Midterm 1 Flashcards
1
Q
Light microscopy
A
Used to see live cells, colour, and whole tissues
Can target what you want to see
Whole tissues can be shown
Resolution limit = 0.2 nanometres (usually)
2
Q
Transmitted Light Microscopy (TLM)
Bright Field Light Microscopy
A
Light passes through specimen and viewed
Optical techniques to increase the contrast of unstained living cells
Tissues must be cut into thin sections to see cells in them
3
Q
types of Transmitted Light Microscopy (TLM)
A
Bright field
Dark field
Phase contrast: microscope shifts light and produces more contrast for more detail
Differential interference- contrast (DIC)
4
Q
Emitted (Fluorescence) Light Microscopy (FLM)
A
Fluorescent molecules absorb light at specific wavelength (coloured), and emit light at a different wavelength which is viewed
Purpose is to visualize certain molecules or structures in cells
Molecules or structures are fluorescently labeled
Epifluorescence microscopes are used to illuminate the whole sample with a light source and the emitted light from the fluorescent label is detected
5
Q
Fluorescence Light Microscopy (FLM) Structures inside can be label using:
A
- Fluorochromes: aka fluorophore, fluorescent chemical compounds
- Fluorochrome-linked antibodies: yellow fluorescent proteins (YPF), many different colours
- GFP and GFP variants (famous protein from jellyfish): green-fluorescent proteins (GFP)
6
Q
types of Fluorescence LM
A
Immunofluorescence
Epifluorescence
Confocal
7
Q
Direct Fluorescent Labeling: some fluorescent dyes can bind to structures and label:
A
membrane
Nucleus (binds DNA)
Mitochondria
Cytoskeleton
8
Q
Indirect stain: immunofluorescence
A
Localizes proteins of interest in a cell using primary antibodies
Secondary Antibodies covalently linked to a fluorescent molecule recognize the primary antibody; provides signal amplification
9
Q
Confocal fluorescence microscopy
A
use of lasers and optical sectioning removes out of focus light (increases resolution of light microscope).
Incoming light is focused on a single plane
Out-of-focus fluorescence form the specimen is excluded
Confocal microscopy cuts optical slices through sample
10
Q
Advantages/disadvantages of Light Microscopy
A
Advantages: Can use color Can use live, whole cells Can track cells Cheap and easy to use
Disadvantages:
Can’t see smaller structures (organelles, ribosomes, etc)
Lower resolution
11
Q
Electron Microscopy (EM)
A
Resolution limit = 0.2nm
higher resolution
Images are often black and white
12
Q
Scanning Electron Microscopy (SEM)
A
The sample is coated with metal
Electron beam is focused on the specimen
Secondary electrons are knocked out of the specimen
A detector collects these shattered secondary electrons to build an image
13
Q
Advantages/disadvantages of SEM
A
Advantages:
Can view surfaces (images appear 3D)
Disadvantages:
Cells must be dead
Complex specimen preparation; heavy metals- bit toxic
Microscope is expensive
14
Q
Transmission Electron Microscopy (TEM) Advantages/disadvantages
A
Prepared specimens are sliced very thinly
Advantages: details of cytoplasm can be seen
Disadvantages: Cells must be dead Complex specimen preparation Difficult to know 3D shape of structures Plane of section: things look different from how you cut it High resolution
15
Q
Features of Biological Membranes
A
- The membrane is a bilayer
- Biological membranes are made up of the phospholipid bilayer, and also have lipids, proteins, sterols, glycolipids, carbs, etc.
- All biological membranes are phospholipid bilayers but not all phospholipids are biological membranes - The membrane is organized but fluid- lipid
- The membrane has different permeability for different types of molecules
- The membrane is asymmetric
16
Q
4 kinds of lipids:
A
Fatty acids, cholesterols/sterols,
Phospholipids,
Triaclyglycerols
17
Q
Fatty acids
A
(micelle)
Fatty acids have a single tail & hydrophilic head group. They form micelles instead of bilayers or liposomes because of their shape. The hydrophilic heads face the aqueous environment.
