Euchromatic
Organized in a structure that resembles beads on a string
o Nucleosome
Each structured with a core particle that contains 147 bp
Histone octamer
o Contains 2 copies of histones
H2A. H2B, H3 and H4
Nucleosomes are linked to H1 rod histone
o Heterochromatin
Repressed and compaction of chromatin into 30 nanometer fiber
o Each core histone contains N-terminal tail that extends from the DNA histone core and can be
covalently modified
The modifications include; methylation, phosphorylation, acetylation and more
All modifications are reversible
Histone modifications
o HATs
Add acetyl groups to specific lysins
o HDAC
Remove acetyl groups
Methylation
o Diagram
Cytosine ring that DMNT (DNA methyltransferases) add the methyl groups
Covalent modification of DNA regulates memory formation
o Teaching fear (fear conditioning) to the mouse; specifically, auditory fear conditioning
Accompany the tone with the shock
Creating a fear response; freezing behaviour
To show that they have learned the behaviour, we place the animal in the box and
play the same auditory sound but have no shock accompany it
If it has learned, they will fear
o Dependent variable; freezing behaviour
Percent of time frozen
o The addition of DMNT and then doing another testing, was to see if without DMNT inhibitor,
it changed the memory
VEH = saline
o Saw that fear conditioning increased DMNT mRNA 30 minutes after training
Thus, if we inhibited DMNT, we would not get the increase in mRNA
o Timing is important
In order for long term memory to form, transcription must occur immediately after
learning a stimuli
Acetylation
o Histone acetylation is regulated by opposing actions of 2 different classes of enzymes
HATs and HDACs
Balancing act that regulates transcription
CBP Histone acetyltransferase activity is a critical component of memory consolidation
o Novel objection recognition
Rodents are more likely to explore novel objects rather than familiar objects
Must acclimatize to the environment (habituation phase)
o If the animal remembers what it explored previously, they will know which is familiar and
novel object
Spend more time with novel object
o Graphs
Found that mice with HAT domain of CBP eliminated, show deficit in object
recognition and spatial memory
Go back to the familiar object (don’t prefer the novel object)
But if their HAT activity is recovered (given treatment), then their memory deficits
are reversed back
o Ultimately, messing transcription means reducing translation
Translation
o tRNA which also has 3 nucleotides
Match the AA to the mRNA codons
The matching set of codon from the tRNA strand is the anticodon
o On the tRNA molecule, there are enzymes called aminoacyl-tRNA synthetases
The TRNA synthetases will attach the AA to the actual tRNA molecule thru a
covalent bond
Initiation
o Initiation step is the rate limiting step
Involves 12 protein factors and commonly know as eukaryotic initiation factors (eIF)
o Binding of tRNA to the AUG codon initiates translation
(1) B4 that happens, there needs to be part of preinitiation complex
The initiator tRNA binds to a small ribosomal unit with some of the eIFs
o Together forms the preinitiation complex
The GTP attached to the eIF2 will find the initiator codon (AUG)
(2) Next, the whole subunit binds to the 5’ cap mRNA and move forward to find the
AUG
(3) The binding of large ribosomal subunit occurs and helps to grow the polypeptide
chain
o Why is initiation the rate limiting step?
eIF2 phosphorylation by several kinases
Leads to a decrease in general translation
o Biggest consequence of eIF2 phosphorylation is down regulation of
global translation and stimulation of translation of some mRNA
Increases gene specific translation
o Specifically of mRNAs that contain the uORF
Example; CREB repressor
Elongation
o Each ribosome has 3 binding sites for the tRNA
o Diagram
Step 1
tRNA binding to the A site
Step 2
Followed by forming the peptide bond in the P site
o Polypeptide chain is forming
Once the peptide is added it moves onto the next tRNA molecule
Step 3
There is translocation of the large subunit (moves over) so that tRNA that has
pasted on its AA is in the E site
o The moving over of large subunits signals the bottom small subunit
to also move over
Step 4
The moving over ejects the tRNA molecule in the E site
o That gives the space to start all over again
Termination
o Stop codons signals the end of translation
o Diagram
Instead of a new tRNA molecule coming in, instead there is binding of a release
factor to the A site
When that elongation step tries to continue, the protein is released
o This occurs via a chemical process whereby a water molecule is
added to the end of the peptidyl tRNA
The water molecule frees the C terminal end of the
polypeptide chain from the tRNA
This causes a whole dissociation of the entire complex
Large ribosomal and small subunits will detach as well as the release factor
IEGs
o In response to some environmental stimulus, these genes are the first to be transcribed
o Presence of IEGs = activity marker
C-Fos
o C-Fos works alongside Jun
Its transcription is stimulated by activation of CREB
o C-Fos the gene and Fos is the protein is produced by the translation of C-Fose gene
o Steps
(1) The c-Fos promoter recruits SRF
(2) SRF recognizes the SRE consensus sequence at the c-Fos promoter
(3) This recruits CBP
Removes the HDAC molecule
(4) Initiates transcription at C-Fos site
Week 4:
What is a NT?
