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402 Cards in this Set

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System (Bioenergetics)
Restricted portion of the universe being studied.
State of system defined by variables.
Study changes by intial->final states.
Calorie (cal)
Amt of E to heat 1g of water 1 degree Celcius at 1 atm.

Joule (J)=0.239cal/mole
dE equation
E prod's. - E react's.
neg. dG
Exergonic
Spontaneous
pos. dG
Endergonic
Nonspontaneous
Entropy (s)
A measure of randomness or disorder.
ds = change in entropy w/ rxn.
higher entropy=more disorder
Enthalpy (H)
Heat content
dH can approximate dE
dH = H prod's. - H react's.
neg dH
exothermic - heat released
pos dH
endothermic - heat absorbed
Irreversible Inhibition
Inhibitor binds covalently to active site.
Michaelis-Menton kinetics
Rate increases w/ increase in substrate conc.
With each increase-smaller increases in rate.
Can alter rate by increasing [E].
Enzyme Kinetics
Rate of rxn.
Enzyme activity depends on conc. of substrates, products, and inhibitors.
Measured in terms of initial rxn rate - before substrate decreases and product accumulation is small.
Active site
Usually groove or pocket.
Part of 3 prime configuration.
Catalysts
Inc. rate of rxn by lowering activation E.
Forms transient complexes - facilitates interactions b/t reactants.
Changes ONLY rate at which equilibrium is reached.
Activation Energy
Specific to every rxn.
Minimun E amt reactants need to be converted to product.
Protein Secondary Structure
Regions of polypeptide coil into a helix.
2 structural patterns: alpha helix and beta sheet.
Alpha helix
A form of secondary structure.
Spiral in shape, consisting of a backbone of peptide bonds w/ specific R groups of a.a. residues jutting out from it.
Each turn of the coil stabilized by H2 bonds b/t the CO and NH groups of one peptide bond and those of the peptide bonds 4 a.a. away in each direction.
Beta sheet
Protein secondary structure.
An extended sheetlike conformation w/ successive atoms in the polypeptide chain located at the peaks and troughs of the pleats.
R groups of the a.a. jut out on alternating sides of the sheet at each fold.
Stabilized by H2 bonds b/t the CO and NH groups of peptide bonds in adjacent polypeptide regions.
Protein Primary structure
Sequence of a.a. linked together by peptide bonds forming a polypeptide (covalent bonds).
Why is carbon so important to biological molecules?
Specific bonding properties.
"valence of 4" prop. - can bond w/ each other.
Forms covalent bonds.
Stable organic cmpds have 4 covalent bonds for every C atom, giving C-containing molecules great structural and functional diversity.
Bond Energy
Amt of E required to break 1 mole (6x10^23) of a bond.
Expresses stablility.
Why is the polarity of water so important?
It accounts for its cohesiveness - Forms H2 bonds.
Temp. stabilizing capacity - due to its extensive H2 bonding, buffers soln's against large temp. changes.
Excellent solvent - can form spheres of hydration.
Vacuole
Storage vescicle
May hold food molecules.
In plants - maintain turgor pressure.
Ribosome
Protein synthesis.
Not an organelle.
2 subunits