18
Q
cholesterols/sterols
A
Not a lot of opportunities for hydrogen bonding
Sterols are big and bulky carbon rings, with a little hydrophilic hydroxyl group.This means that they will form a layer on the water surface with the OH groups facing the water
19
Q
Phospholipids
A
two fatty acids tails and hydrophilic head groups are necessary for formation of these structures in water. The polar heads face the water while the fatty acid tails form a hydrophobic core
20
Q
Triaclyglycerols (triglycerides or triacylglycerols)
A
have three fatty acid tails, but do not form layers as they lack a polar head group
3 fatty acids esterified to a glycerol
Storage form of fatty acids
Neutral fats form oil droplets, not bilayers
Not strong hydrophilic group; not amphipathic
21
Q
Thermodynamics of the hydrophobic effect
A
Minimum energy conformation (most stable) achieved by minimizing exposure of hydrophobic groups to water
Free energy of the system is minimized if the hydrophobic region (lipid tails) cluster together to limit contact with water, increasing the motional freedom of water
Water likes to form hydrogen bonds with other water molecules (energetically favorable- entropy of hydrophobic molecules decrease, but entropy of water increases)
These hydrogen bonds are continually breaking and re-forming; water molecules are constantly rotating as well
22
Q
To form bilayers, lipids need to be _______ and the _________
A
amphipathic, right shape
23
Q
Membrane fluidity
A
Lateral diffusion (2D movement) Both proteins and lipids can move within the 2D plane
24
Q
How could a cell change its lipids to maintain appropriate fluidity?
A
- Degree of unsaturation in lipids; fatty acid saturation
- Higher number of saturated lipids; more tightly packed; more Van der Waals interactions; less fluid
- Higher number of unsaturated lipids; more kinks in the fatty acid tails due to double bonded structure; more fluid - Fatty acid tail length
- Shorter tails (<18) are more fluid
- Phospholipids with shorter fatty acid chains have less surface area & therefore fewer van der waals interactions - Amount of sterol in the membrane
25
How does the amount of sterols change membrane fluidity?
- At low temperatures, sterols can increase fluidity by preventing tight packing of fatty acids (fewer Van der Waals interaction)
Sterols will make spaces in the membrane and increase fluidity
- At high temperatures, the ring structures of sterols act to ‘stiffen’ the cell membrane. This is because the sterols provide more surface area to form more van der waals interactions with the fatty acid tails
26
Lipid rafts
Microdomains in the plasma membrane rich in specific types of lipids (sphingomyelin & cholesterol)
Lipid rafts are thicker than other regions of the cell membrane- much more ordered
27
How does attachment to structures inside/outside the cell/membrane protein adhesion to other molecules affect membrane fluidity?
The important take-away is that the attachment of membrane proteins to the cell cortex, ECM, etc will result in these becoming anchored proteins. As such, they are no longer going to be able to move laterally in the membrane bilayer
28
Integrins
proteins embedded in the membrane attached to fibronectin which make sure integrins stay stabilized
Adhesion to protein outside the cell (ECM = extracellular matrix)
29
cadherins
Adhesion to neighboring cells- attach together and stay together
Cell to cell adhesion molecules (cadherins) linking the plasma membranes of neuronal cells
30
Tight junctions
Barriers to diffusion: Tight junctions are adhesions between neighboring epithelial cells that form ‘kissing points’ between the two cells so nothing leaks in between the cells
31
Membrane permeability is dependent on the properties of the molecules
```
Gases and hydrophobic molecules diffuse freely across lipid bilayer- no problem interacting with hydrophobic core
Small uncharged polar molecules diffuse well across lipid bilayers
Diffusion of large uncharged polar molecules across lipid bilayers is negligible
Charged substances (ions) cannot diffuse across the lipid bilayer
```
32
Primary Structure - sequence of amino acids (peptide bonds)
Start with N-terminal
end with C terminal
Peptide bonds (covalent bonds between amino acids)
Read in groups of 10
Amino Acid Residue - amino acid has been incorporated into a primary structure
33
Secondary Structure- backbone interactions (H-bonds): alpha-helix
H-bonds are:
Are repetitive all the way along the backbone of the alpha-helix
H- bonds from one amino (n) group and carbonyl (n+4) that is 4 positions away
These interactions do not involve side chains/R-groups; many sequences can adopt helical structure
Parallel to the long axis of the helix
R- groups project outwards
Nothing can travel through alpha-helix “pore”
Too tight and no space for molecules to travel through
34
Secondary Structure Backbone interactions (H-bonds): beta-sheets
R groups point up & down alternating away from the peptide backbone
R groups are above or below the plane of the sheet; thereby have different properties from one side to the other
Beta sheets are usually twisted and not completely flat
Can be parallel and antiparallel on the same sheet
35
Protein domains: how proteins are functionally organized
Secondary structure elements fold into domains within a tertiary structure
36
Examples of protein domains:
```
Transmembrane domain- part that functions to be part of a membrane
DNA binding domain- helps enzymes bind to DNA with domains
Catalytic domain (which carries out enzymatic activity)
cAMP(cyclic-AMP) binding domain- specific site or domain cAMP an important signal molecule
```
37
Tertiary & quaternary structure depend on....
