last class: gene regulation 1. dna-protein interaction, different motifs, techniques to study...
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Last Class: Gene Regulation
1. DNA-protein interaction, different motifs, techniques to study DNA-protein interaction
2. Gene regulation on DNAs, gene activators (acting on promoter, enhancer, chromatin etc), repressors
3. regulation of gene activators/repressors4. integrated response.
Posttranscriptional Regulations
Posttranscriptional regulation possibilities
Alternative RNA Splicing
Alternative Splicing of RNA of the Drosophila DSCAM gene (axon
guidance receptors directing growth cone localization)
Negative and Positive Controls of Alternative Splicing (active regulation)
sometimes, the splicing sequence is ambiguous, so constitutive random splicing
Antibodies (membrane-bound, secreted) in B
lymphocytecleavage regulated RNA
processing (CstF)
RNA Editing (inserting Uracil at different sites and change encoding sequence)
Nuclear Export
Rev binds to rev response elelent (RRE), which
binds to nuclear export receptor (exportin 1) to regulate nuclear export even without splicing
a delay of virus infection symptom
Localization in cytoplasmalso determine the fate of RNAs
3’ UTR (untranslated region)
3’ UTR in regulating LocalizationRed: intact 3’ UTR,
Green: 3’ UTR deleted
Translational Regulation
Negative Translational Controlaconitase inhibits ferritin production
Phosphorylation RegulationeIF-2B serving as GEF for eIF-2 and promote translation initiation
phosphorylation locked eIF-2 in inactive form
Internal Ribosome Entry Site (IRES)
Two Mechanisms of mRNA decay
1. regular 3’ tail shortening followed by ‘5 decapping
and degradation2. endonucleolytic cleavage
and fast decapping and degrading
The competition between mRNA translation and decay
initiation machinery and deadenylation proteins are all associated with 5’ and 3’
With Iron, aconitase release enhances the production of ferritin to bind iron while destabilizes transferrin receptor mRNA to reduce the
transportation of more iron intracellularly
Stop Codon CheckingShould after all the exons (nonsense-mediated mRNA decaying)
SiRNA MechanismRNase, ATP hydrolysis
and RNA helicase
Summary• Premature termination
• Alternative RNA Splicing
• 3’ cleavage and Poly A’ addition
• RNA editing
• Necleus transportation
• Localization of RNA at the cytoplasm
• Translational initiation
• Degradation
• Cell Membranes
• Lipid Bilayers
• Cell Membrane
Cell Membrane Views
Phospholipid Molecule
Hydrophilic and Hydrophobic
Molecules interacting
differently with water
Wedge-shaped lipids form micellesCylinder-shaped lipids form bilayers
Spontaneous sealing of phospholipid bilayer
LiposomesProved the self sealing
process
Phospholipid mobilityLateral diffusion:
Diffusion coefficient 10-8 cm2/sec
Migrate in seconds to cover the whole
surface
Flip-Flop: phospholipid translocators
Cis-double bonds affect packing
Saturated: packed and thick
Unsaturated : loose and thin
Phase transition: liquid to solid
Cholesterol and Glycolipids
Function of Cholesterol
1. Provide structural support, prevent small molecule to pass
2. Prevent tight packing and transition
Phospholipid types
Microdomains on plasma membrane
Lipid rafts (~ 50nm)
ChoresterolSphingolipids (long saturated chains)Other proteins
Asymmetrical distribution of
phospholipids and glycolipids
Protein kinase C (PKC) binds to
negatively charged
phosphotidylserine to be functional
Phospholipids in cell signaling
PKCCalcium
GEF, AKT, migrating front
Phospholipase Cleavage sites
Phosphotidylserine exposed on outer surface as apoptosis signal
1. Phospholipid translocator2. Scramblase
Glycolipids
Gm1 Ganglioside with charge can serve as signal for lipid rafts, binds to Cholera toxin
Summary•Lipid molecules: phospholipids, cholesterol, glycolipid, all amphipathic•Lipid bilayer, hydrophobic inside and hydrophilic outside•Subdomains on membrane, asymmetry important for functions•Phospholipids as signals
Membrane Proteins
Glycosylphosphatidylinositol (GPI) anchor protein
Integral membrane proteinPeripheral membrane protein
Fatty acid chain (acyl, N-terminal) or prenyl group modifications (C-terminal)
Or geranylgeranylOr palmitic acid
Hydropathy PlotsIndex of hydrophobicity
Membrane proteins are glycosylated
Sugar are added in the lumen of the ER and
Golgi apparatus, therefore, sugar are
outside of cell surface
Cytosol has reduced environment,
preventing disulfide bonds
A detergent micelle
Detergent to solubilize and purify membrane proteins
Solubilize membrane proteins with detergent
Different DetergentsIonic (strong) or nonionic
(weak)
Mild detergent for the solubilizing,
purification, and reconstitution of
membrane protein functions to study the
functions of membrane proteins in simplified
environment
The study of membrane proteinsMost prominent example
red blood cells
No nucleus or internal organelles
The preparation of red blood cell membranes
15 major membrane proteins in red blood cells
Label them with impermeable dyes can
determine the location on layers
Membrane proteins are diffusible
Techniques to study protein motion on membrane(Fluorescence Recovery After Photobleaching
(Fluorescence Loss In Photobleaching)
Proteins restrictionsTight Junction is one kind of them
Proteins and lipids on the outer layers can’t move to other compartments
Protein distributions in a guinea pig sperm cell
4 ways of protein restrictions1. self-assembly2. Tethered to macromolecules outside3. Tethered to macromolecules inside
4. Cell-cell adhesion