Microtubules
Organize cytoplasm (cell shape).
Cilia and flagella.
Largest; mitotic spindle fibers.
Hollow cylinders of tubulin subunits.
Microfilaments
Muscle fibriles (locomotion).
Smallest.
Actin polymers (G-actin).
Peroxisome
Generates and degrades H2O2.
In animals, found in liver and kidneys - detox and catabolize, breakdown of fatty acids.
H2O2 -> H2O + O2
Secretory vescicles
Molecules for export.
Synthesized in rough ER.
Packaged in Golgi.
Export via exocytosis.
Lysosome
Vescicle w/ hydrolases.
Recycling.
Enzymes synthesized on rough ER and packaged in Golgi.
Golgi Complex
Stack of flattened vesicles.
Processes and packages secreted proteins - accepts vescicles from rough ER.
Synthesizes polysaccharides.
Endoplasmic Reticulum (ER)
Membrane Network - interconnected cisternae - tubular membranes.
Continuous w/ outer nuclear membrane.
Rough ER b/c of ribosomes.
Smooth ER - lipid synthesis, detoxification enzymes.
Chloroplast
Photosynthesis in plants and algae.
Numerous in plants.
Dark rxns - cell makes sugars.
Double Membrane - also 3rd membrane = thylakoid sacs.
Own DNA and ribosomes.
Makes ribosomal RNA?
Nucleolus
Bacterial genetic material
Circular DNA molecule.
Some proteins.
Nucleoid
In prokaryotes.
Folded genetic material.
Usually attached to cell membrane.
Protein functions
Enzymes - catalyst for rxns.
Structure - support and shape.
Motility - contraction and movement (actin and myosin).
Regulatory - control, coordination of cell processes.
Amino Acids (a.a)
Monomer of proteins.
20.
All have central carbon.
Side chains vary in biochemistry.
Phospholipid structure
Polar head - hydrophilic.
2 non-polar hydrocarbon tails - hydrophobic.
Polarity of the hydrophilic head is due to a neg. charged phosphate group often linked to a pos. charged group (amino group).
Amphiphatic molecule
Have both hydrophilic and hydrophobic regions.
Membrane phospholipids are amphipathic.
Phospholipid structure
Polar head - hydrophilic.
2 non-polar hydrocarbon tails - hydrophobic.
Polarity of the hydrophilic head is due to a neg. charged phosphate group often linked to a pos. charged group (amino group).
Amphiphatic molecule
Have both hydrophilic and hydrophobic regions.
Membrane phospholipids are amphipathic.
Polysaccharides
Polymers of sugar - usually 1-2 types per polymer.
Used for storage and structure.
Starch, glycogen, cellulose.
Monosaccharide
Monomer of polysaccharides
Usually 3-7 C's.
General formula Cn H2n On
Glucose most common.
Most common monosaccharide in biosphere/cells?
Glucose
Nucleic acids
RNA, DNA
Storage, transmission, expression of genetic info.
Differ by sugar molecule and one base.
Nucleic acid monomer
Nucleotides
Lipids function
Energy storage
Membrane structure
Other specific functions
Steroid structure
4 fused rings
Differ by # and position of double bonds
Steroids
Only in eukaryotic cells
Cholesterol most common
Cholesterol
Most common steroid
Starting pt in synthesis of sex hormones - glucocorticoids and mineralocorticoids.
Found in cell membranes
What allows proteins to shift conformations?
Weak interactions such as H2 bonds.
Peptide bond
A covalent C-N bond linking 2 a.a.