| and types of interactions include..........
```
side chain interactions (R-groups)
Types of interactions:
Hydrogen bond
Ionic bonds
Van der waals interactions
Disulfide bonds
```
38
Bilayer and protein structure formation is similar as both are driven by thermodynamics in terms of
Non-covalent & covalent interactions ensure the most stable final conformational state
Increase the stability of the system
R-groups in the right position to facilitate H-bonds
39
Integrated proteins
proteins directly attached to the membrane; amphipathic. Can monomeric or multimeric
Asymmetry: the orientation of transmembrane proteins matter; the leaflet of attachment matters
Transmembrane protein - through membrane
Monolayer- associated with one layer of the membrane
Lipid-linked - covalently attached to lipid and the lipids are directly attached to the bilayer
40
Peripheral proteins
bound to membrane surfaces through non-covalent association with other membrane proteins
Asymmetry: different proteins attach to different sides
Attached to membrane indirectly
41
Transmembrane domain
part of a membrane protein that passes through the lipid bilayer
Most transmembrane domains are alpha helices, though some are beta barrels (larger pore)
To make pore, need multiple alpha- helices or a beta-barrel
42
The inner core of the beta barrel:
Hydrophilic and polar
In this environment facing inwards, there’s water. They need to interact with water and form H-bonds
The R-groups have to interact with the aqueous environment. The ability to form H-bonds with water molecules will stabilize this protein, and hydrophilic/polar R-groups will facilitate formation of these H-bonds.
43
The outside of the beta barrel:
Hydrophobic and nonpolar
They are interacting with fatty acids and lipid tails
The R-groups have to interact with the strongly hydrophobic environment made up of the fatty acid tails. The ability to form Van der Waals interactions will stabilize this protein, and hydrophobic/nonpolar R groups will facilitate this
44
At the top and bottom of the barrel
Hydrophilic and polar
The R groups have to interact with the aqueous environment. The ability to form H-bonds with water molecules will stabilize this protein, and hydrophilic/polar R-groups will facilitate formation of these H-bonds. Plus, there are some hydrophilic head groups from the phospholipids that will play into this too
45
what kind of bonds stabilize primary sequence?
covalent bonds between the backbone atoms
46
what kind of bonds stabilize secondary (helices, sheets)?
non-covalent bonds between backbone atoms
47
what kind of bonds stabilize tertiary (protein fold)?
covalent bonds between r-groups
non-covalent bonds between backbone atoms
non-covalent bonds between backbone and r-groups
non-covalent bonds between atoms in two R groups
48
what kind of bonds stabilize quaternary?
covalent bonds between r-groups
non-covalent bonds between backbone atoms
non-covalent bonds between backbone and r-groups
non-covalent bonds between atoms in two R groups
49
How are the two halves (leaflets) are the membrane are different from each other; asymmetric
Lipid composition of outer leaflet is different than that of inner leaflet of the bilayer
Non-cytosolic face (faces the extracellular space):
Phosphatidylcholine - choline head group
Sphingomyelin - part of lipid raft
Carbohydrates linked to membrane on extracellular leaflet
I.e. glycolipids (glycoproteins- proteins with sugar molecules attached not shown here)
Cytosolic Face: different head groups
Phosphotidylserine
Phosphotidylethanolamine
phosphotidylinositol
50
smooth ER makes....
lipids
51
flippase
Enzymes called flippase transfers phospholipids to other half of bilayer for symmetric growth of both halves of bilayer.
Asymmetry is established by flippase enzymes (or some are called scramblases or flopases) because they choose which lipids are being flipped and end up with different assortment
52
flippases vs. scramblases
Flippases: specific transfer direction from a leaflet to other leaflet
Scramblases: non-specific transfer from either leaflet
53
Bioinformatics approach: hydropathy plots
To predict the number of transmembrane segments & orientation
Not definite but very likely prediction
Bioinformatics tool to predict alpha-helical transmembrane domains
Computer generated
Based on properties of the amino acids in the peptide sequence
Adobe the dotted line indicates stretches of hydrophobic amino acids that can form transmembrane domains (~18-20 a.a); threshold
Any point above 0 are considered hydrophobic & any below are considered hydrophilic, on average relative to surrounding regions too
There will be alternating hydrophobic & hydrophilic residues in the beta barrel sequence resulting in near zero hydropathy plot
We use the primary sequence to predict the secondary structure of unstudied proteins
54
Protein purification & biochemical experiments
To determine membrane protein type, components & orientation
Step 1: Isolate your protein
- Peripheral proteins: High salts tends to weaken protein-protein interactions by disrupting electrostatics bonds
-Integral Proteins: held by van der waals interactions
Detergent: to solubilize integral proteins, we need to use harsh treatments: replace the membrane with detergents
Step 2: unfold/linearize proteins by adding SDS & break disulfide bonds
- They dissociate quaternary structures too and separated peptides
Step 3: Separate proteins by size with a polyacrylamide gel matrix
-From negative to positive move down
-Larger molecules are closer to the top if the gel
-Smaller molecules are closer to the bottom of the gel
-The SDS added gives the proteins an overall negative charge which helps to draw it to the anode with similar shape and mass ratio
55
Gel electrophoresis:
Sample applied at negative end of gel
Nucleic acids (DNA, RNA)
Equal charge to mass ratio throughout the length of each molecule (from what chemical group?)
Separates according to size
Generally separated on a horizontal agarose gel
Phosphate backbone has negative charge which allows them to separate by size
protein
Different charges and shapes because of amino acid sequence
A strong ionic detergent (SDS) helps to equalize the charge to mass ratio and also denatures proteins so that they also separate according to size (instead of charge and shape)
Generally separated on a vertical polyacrylamide gel
56
proteases (e.g. trypsin)
trypsin: which are enzymes that can cleave peptide bonds
The enzyme cannot cross the lipid bilayer
The enzyme will only digest the parts of the membrane proteins that are exposed to the enzyme
57
function of nucleus
Separation of transcription and translation (major role)
Protection of the coding material (minor role)
Nucleus continuous with rough ER and grooves are called cisternae
58
Nuclear lamina
structure responsible for maintaining the nuclear membrane & plays a role in DNA replication, transcription & gene regulation
Nuclear lamina is made up of strong nuclear lamin proteins. Proteins in the inner membrane can be anchored to the nuclear lamina
Made up of intermediate filament proteins called lamins that prevents collapse into a single layer for which is favoured due to higher thermodynamic stability
SEM image of nuclear lamina
Nuclear lamina aids in disassembly & reassembly of the nuclear envelope during cell division
59
Phosphorylation
covalent attachment of a phosphate group
Phosphorylation of nuclear pore proteins to disassemble the nuclear envelope for cell division
Then dephosphorylation of nuclear pore proteins and lamins to reform envelope
60
Nucleolus
specialised chromosomal region of nucleus
Not a membrane bound organelle
Function: site of rRNA synthesis & assembly of ribosomal subunits made from rRNA & ribosomal proteins
Ribosomal subunits come together for the final package before sent out to the cytosol
Chromosomal regions that form the nucleolus have 100-200 copies of rRNA genes; we need a lot of ribosomes which are important for protein synthesis
61
Ribosome:
made of rRNA and protein
rRNA is transcribed in the nucleolus
Ribosomal proteins are translated in the cytosol and comes back into the nucleus
rRNA & ribosomal proteins are assembled in the nucleolus into large & small subunits
Ribosomal subunits (large & small) come
62
Ribosomal subunits (large & small) come together as ribosomes during ______
translation in the cytoplasm
63
The number of nucleoli in a human cell depends on
the cell cycle.
In M phase, when the chromosomes condense, the nucleolus fragments and then disappears
Then, in telophase the tips of the 10 chromosomes reform 10 small nucleoli which progressively fuse into a single nucleolus
Number of nucleoli range from 1-10 depending on the cell cycle
64
Examples of molecules entering the nucleus:
```
Histones
Ribosomal proteins
Nuclear lamins
Polymerases
Transcription factors
Nucleotides ATP
```
65
Examples of molecules leaving the nucleus:
Ribosomal subunits (ribosomal proteins +rRNAs) (leave to function as ribosome)
nRNA-protein complexes
tRNAs (allow amino acids to be brought to ribosomes for translation)
66
Nuclear pore
multi-protein subunit complex that acts as a nuclear gate
Pore complex has a diameter of ~90-120nm
Predicted pore/opening = ~9nm in diameter, but active transport can move much larger molecules, ~35-40nm in diameter
Bi-directional movement
Cross-section of nuclear pore
Nuclear basket made of nuclear fibrils on nuclear side of nuclear pore
67
Diffusion
ions & small macromolecules (<5kDa or ~9nm in diameter) can pass freely and non-selectively through the nuclear pore complex (NPC)
68
Active transport
energy required to transport large molecules (> 5kDa or 9nm in diameter size of opening of nuclear pore) transported into (& out of) the nucleus
Proteins must have special amino acid “targeting/signal sequence” called Nuclear Localization Signal (NLS) - which directs them into the nucleus for active transport
Histones, Polymerase, lamins all have to get into the nucleus
69
Targeting signal:
signal that is present in protein that allows it to be recognized
IMPORT: Nuclear Localization signal (NLS)
EXPORT: Nucleus export signal (NES)
Targeting signals are encoded within proteins
Targeting signals direct the protein to a specific organelle
Targeting signal must be present for protein to leave the cytosol compartment
70
Nuclear transport receptor
bind to nuclear signal sequence and can dock onto proteins of the nuclear pore
IMPORT: Nuclear import receptors (NIR)
EXPORT: Nuclear export receptors (NER)
71
nuclear import model
NLS of a protein binds to NIR (nuclear import receptor) creating complex
Complex binds to fibril on annular ring
Fibril guides the protein into the pore
Protein is moved through the pore (GTP-driven process)
GTP needed to remove the cargo protein from nuclear transport protein inside the nucleus
The hydrolysis of GTP (step 4 in diagram) is required, and provides the energy to allow for nuclear import
72
Proteins with no localization signal in their primary sequence will end up localized in the____
cytosol
Exception:
- proteins produced by ribosome located in the chloroplast & mitochondria; no need to travel anywhere so no localization signal needed
- Proteins that are super small (<9nm) and can pass through nuclear pore passively through diffusion without NLS; not common
73
Components required for import/export from the nucleus
1. Cargo proteins: (prospective nuclear protein) signal sequence NLS=”KKKRK”
2. Receptor proteins: recognizes signal sequence, brings to pore & binds cargo protein
3. Energy: GTP helps contribute to directionality of transport; with GTP out of nucleus; without when going into nucleus
4. Nuclear pore complex
74
How to identify & study a targeting sequence?