o The specific amounts will determine the effect it will exert onto the postsynaptic neuron or
some specific target
o The NT never sits in the synaptic cleft forever
Types of NT
o Neuroactive peptides
Bigger sized NT = packed in larger dense-core vesicles
Biogenic amines (produced and synthesized by the organism itself without any outside help) vs amino
acids (formed from carbohydrate substrates)
o NT can be classified as these 2 types
Catecholamines (most important function is motor control)
o Step 1
Tyrosine hydroxylase converts Tyrosine into L-DOPA
o Step 2
L DOPA is decarboxylated by L-DOPA decarboxylase into dopamine
o Step 3
DOPA beta-hydroxylase converts dopamine into norepinephrine
The presence or absence of DOPA beta-hydroxylase determines whether a
neuron becomes dopaminergic or norepinephrine
o Determining enzyme
Without DOPA beta-hydroxylase = dopamine neuron
o Step 4
Phenyltethanolamine-N-Methyltransferase helps to make the norepinephrine into
epinephrine
Regulation of TH
o There are different levels of catecholamines necessary for different type of behavioural
stimulus
o Stress causes rapid firing
Rapid firing could cause a depletion of NT pool
o What regulates the catecholamines?
Tyrosine and Tyrosine hydroxylase (TH) regulate its amount produced
At baseline, the TH is completely saturated by tyrosine molecules
o If we were to increase the level of tyrosine, it will not change the rate
of TH activity
Therefore, if we wanted to increase dopamine synthesis, and
give more tyrosine, it will not increase the amount of
dopamine produced
Because TH is completely saturated
o Rate limiting step (bottleneck)
o TH is controlled in 3 ways (can actually change how much dopamine or epinephrine is
actually produced)
(1) Phosphorylation of TH
(2) End-product inhibition of TH
Catecholamines can inhibit the activity of TH via competition of co-factors
that is needed
(3) De novo TH synthesis (increased demand for catecholamine synthesis)
This can be met by producing more TH molecules
(1) De novo TH synthesis
o Diagram
CREB TF can bind to the TH promoter to increase TH expression
Activation causes a cascade of reactions that leads to phosphorylation of
CREB, leading to increased gene expression of TH
(2) End-product inhibition
o BH4 is required for high TH activity (facilitates TH activity)
o Diagram
When catecholamine binds to the flexible loop of TH, it causes a conformational
change
Makes it less available for BH4 cofactor binding
o Competitive inhibitor
(3) Phosphorylation
o Different phosphorylation sites cause different effects
Diagram
Generally, phosphorylation of the R domain of Th moves it from a position
of obstructing access to the active site to allowing the access site to be more
accessible
o However, its more complicated
Depolarization (stimuli) causes increased in activity of PKC, PKA and CaM kinases
that affect TH in different types of phosphorylation
Wherever within the flexible loop, the phosphorylation occurs, there is a
different effect
o Phosphorylation onto Ser40 by PKA
o Phosphorylation onto Ser31
o Phosphorylation onto Ser19 by CamKII
Serotonin
o Here, the availability of tryptophan is the rate limiting step for serotonin (main difference
between catecholamines and serotonin)
Other than that, the basic outline mechanism is similar to the catecholamines
GABA
o Its not biogenic amine NT
There needs to be a glucose metabolism
Glucose is the #1 precursor
o GABA shunt (Diagram)
Again, glucose is the precursor to GABA
There are other precursors
Step 1
Transmission of GABA T
o Changes the glucose (alpha-ketoglutarate) into glutamate
Step 2
GAD catalyzes the decarboxylation of glutamate to get GABA
o The steps can be backwards as well
GABA can be metabolized by GABA T to form succinic semialdehyde
Recycling loop
o Since GAD is the final step
Presence of GAD can indicate the presence of GABA
Only appears in GABA neurons (specificity)