Formed by dehydration.
What is a dimer?
A multimeric protein composed of 2 polypeptides
Starch
Storage polysaccharide found in plant cells.
Glycogen
Storage polysaccharide found in animal cells, also bacteria
Energy
The capacity to cause specific physical or chemical changes.

The capacity to do work.
Cellular activities which give rise to specific physical and chem. changes (6).
Synthesis
Mechanical - Change in location
Concentration
Electrical
Heat - by product of rxns
Bioluminescence - light
Synthesis
Biosynthesis
Change in chemical bonding
Formation of new chem. bonds.
Maintaining chem. and cellular structures.
Photosynthesis
Mechanical work
Positioning/orientation
Change in location
Use of cilia and flagella.
Muscle contraction
Concentration work
Changes in chem. concentration across membrane.
Movement of molecules across a membrane against a concentration gradient.
Accumulate or removal of molecules.
Electrical work
Movement of ions across a membrane against a electrochemical gradient.
Maintaining electrical potential.
Nerve impulse transmission.
ATP production.
Heat
An incr. in temp. that is useful for warm blooded animals.
Regulation of body temp.
Enzymes function at 37 C in our bodies.
Bioluminescence
Production of light.
Generated by rxn of ATP w/ specific luminescent cmpds.
Phototrophs
Can obtain E for cellular needs by capturing light E from the sun using specific pigment systems.
Transform this E to chem. E and store the E in the form of ATP.
Chemotrophs
Rely on oxidation of chem. bonds in organic or inorganic (chemoautotrophs) molecules for E.
Oxidation rxns release _____?
Energy
DIC treatment?
**Identify and treat the underlying disease!
Also transfusion (especially fresh frozen plasma)
Spontaneous heat transfer
Always occurs from the hotter place to the colder place.
First Law of Thermodynamics
The law of conservation of E.
In every physical or chem. change, the total amt of E in the universe remains constant, although the form of the E may change.
E cant be created or destroyed
Internal Energy
The total E stored w/in a system.
Enthalpy
Heat content
Represented by H
Second Law of Thermodynamics
Law of thermodynamic spontaneity.
In every physical or chem. change, the universe always tends toward greater disorder or randomness (entropy).
Allows us to predict in what direction a rxn will proceed under specified conditions.
Thermodynamic spontaneity
A measure of whether a rxn or process can go, but it says nothing a/b whether it will go.
Directionality important
Two alternative means of assessing thermodynamic spontaneity.
Entropy
Free energy
Entropy (S)
A measure of randomness or disorder.
Can't quantify directly, but can get some feel for it.
Increases: Melting, evaporation of gasoline, burning paper
Link b/t spontaneous events and entropy changes
Whenever a process occurs spontaneously in nature, entropy of the universe increases.
dS is positive for all spontaneous rxns/events.
Free energy (G)
Measure of spontaneity for a system alone, not including the universe.
Related to enthalpy and entropy
dH is positive when?
Endothermic rxns
dH is negative when?
Exothermic rxns
Second Law of Thermodynamics and Free Energy
All processes that occur spontaneously result in a decrease in the free energy (dG) content of the system.