Remove parts of the protein and see if it can still be targeted to its final location:
- Deletion of parts of the protein
- Partial digestion of protein
Mutate amino acids and see which changes affect targeting
Fuse different regions of the protein to a cytosolic protein and see if location of the cytosolic protein is affected
75
Loss of function experiments:
Remove from system and observe what happens (e.g. deletion, mutation)
This type of experiment asks if the component removed is necessary
76
Gain of function experiments:
Add a component that is not normally present and observe what happens
This experiment asks if the component is added is sufficient
77
Export from the Nucleus
Proteins are exported in a similar way that import happens
I.e. nuclear export signal (NES) in protein is bound by protein receptor, which aids in active export of the protein out of the nuclear pore
Mature RNA ready for export MUST be bound by proteins
NES is an amino acid signal, which means RNA cannot possess it
78
Chromatin territories:
Interphase chromosomes are spatially organized
| Specific regions of the chromosomes are attached to either the nuclear envelope or the nuclear lamina
79
Heterochromatin
Densely darker staining regions
About 10% of an interphase chromosome
Densely packaged
Concentrated around centromere & telomeres of the chromosomes
Not many active genes included heterochromatin
80
Euchromatin
Lightly staining regions
Contains less condensed DNA
Genes within this region are actively being transcribed
81
Histone octamer complex
a complex comprising of 2 copies of 4 polypeptides that form primarily due to hydrophobic interactions between their tertiary structures
- Hydrogen bonds & ionic bonds allow for DNA to wrap around the histone octamer
Phosphate groups have negative charge on DNA which interact with histone octamer
82
History octamer & DNA assembly:
Alpha Helices joined by loops; two molecules come together as a dimer (H3-H4 dimer; H2A-H2B dimer)
dimerization= two polypeptides coming together to form a dimer
Hydrophobic residues in polypeptide that help form dimers due to hydrophobic effect
H3-H4 Tetramers formed from dimers which are then wrapped by DNA to the hydrophilic regions outside the tetramer.
The two H2A-H2B dimers bind to H3-H4 tetramer
The result is the histone octamer
83
Naked DNA
Very rare as they are very vulnerable to damage
2nm
~2m long
84
“Beads on a string” form DNA
Only seen experimentally
10-11nm fiber (nucleosomes)
Linker DNA is between histone wrapped DNA
Nucleosomes (11nm fiber)
Nucleosome core histones: 2x H2A, H2B, H3, H4A
85
Interphase chromatin
Can form further loops/ higher ordered loops of the 30nm fiber
Chromatin can form chromosomes
Histone H1= linker histone
Pulls nucleosomes together into the 30nm fiber
Chromatin (30nm)
Formation of heterochromatin
86
Histone H1
linker histone
Pulls nucleosomes together into the 30nm fiber
Tighter packing
Details under investigation
H1 linker histone binds DNA and pulls the nucleosomes into a repeated, twisted array
It is not fully understand how this complex forms
87
Mitotic chromosomes (>300nm)
Large enough to see with light microscopy
Formed by non-histone chromatin proteins that fold a scaffold
Each DNA molecule has been packaged into a mitotic chromosome 10-50,000x shorter than its extended length
88
Dividing cell vs. Nondividing cell/ interphase:
Dividing cell: packaged carefully and tightly so when the cell divides, the chromosomes all go to the right place
Nondividing cell/ interphase: much looser and less tightly organized
89
Chromatin remodeling complexes
Chromatin remodeling complexes that physically move nucleosomes closer or farther apart
Nucleosome sliding to loosen fiber which uses ATP and converts it to ADP
Density of interphase chromatin is regulated by recruiting chromatin remodeling complexes
The complex binds histones/DNA to slide the nucleosome, revealing more “free” DNA
Chromatin remodeling complex can slide or remove histones so that the DNA wrapped around them can now be accessed by the transcription machinery
Chromatin remodeling factor can bind to TATA box which loosens the packing or even remove the histones
90
Histone modifications (of tails)
Acetylation
Methylation
Phosphorylation
Different combinations serve as docking sites to recruit proteins that promote DNA packing/unpacking
Histone modifications can be added on residues to different histone tails
91
Binding of transcription regulators
transcription regulators can further destabilize nucleosomes & create open DNA regions
92
Regulatory region
upstream and it is comprised of enhancer, promoter
| RNA is the gene product of transcription
93
RNA is made from ____, therefore the template strand is read from________
RNA is made from 5’ to 3, therefore the template strand is read from 3’ to 5’
94
Promoter:
Regulatory region of DNA near the transcriptional start site
| Binds RNA polymerase and general transcription factors
95
Enhancers/repressors:
Bind activators and repressors to control transcription
| May be far away from the actual gene
96
Eukaryotic RNA is transcribed & processed___________ .
