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/repressors 4. 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 lymphocyte cleavage 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 cytoplasm also determine the fate of RNAs 3’ UTR (untranslated region)

3’ UTR in regulating Localization Red: intact 3’ UTR, Green: 3’ UTR deleted

Translational Regulation

Negative Translational Control aconitase inhibits ferritin production

Phosphorylation Regulation eIF-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 degradation 2. 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 Checking Should after all the exons (nonsense-mediated mRNA decaying)

SiRNA Mechanism RNase, 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 micelles Cylinder-shaped lipids form bilayers

Spontaneous sealing of phospholipid bilayer

Liposomes Proved the self sealing process

Phospholipid mobility Lateral diffusion: Diffusion coefficient cm 2 /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) Choresterol Sphingolipids (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 PKC Calcium GEF, AKT, migrating front

Phospholipase Cleavage sites

Phosphotidylserine exposed on outer surface as apoptosis signal 1.Phospholipid translocator 2.Scramblase

Glycolipids G m1 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 Glycosylphosphatidylinosi tol (GPI) anchor protein Integral membrane protein Peripheral membrane protein

Fatty acid chain (acyl, N-terminal) or prenyl group modifications (C-terminal) Or geranylgeranyl Or palmitic acid

Hydropathy Plots Index 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 Detergents Ionic (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 proteins Most 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 restrictions Tight 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 restrictions 1.self-assembly 2.Tethered to macromolecules outside 3.Tethered to macromolecules inside 4.Cell-cell adhesion