An exergonic rxn.
Exergonic
An energy-yielding rxn.

Decrease in free energy (G).
Endergonic
An energy-requiring rxn.

Increase in free energy (G).
Spontaneity
Tells us only that a rxn can go.
Says nothing a/b if it will go.

A rxn can have a -dG and no proceed to any measurable extent.
Keq
Equilibirium constant.
Ratio of product conc. to reactant conc. at equilibrium.
What drives every rxn?
Tendency to equilibrium
The amt of free E available from a chem. rxn depends on what?
How far the components are from equilibrium.
dG calculates what?
Energy needed to reach equilibrium.
K'eq > 1.0
dG' neg.
Rxn will proceed to rt under standard conditions.
Products predominate over reactants at eq.
K'eq < 1.0
dG' pos.
Rxn will proceed to lft under standard conditions.
Reactants predominate over products at eq.
2 ways to incr. the proportion of molecules w/ sufficient E to overcome Ea.
Incr. avg E content of all molecules - Input of heat

Lower Ea requirement - catalyst
Catalysts
Enhances the rate of rxn by lowering Ea.

Ensures that a higher proportion of the molecules possess suffiecient E to undergo rxn w/out the input of heat.
Do NOT change position of eq.
a.a. common in active sites
Cysteine
Histidine
Serine
Aspartate
Glutamate
Lysine
Prosthetic groups
Specific non protein components sometimes on an active site.
Usually either metal ions or coenzymes.
Frequently func. as electron acceptors (a.a. not good acceptors)
Coenzymes
Small organic molecules derived from vitamins.
3 mechanisms of substrate activation
Bond distortion
Proton transfer
Electron transfer
Bond distortion (substrate activation)
Change in enzyme conformation by initial substrate binding to the active site.
Distorts 1 or more bonds.
Makes more susceptible to catalytic attack.
Proton transfer (substrate activation)
Enzyme may accept or donate protons.
Inc. chem. reactivity of the substrate.
Via charge a.a. in active site.
Electron transfer (substrate activation)
Enzymes may accept or donate electrons.
Forms temporary covalent bond b/t enzyme and substrate
The catalytic event
Substrate collides w/ active site forming temporary bond.
Changing in enzyme conformation for better fit.
Products formed and released.
Active site available for another substrate molecule.
Initial rxn velocity (v) depends on what?
Substrate concentration
Maximum velocity (Vmax)
As substrate conc. becomes large, value of v reaches a max.
Depends on # of enzymes, can only be incr. by adding more enzyme.
Saturation
Inablility of incr. substrate conc. to incr. rxn velocity.
Saturation
All available enzyme molecules are operating at max. capacity.
Km
Concentration of substrate that gives exactly half the max. velocity.
Low Km
The lower the Km value for a given enzyme and substrate, the lower the substrate conc. range in which the enzyme is effective.
Enzyme regulation
Incr. product decr. rxn rate.
2 mechanisms: allosteric regulation and covalent modification
peu importe
never mind, it does not matter
Allosteric enzymes
Have 2 forms
High affinity for substrate or little to none.
Forms are interconvertible.
Allosteric effector
Small organic molecule that regulates the activity of an enzyme for which it is neither the substrate nor immediate product.
Allosteric effector mechanism
Binds to one of the two forms of an allosteric enzyme, stabilizing it.
Binds to allosteric site which is distinct from active site.
May be inhibitor or activator.
Cooperativity
Property of allosteric enzymes.
Positive or negative
As the enzyme binds substrate, the enzyme changes conformation which affect the affinity of remaining substrate sites.
Covalent modification
Add or remove chem. groups
Proteolytic cleavage
Phosphorylation/dephosphorylation
Neutral glycolipids
Cerebrosides
Neg. charged glycolipid
Ganglioside
Gangliosides
A glycolipid
Func. as antigens recognized by antibodies in immune rxns.
Blood typing
Important to brain and nerve cells
Glycolipids (cerebrosides, gangliosides)
Common membrane saturated F.A.
Palmitate
Stearate
Common membrane unsaturated F.A.
Oleate
Linoleate
All unsaturated F.A. in membranes are in what conformation?
cis
Sharp bend, or kink, in the hydrocarbon chain at every double bond.
Do not pack tightly as a result.
Flippases
Smooth ER
Phospholipid translocator
Catalyze the flip-flop of membrane lipids from one monolayer to the other.
Tm
Membrane transition temp
Membrane must be above Tm value, or in its fluid state, to func. properly
Low Tm
More fluid
Tm decreases w/ incr. in double bonds, or unsaturation.
Cholesterol and Tm
Decr. membrane fluidity at temp above the Tm
Incr. fluidity at temp. below the Tm.
Homeoviscous adaptation
Regulation of membrane fluidity.
Important for org. that control body temp.

Alteration of membr FAs w/ change in temp
Temp drop  act’n enzyme  cleave C’s from FA’s  shorter chains  lower Tm  more fluid
Temp drop  incr’d desaturase enzyme  intro db into FA’s  decr’d sat’n  lower Tm  more fluid
Homeoviscous adaptation and O2
Desaturase also responds to O2 changes with temp
O2 is substr of desaturase
Temp drop  incr’d O2  incr’d desaturase activity  intro db into FA’s  decr’d sat’n  lower Tm  more fluid
Impt to plants, hibernating animals
Membrane protein categories
Integral
Peripheral
Lipid anchored
Integral membrane proteins
Amphipathic
Hydrophobic regions embedded w/in the memb.
Difficult to remove
Molecules transported via simple diffusion
Small nonpolar molecules
Oxygen CO2, ethanol
Transport proteins
Integral memb. proteins that recognize substances w/ great specificity and speed their movement across the memb.
Faciliated diffusion
Transport proteins move solutes down their free E gradient in the direction of thermodynamic equil.


Also passive transport
The movement of an ion is determined by its what?
electrochemical potential.
Electrochemical potential
The sum of its conc. gradient and the charge gradient across the memb.
Faciliated diff - exergonic movement
Active transport - endergonic movement.
Neg. Vm
Most cells have a neg. membrane potential (Vm).
Excess of neg. solutes inside the cell.
Osmosis
Water tends to move from regions of lower solute conc. (higher free E) to regions of higher solute conc. (lower free E).
Direct A.T.
Accumulation of solute molecules or ions on one side of the memb. is coupled directly to an exergonic chem rxn, most commonly the hydrolysis of ATP.
Indirect A.T.
Simultaneous transport of 2 solutes.
One solute drives the unfavorable movement of other solute up its gradient.
Symport or antiport.
V type ATPase
H+ pumped into vesicles, Golgi
Keeps pH low activating hydrolytic enzymes
What organelles compose the endomembrane system of a eukaryotic cell?
Endoplasmic reticulum, Golgi complex, endosomes, lysosome (but NOT peroxisomes).
Endoplasmic Reticulum (ER)
A cont. network of flattened sacs, tubules, and associated vesicles that stretch throughout the cytoplasm.
50-90% total mammalian membrane.
Important for plasma memb. synthesis.
Synthesis of proteins for export from the cell.
Biosynthesis of lipids.
Rough ER
Ribosomes on cytosolic side.
Subdomain = transitional elements - shuttle lipids and proteins from the ER to Golgi.
Transitional elements
Subdomain of rough ER.
Important role in formation of transitional vesicles - shuttle lipids and proteins from ER to Golgi complex.
Resemble smooth ER.
Are the rough ER and smooth ER seperate organelles?
No.
Their lumenal spaces are cont.
Material can travel b/t the rough and smooth ER w/o vesicles.
ER cisternae
Membrane-bound sacs w/in the ER.
Enclose a space called the ER lumen.
ER lumen
Space enclosed by ER cisternae.
Of the total memb. in a mammalian cell, up to 50-90% surrounds the ER lumen
Rough ER functions
Ribosomes attached to cytosolic side synth. membr-bound and soluble proteins for the endomembrane system.
Initial steps of addition of carbohydrate groups to glycoproteins.
Folding of polypeptides, removal of misfolded polypeptides.
Smooth ER functions
Drug detoxification
Steroid biosynthesis
Carbohydrate metabolism
Calcium storage
Drug detoxification
Smooth ER
Rxn common to most pathways for drug detoxification and steroid biosynth. is hydroxylation.
Catalyzed by cytochromes P450 proteins.
Addition of hydroxyl groups to organic acceptor molecules making them more soluble and easier to excrete from the body.
Hydroxylation
Drug detox and steroid biosynth. in smooth ER.
Enzyme catalyzed addition of hydroxyl groups makes more soluble and easier to excrete.
Catalyzed by cytochrome P450 proteins.
e- transport system transfers e-'s from either reduced coenzymes NADPH or NADH to a cytochrome P450 protein.
This reduced P450 donates e- to O2.
Carbohydrate metabolism
Smooth ER function
Lots of glu.-6-phosphatase in hepatocyte smooth ER memb.
Liver important to maintaing blood glu. levels.
Enzymatic breakdown of stored glycogen
Carbohydrate metabolism rxn
glu.-6-phosphate + H2O --> glu. + Pi
Carbohydrate metabolism mechanism
When glu. needed by the body, liver glycogen is broken down by phosphorolysis producing glu.-6-phosphate.
Memb. impermeable to phosphorylated sugars so must be converted to free glu. by glu.-6-phosphatase.
Free glu. leaves the liver cell via GLUT-2.
Glu.-6-phosphatase activity present in liver, kidney, intestine, but NOT muscle or brain cells - retain glu.-6-phosphate for their own E needs.
Specializes in calcium storage
sarcoplasmic reticulum found in muscle cells.
type of smooth er
steroid biosynthesis (p.p)
smooth er
many pathway enzymes are p450's.
much smooth er in adrenal, leydig cells, liver, ovary, plastids in plants
carbohydrate metabolism (p.p)
much glu.-6-phosphatase in hepatocyte smooth er memb.
liver impt to maintaining blood glu. levels.
GLUT2 glu. transporters
steroid biosynthesis (book)
smooth er site of biosynth. of cholesterol and steroid hormones: cortisol, testosterone, estrogen.
Hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase) is committed step in cholesterol biosynth.
present in large amts in liver cells.
this enzyme targeted for inhibition by statin drugs.
p-450 monooxygenases impt in synth of cholesterol and its conversion to steroid hormones by hydroxylation
HMG-CoA reductase
hydroxymethylglutaryl-CoA reductase
committed step of steroid biosynthesis.
targeted for inhibition by statin drugs.
Calcium storage (p.p)
sarcoplasmic reticulum - specialized smooth er in muscle cells.
much ca-binding protein in lumen
atp dependent ca-ATPases pump ca into er - signals from neurotransmitter binding
Most membr. lipids and proteins synthesized where.
endoplasmic reticulum
Membrane biosynth (p.p)
most membr lipids and proteins synth. in er.