In eukaryotic cells, translation does not occur at the same time as transcription. At the end of rRNA at the 5’ ends are proteins involved in _________
simultaneously.
RNA transcript processing
97
rRNAs do not require specific _____ for export out of the nucleus ( as they are exported as part of the ribosomal subunit), but mRNAs that do need to exit the nucleus will require those______ to be added later on
5’ caps
98
summarize transcription:
Either strand of DNA can act as a template for transcription (BUT the template is always read in 3’ to 5’ direction). Which strand will be used depends on the promoter, which has polarity and allows binding of polymerase on only one of the strands
The process of transcription requires ribonucleoside triphosphates, RNA polymerase, general (=basal) transcription factors and transcriptional regulators
The mechanism of transcription involves binding of general transcription factors and RNA polymerase to the promoter region (=transcription initiation), elongation, and termination
Transcriptional regulators control the process of transcription and determine if gene transcription is turned on or off
99
General transcription factors
bind to promoter to help recruit RNA polymerase
100
Other proteins bind to regulatory regions to..
help promote or repress transcription
101
DNA-binding proteins (transcription factors)
specifically recognize the correct binding site on DNA by making specific non-covalent interactions with the sides of the base-pairs in the major or minor groove of that sequence.
Assembly & stability of the general transcriptional machinery depends on the binding of other regulators
102
Transcription regulators can inhibit transcription by:
1. There may be competitive DNA binding
2. Masking the activation surface
3. general transcription factors
103
Transcription regulators control gene expression because of
chromatin remodeling & histone modifiers
| Chromatin remodeling complexes & histone modifications help expose DNA for transcription
104
RNA modification prior to nuclear export
Transcript processing:
- 5’ capping & 3’ polyadenylation
- mRNA splicing by spliceosomes
- alternative splicing
105
Signals required for processing of macromolecules are usually _____________
encoded within the primary transcript or sequence
106
The 5’-Cap:
Helps stabilize the transcript
(to protect from nuclease degradation)
Helps in binding protein for explore from nucleus
Helps in ribosome recognition & binding (to initiate translation in the cytopolasm)
5’cap is added to eukaryotic pre-mRNA shortly after initiation of RNA synthesis
Unusual guanine with an added methyl group
107
The 3’-PolyA tail:
a sequence in the pre-mRNA transcript signals for cleavage & then repeated adenines are added
Helps in stabilizing the transcript
Helps translation termination
108
Transcript processing: mRNA splicing by spliceosomes
Exons are protein coding regions- kept
Introns are non-coding regions- removed
Special recognition sequences on the pre-mRNA are located at the intron-exon junctions & within the intron
These sequences are recognized by SnRNPs = small nuclear ribonucleoproteins
Complementary base pairing between snRNA & mRNA
109
Transcript processing: alternative splicing
Alternative splicing: a gene can be spliced different ways to produce variants of the same protein
Regulated by proteins that can bind to the primary transcript:
Includes splicing activators (promotes usage of a particular splice site)
Splicing repressors (that reduces usage of a particular splice site)
Other possible mechanism: CpG DNA methylation to regulate exon skipping
In this case, exon skipping allows cells to ‘skip’ over mutated exons that results in a disrupted reading frame
110
Duchenne Muscular Dystrophy (DMD):
a disease in which skeletal muscles weaken & break down over time
DMD is particularly severe form of muscle dystrophy
X-linked recessive trait
Signs & symptoms include muscular wasting, scoliosis, inability to walk, difficulty in respiration
Due to the loss of function dystrophin protein
Depletion of exons 45-50 is the primary cause of this disease
The disruption in of the reading from due to a frameshift mutation produces premature stop codons
Exon skipping as possible treatment for DMD
AON (antisense oligonucleotides) mask exon 51, allowing a partially functional dystrophin to be produced
Exondys 51 is administered as intravenous infusion given on a weekly basis