Glutamate
o Glutamate can also be synthesized via glial cells
Basically, 2 ways
Conversion via GABA to glutamate and glial cells that convert glutamine
into glutamate
ACH
o Involves only 1 enzymatic action
Catalyzed by choline acetyltransferase (ChAT)
ChAT determines whether there is ACh or not
o Rate limiting step
o ChAT combines acetyl CoA and choline into acetylcholine
Acetyl CoA comes from our diet (glucose metabolism)
Acetyl CoA comes from the mitochondria
o Thus, it must be transported into the cytoplasm to be used
Rate limiting step as well
Need enough availability
Vesicular transporters
o Table
VMAT is for catecholamines
VaChT is for acetylcholine
VGAT is for GABA
VGLUT is for Glutamate
VMAT
o Most transporters are stochiometric antiporters
Involves charged particles and the electrical gradient
o For every 2 H+ ions going out of the vesicle, it leads to transportation of 1 NT into the vesicle
This is maintained via the hydrogen ATPase
Allows via energy consumption, lots of hydrogen to stay inside the vesicle to
encourage moving out
o Blocking or inducing the ATPase (increasing or decreasing)
Effect = how much VMAT is able to bring in NT into the
vesicle
Can be used to change the amount of NT moving into a vesicles
2 methods of inactivation
o Diffusion
Can drift away
Can also be diluted by extracellular fluid whereby the concentration is non detectable
or is not useful in that condition
o Uptake
Involves transporter molecules
2 functions
o Either reuptake NT into storage vesicles
Recycling method
o Or transporter causes enzymatic inactivation
Breakdown of molecule into inactive components
Reuptakes
o All based on the SLC transporter molecule
Reuptake
o There is different transporter for specific and different NT that they are dealing with
For monoamines, there’s SERT (serotonin), NET (norepinephrine) and DAT
(dopamine)
For GABA/glycine, there’s GATs and GLYTs
Location varies
Reuptake - DAT
o Energy dependent
Therefore, depends on the sodium co-transport and requires extracellular Cl-
Diagram
o Sodium and dopamine go in at the same time (have to at the same
time)
Causes a conformational change that will lead to dopamine
being transported inside
o General structure
During splicing, there is different versions of N-terminals and C-terminals being
made
Depending on how its spliced, it leads into different transporters for different
molecules
o In splicing is basically when we differentiate the different
transporters from each other
DAT: regulation
o Reuptake is also closely regulated by all mechanisms we have discussed b4
ERK pathways
If activated by secondary messengers, it causes upregulation of DAT surface
levels
o Increasing transporter capacity = more reuptake
PKC
Causes reduced transport meaning PAC will not internalize dopamine back
o There is less reuptake, causing enhanced internalization (not open for
reuptake essentially)
Enzymatic inactivation
o MAO
o COMT
Diagram
o Use legend to look for Abcde (enzymes)
Dopamine
Dopamine can also be broken down by COMT and then subsequent enzymes
into homovanillic acid
o However, another way is that is can be first converted into
norepinephrine and then broken down by MAO
Subsequent enzymes allow into homovanillic acid
o Another way is being converted then into epinephrine and then by
MAO
To get vanillylmandelic acid
o Memorize the end products and starting products as well where enzymes MAO and COMT
are
No need for intermediaries
ACh
o AChE (breaks down ACh)
It promotes ACh hydrolysis by forming Acetyl-AChE intermediary which releases
the choline
Thru hydrolysis; get back to initial products = Choline and acetyl-CoA
o Diagram
ACh is first broken down and then it is recycled back into the cell
ACh inactivation takes place extracellular
Other enzymes can be first reuptake into the cell and then inactivated but
ACh is inactivated outside the terminal
Week 6: