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