Slides 2 Flashcards
Localization and function of proteins is regulated by (3)
- transcription
- translation
- post-translational modification
Transcription =
DNA information is copied into mRNA
Translation =
mRNA is used as a template to synthesize proteins
RNA splicing and editing in the nucleus change:
> Made of what?
Their sequence and the relative amount of resulting proteins
>exons
What are Non-coding RNA (ncRNA) that exert various regulator functions called?
Introns
What is pre-mRNA?
the mRNA that includes introns and exons
What is chromatin made of?
tightly packed DNA wound around proteins (histones) known as nucleosomes
Rosalind Franklin ans Raymond Gosling used X-ray diffraction to discover what?
That DNA exists in 2 forms:
- A the dry A form that holds less water
- The wet B form where water molecules cling to DNA, causing it to stretch out
How many layers are in every twist of DNA?
10
What bonds bind the 2 strands of DNA?
Hydrogen bonds
What does it mean to say that DNA is anti-parallel?
5’ end of one strand is paired with the 3’ end of its complimentary strand
What is the most important energy source in the brain?
Oxygen –> brain used 50% of oxygen that goes through it
> only 10% of glucose
the highest energy yield from glucose comes from what?
NADH from glycolysis in mitochondria
Pathways linked to the tricarboxylic acid (TCA) pathway and oxidative metabolism are essential for: (6)
- NT turnover
- synthesis of excitatory and inhibitory NT
- mRNA, protein, lipids, and organelle turn over
- Axonal transport
- Ion pumping to maintain and restore ionic gradients
- Synthesis of amino acids
The brain is not able to store any oxygen, what does that mean for the supply?
It needs to be continuous to meet the energy needs
Do oxygen and glucose require energy to pass through the BBB?
Oxygen passes right through, no energy needed
Glucose passes through channels (indirectly need energy to make channels)
What are the functions of the glucose channels Glut 1, Glut 3, and Glut 5?
Glut 1 = astrocyte taking glucose from blood
Glut 3 = astrocyte transferring glucose into neuron
Glut 5 = microglia
How much glucose is transported into the brain?
~3 times more than can be used
> good because none can be stored
What is the energy yield from glucose –> pyruvate?
2 ATP
2 NADH
Where is most of the energy of a cell produced?
In the mitochondria by oxidative phosphorylation
What are different energy substrates?
- Carbohydrates (glucose after being converted into pyruvate)
- Lactate
- Ketone bodies
- Fatty acids
What are the waste products of oxidative phosphorylation?
Carbon dioxide and water
ATP is synthesized via what enzyme?
ATP synthase
Methylation of DNA
Silencing
Closed chromatin
A form
Acetylation
Expressing
Open chromatin
B form
Promotor selection is determined by what?
The interaction of one or more transcriptional activators(s) with recognition sites (on DNA) near target genes
Activators recruit what?
Components of the transcription machinery
Activation of gene expression is induced by what?
A certain sequence of large proteins co-activator complexes
Activators also recruit APT-dependent nucleosome remodelling complexes, which do what?
Move or displace histones at the promoter
What epigenetic changes happen to the BDNF gene during stress?
Methylation –> reducing expression
How do anti-depressants work on the BDNF gene?
Reverses the suppression of BDNF through increased histone acetylation
What are the functions of chromatin?(3)
- package DNA into chromosomes
- control replication *
- control expression **
Def of epigenetics:
Chromatin regulation outside the genetic code that can control gene expression
Unwound protein-chromosome complexes are called:
Chromatin
What is the basic unit of chromatin organization
147 base pairs
wrapped around a core of histone proteins
Packaging of chromatin affects:
gene expression
What is the difference between euchromatin and heterochromatin?
Euchromatin = transcription can occur because it is open Heterochromatin = transcription cannot occur because it is closed
What are HAT and HMT and what do they do?
Histone (Acetyl or methyl) Transferases
> They moderate the amount of methylation or acetylation on chromatin
What do SWI/SNF do?
Opens up DNA to allow things to bind (transcription machinery, transcription factors and co-activators)
HDAC do what?
De-acetylate histones (make DNA more tightly packed)
What are 2 main benefits to the regulation of gene expression through epigenetics?
- It conserves energy - only have the proteins made when needed
- Having chromatin tightly packed saves space
What are the functions of methylation and acetylation
Methylation : makes brain regions stable
Acetylation : makes brain plastic
histones are made up of how many proteins?
8
First level of organization or packaging of DNA is:
Winding DNA strands around histones
Second level of organization of DNA:
Histones package together to form nucleosome complexes
Third level of organization of DNA:
Supercoiling (negative and positive charges attraction)
How many histones make up a nucleosome
4
Histone modification occurs at which amino acids?
Lysine amino acid
It is the level of acetylation of methylation that determines how tightly packed DNA is
Acetylation
Hyperacetylation
Chromatin is relaxed and accessible to the transcriptional proteins
> increased gene transcription
Hypoacetylation
Chromatin is condensed preventing access of transcriptional proteins
> gene silencing
What could be a function of inhibiting HDAC?
Inhibit the enzyme that breaks down acetylation
|»_space; Increase acetylation
What does DNMT do?
Add methyl groups to DNA
What are MBD
Methyl binding domains - have repressor proteins (HDACs)
How does methylation directly silence a gene?
Doesn’t allow general or specific transcription factors to bind to gene
What are the transcriptional machinery?
RNA polymerases
How can hypomethylation be problematic (cancer)
Methylation exists to control cell division (reduce transcription), so a lack of methylation would result in unregulated growth (cancer)
How do drugs affect epigenetics?
They lead to the activation or inhibition of transcription factors and chromatin regulating proteins
How does alcohol withdrawal reduce BDNF levels and decreased dendritic density (anxiety behaviours)
HDAC activity increases and DNA becomes more tightly packed
Homodimer
protein complex with one subunit
Heterodimer
Protein with more than one subunit
What is the final step in assembling the Initiation Complex
The addition of a phosphate group to the CTD on RNA Polymerase II
What is Polymerase II able to do once it is phosphorylated?
Leave the PIC and begin synthesizing RNA
How is TFIIH like DNA helicase?
Can separate strands of DNA
What important thing happens after TFIIH separates the strands of DNA?
RNA Polymerase II can bind to the DNA and start RNA synthesis
What is elongation regulated by?
Developmental or cell signalling stimuli
What is the 5’ capping?
7-methylguanosine cap is added to the 5’ end of mRNA during elongation
What is the Poly (A) tail?
A series of As that are added to the 3’ end of the mRNA one elongation is complete
What is the purpose of the 5’ cap and poly (A) tail?
To protect the mRNA from degradation once it exits the nucleus, helps export DNA, binding to ribosome
Exons def
protein coding-sequences
introns def
intervening sequences that are removed from the pre-mRNA
|»_space; Do not encode functional proteins
Why is it good to remove introns?
Because the extra non-coding mRNA would slow down transcription
If introns don’t code, why do that exist?
To increase the repertoire of the proteins that can be made from a single gene
How is pre-mRNA converted to mature mRNA?
By removing the introns from the pre-mRNA and ligation of exons by the spliceosomes
What are the splicosomes composed of?
5 subunits called snRNP (small nuclear ridonucleoparticles)
How do spiceosomes know how to identify introns?
snRNAs recognize motifs in DNA sequence that define the intron
How to splicosomes splice?
They cleave the sugar phosphate backbone at the G at the start of the exon and then “glues” it to the A at the start of the other exon
Are miRNA more often negative feedback-ing for excitation or inhibition
Inhibition
Is CREB and general or specific transcriptional factors?
Specific
What are characteristics of G-Protein linked signals?
- slow neural response (ms to hours)
- Modulates many things
- signal is amplified
- Allows for plasticity
How does receptor binding enhance or inhibit expression of target gene?
- Direct interaction with ligand-activated GR with glucocorticoid response elements
- Interaction of the activated receptor with other transcription factors
How does chronic stress inhibit neurogenesis
CORT- activated glucocorticoid receptors may inhibit CREB activity
Transcription is the process that ensures the neuron maintains ____ and adequately responds to _____ _____
Homeostasis
Environmental Signals
Gene expression is regulated by the presence of:
- Cis-regulatory sequences (where specific transcription factors bind)
- Protein complexes (transcription regulators)
What are general transcription factors?
Proteins that help position Pol II on the promotor
> pull apart DNA and move Pol II into elongation mode
Specific transcription factors
COntrol the expression of specific genes
General vs Specific transcription factors
General = Are basal and used for every gene Specific = express only certain genes
How is CREB activation initiated?
By translocation of calcium sensor or by activating kinases
Calcium sensors supports CREB phosphorylation through the activation of what?
CaMKIV
What is the 5 step process of gene transcription regulated by second messengers?
- Ligand binds to G-protein
- Activates adenylyl cyclase which increases levels of cAMP
- Increase in cAMP activated PKA in cytosol
- Released subunits travel into nucleus
- Subunits phosphorylate CREB
- pCREB recruits co-activator CBP which stimulates gene transcription
How is CREB phosphorylated?
- CREB binds to cAMP response element (CRE)
2. Protein kinase A and CaMK2 stimulate phosphorylation
CREM is part of the _____-zipper which means they do what
They can unwind DNA
Leucine-zipper proteins bind to what DNA sequences
cAMP response element (CRE)
What is the key enzyme that mediates transcription?
RNA Polymerase II
What do polymerase 1 and 3 do?
1 = transfer RNA 3 = Ribosomal RNA
RNA Polymerase II catalyzes transcription for what? (2)
- All protein coding genes
2. snRNAs
What bond types does RNA polymerase II form?
Phosphodiester bonds
What binds to the TATA box?
General transcription factors (TFIID) for Polymerase II
Where is the TATA box located?
25 base pairs from the transcription start site
Which part of the TFIID protein binds to the TATA box?
the TATA-Binding Protein (TBP)
What does the TATA-Binding Protein do
It bends the DNA to alloe the transcription factors and RNA Polymerase to closer
After TFIID have attached to the core promoter, how is the Pre-Initiation Complex formed?
By the attachment of the remaining general transcription factors
What is the order the transcription factors bind in?
- TFIIA
- TFIIB
- TFIIF/RNA polymerase II
- TFIIE
- THIIH
Summary of pre-initiation complex
- TBP attaches and bends DNA as TATA box
- Bend gets TFIIB to put RNA Polymerase II at transcription start site
- TFIIH opens promoter complex
Alternative Splicing
Increases the number of mRNA products any one gene can produce by skipping exons o adding introns
Introns play a role in a wide range of gene expression regulatory functions such as (2)
- Nonsense mediated decay
2. mRNA export
What are the 3 types of non-coding RNA?
Introns
tRNA
Ribosomes (rRNA)
How can miRNA be used to treat obesity?
There are miRNA that inhibit the production of fat cells
Gene expression is regulated by (4)
- Control of transcription rate
- Control of splicing and processing of mRNA
- Control of transport and localization of mRNA
- Control rate of degradation of transcripts
What does it mean to say that transcription factors are constitutively present?
They are present all the time, under basal conditions
What does it mean to say that proteins are inducible?
They need to be transcribed, not present under basal conditions
CREB activates which inducible transcription factor? Which is made up of what sub-units?
AP-1
Fos and Jun
How long does AP-1 stay around after induction?
It is transcribed rapidly but then it goes away
AP-1 is an ________ early gene
Immediate early gene
AP-1 is a heterodimer made of what sub units?
C-Fos and C-Jun
AP-1 is a _______ zipper
Leucine
Are Fos and Jun present under basal conditions?
No, they must be transcribed
What happens if C-Fos goes back into the nucleus by itself?
It is a negative feedback loop that will repress inducible transcription
Intracellular signal transduction
A chain of reactions stimulated by a signal from the cell surface
Which AP-1 dimer has leucine zipper properties?
C-Fos
Long term drug use results on the increased expression of what transcription factor?
FosB/DeltaFosB
Is Delta FosB transient or stable?
Stable, it is always present
How is Delta FosB different from C-Fos?
It regulates the expression of different genes
DeltaFosB increases transcription of ARC, which results in what?
Increases dendritic spine growth and makes lots of connections
>possible mechanism of addiction
DeltaFosB sticks around even after you stop taking drugs, what repercussions does that have?
The drug seeking circuitry is still there so it will continue the drug seeking behaviours
What are the 6 main functions of proteins?
- Metabolism
- Cell division
- Immune system defense
- Gene expression and regulation
- Physical structure
- DNA replication, transcription and translation
The unique sequence of amino acids of a protein allow for what?
A unique 3D folded structure
Alpha helix
A coil
Beta pleat
A sheet
What part of the amino acid determines the physical and chemical properties?
R-side chain (functional group)
For a protein, shape determines _____
Function
Polar amino acids have hydro____ R groups
hydrophilic
Non-polar amino acids have hydro____ R groups
hydrophobic
If proteins free float around in the cytosol, the amino acid side chains on the outside will be hydro____
hydrophilic
2 characteristics of water soluble proteins
- non-polar side chains on inside of molecule
- polar side chains on outside of molecule
2 characteristics of membrane bound proteins
- non-polar side chains are on the surface to come into contact with membrane
- polar amino acids will line the interior of the pores
What type of amino acid lines the inside of protein channels?
Hydrophilic amino acids (because the hydrophobic ones are on the outside interacting with the membrane.
The active sites of enzymes depend on the location and distribution of the polar and non-polar amino acids because:
Hydrophobic and hydrophilic interaction can play a role in substrate binding to the active site
how many different amino acids are there?
20
What can happen if there is a mutation that changes the charge of an amino acid?
the protein becomes less stable and it wobbles around.
> can bind with other unstable proteins
What makes the Huntington’s gene diseased?
The repeat of the sequence CAG too many times (more than 37) causing it to misfold
Fibrous proteins
Elongated proteins that are usually structural, sometimes functional
Globular proteins
Compact and tightly coiled
Native conformation of a protein
The tertiary structure that the protein naturally folds into
Do proteins need help folding?
Most do not
Primary structure
Sequence of amino acids
Secondary structure
folding as a result of hydrogen bonding
|»_space; alpha helix and beta pleated sheets
Tertiary structure
Combination of the secondary folding into a 3D shape
Quaternary structure `
The interaction between multiple protein subunits to make a large structure
Are proteins fixed to one structure?
No, they can go back and forth
> usually between 3 and 4
Some proteins require what type of assistance to fold properly?
Post translational modifications or molecular chaperone help
What is the main post translational modification
Phosphorylation
What happens if misfolded proteins start to accumulate?
Leads to the formation of protofibrils, fibrils, and inclusion bodies
Which is the most common secondary structure?
Alpha helices
Why are alpha helices more common than beta pleated sheets?
They have a thermodynamically stable conformation (less wobble, lower energy)
Is high or low energy of a protein ideal at rest?
Low energy
Which composition makes prions infectious?
When they are composed of mostly beta pleated sheets
How do prions spread?
They can make natively formed proteins fold to have more beta pleated sheet (abnormal) conformation
Most proteins are fibrous or globular?
Globular
What do globular proteins typically look like?
Hydrophobic core and hydrophilic outer layer
what type of bond binds the alpha helices and beta pleated sheets?
Disulfide bonds
Attraction between negatively charged _____ groups and the positively changed _____ group stabilize the tertiary structure
Carboxyl group
Amino group
What minor force can exist between non-polar side chains in proteins
Van der Wall forces
What bonds stabilize quaternary structures?
Hydrogen bonding
Disulfide bridges
Salt bridges
What are the 3 subunits of the g-protein called?
Alpha
Beta
Gamma
what happens when a ligand binds to a g-protein?
it changes shape, causing the alpha subunit to break away
What does the alpha subunit do once it breaks off?
Interacts with adenelal cyclase (AC) and converts ATP into cAMP, which activates PKA to phosphorylate CREB
What does phosphorylation do? (4)
1) Regulate cell cycle
2) Growth
3) Apoptosis
4) signal transduction pathway
What does glycosynation do (5)
Effects on protein: Folding Conformation Distribution Stability Activity
What does ubiquitination do?
Catalyzes the degradation of the ubiquinated protein
Why do proteins become uniquinated?
Because it is mis-folded
Phosphorylation activates a protein when it is on _____ and inactivates it when it is on _____
Top
Bottom
Is phosphorylation irreversible?
No
> By phosphytase
What energy state is the protein at when phosphorylated?
Higher energy state
Unfolded or misfolded are in a _____ energy state than the native conformation
Higher
What can happen when there is an unfolded protein (3)
- Protein can refold
- Attract chaperones for disposal
- They can bind to other non folding proteins making aggregates
Aggregations are at an even lower free energy state than the native form, what does this result in?
It makes it irreversible,
protein is no longer functional
Oxygen goes directly where when it enters the cell?
Mitochondria
Glycolysis is what process and it happens where?
When pyruvate is obtained from glucose
>In the cytoplasm
Where is pyruvate made and then where does it go after it is created?
Cytoplasm
Mitochondria
What elements are involved in oxidative phosphorylation?
Electron transport chain
Kreb’s cycle is also known as:
TCA
How much ATP does the Kreb cycle generate?
2 APT
How much ATP is generated from the electron transport chain?
34
How do ions enter the brain>
Ion channels
What passes via membrane transport into the brain?
small lipophilic molecules, like oxygen
Who are energy transport molecules like glucose transported into the brain?
Carrier mediated transport (solute carriers)
Direct vs indirect pathway of a neuron getting energy
Direct: glucose goes into the neuron and ATP is generated there
Indirect: glucose goes into astrocyte and generates ATP and gives it to neuron
Where does dimerization of AP-1 take place?
In the nucleus
For this class, methylation happens on ____ and acetylation happens on ______
Methylation = DNA Acetylation = Hystones
HAT is recruited by what?
Sequence specific transcription factor (TF1)
Acetylation is essential for _________-dependent nucleosome remodelling
SWI/SNF
Drug-induced changes at the chromatin level are extremely _____ and define _____
Stable
Addiction
What are 2 chromatin remodeler complexes?
SWI/SNF
What percentage of the genome code for transcription regulating proteins?
5-10%
What proteins can mediate cAMP mediated transcription?
CREB
CREM
ATF-1
What are the 3 types of post-transcriptional modifications?
- Poly A tail
- 5’ cap
- Splicing
What are the 3 names for the different forms of mRNA?
Transcript Variants
Splice variants
Isoforms
The largest group of amino acids have what type of side chain?
Non-polar