lipids produced and incorporated into inside layer of bilayer
phospholipid translocators (flippases) catalyze transverse diffusion.
particular enzymes for particular phospholipids - membr. asymmetry - cont. when er vesicles merge w/ plasma membr.
Membrane biosynthesis (book)
er is primary source for phospholipids and cholesterol.
biosynth and incorporation of membr phospholipid molecules restricted to monolayer of er membr facing the cytosol.
cellular membr are phospholipid bilayers - so phospholipid translocators, flippases, catalyze translocation of phospholipids thru er membr.
phospholipid translocators
also flippases
specific so type of phospholipid molecules transferred across a membr depends on translocator present - contributes to membr asymmetry.
cytosolic phosphlipid exchange proteins
movement of phospholipids from er to mitochondria, chloroplast, or peroxisome poses a problem - these organelles do not grow by fusion w/ er derived vesicles (endomembr organelles do)
so these proteins convey phospholipid molecules from er membr to outer mitochondrial membr.
specific
also contribute to movement to plasma membr.
golgi complex (p.p.)
linked w/ er
membr and protein trafficking
er glycoproteins further processed
er glycoproteins, membr lipids sorted and packaged for export
flattened, stacked sacs (cisternae)
surrounded by transport vesicles.
golgi complex (book)
endomembr organelle
central role in membr and protein trafficking in eukaryotic cells
series of flattened membr bounded cisternae - disc shaped sacs that are stacked together (3-8 sacs)
intracisternal space is golgi's lumen and part of endomembr system's network of internal spaces.
both er and golgi surrounded by numerous transport vesicles
golgi complex faces
each golgi stack has 2 distinct sides, or faces.
cis oriented toward er (CGN) - vesicles w/ newly synth. lipids and proteins from er fuse w/ CGN membr.
trans (TGN) - proteins and lipids leave in transport vesicles that cont. bud from tips of TGN cisternae, carry lipids and proteins to secretory granules, endosomes, lysosomes, plasma membr.
b/t CGN and TGN - medial cisternae where protein processing occurs.
medial cisternae of golgi complex
b/t cis golgi network and trans golgi network
much of protein processing occurs here.
medial cisternae, CGN,and TGN of golgi complex
biochemically and fucntionally distinct.
each compartment contains specific receptor proteins and enzymes necessary for specific steps in protein and membr processing
biochemical polarity
stationary cisternae model (p.p)
explains movement of lipids and proteins from cgn to tgn via medial cisternae of golgi.
shuttle vesicles move b/t areas
vesicles bud from cisterna, fuse w/ next cisterna, cis tot rans - molecules may remain in lumen
stationary cisternae model (book)
each compartment of golgi stack is a stable structure
trafficking b/t cisternae mediated by shuttle vesicles that bud from cisterna and fuse w/ next cisterna in cis to trans sequence.
proteins destined for TGN are carried forward by shuttle vesicles.
mol. that belong in er and golgi compartments are actively retained or retrieved.
cisternal maturation model (book)
golgi cisternae are transient compartments that gradually change from CGN cisternae thru medial cisternae to TGN cisternae.
transition vesicles from er converge to form CGN
cis cisterna transformed to med. cisterna then to trans cisterna as additional enzymes acquired.
two models that depict flow of lipids and proteins thru golgi complex
cisternal maturation model
stationary cisternae model
which model may depend on cell type
both models: membr lipids and proteins move er - golgi - plasma membr.
both models: TGN forms transport vesicles or secretory granules w/ cargo targeted for various destination beyond the golgi.
anterograde transport
the movement of material from er thru golgi complex toward plasma membr.
retrograde transport
flow of vesicles from golgi cisternae back toward the er
func. to balance the flow of lipids toward plama membr and to ensure a supply of components for forming new vesicles - cell recycles lipids and proteins no longer needed during the late stages of anterograde transport
protein glycosylation (p.p)
addition of CH chains to prot's in er, golgi
N-linked via asparagine
O linked via serine, threonine
protein glycosylation (book)
addition of carbohydrate side chains to specific a.a residues of prot's forming glycoproteins.
N-linked and O-linked glycoslyation
N-linked glycosylation (p.p)
cytosolic er membr surface
dolichol phosphate inserted into er membr.
2 GlcNAc added to dolichol PO4
Core oligosaccharide of 99 monosaccharides
Flippase moves growing CH chain to lumen
more mannose added, glu added - shuttled by dolichol phosphate
core oligosacch transferred to asp
modification - glucosidases, mannosidases
first Glc signals interaction of glycoprot w/ er prot's
proper folding sensed by er glucosyl transferase
terninal glycosylations in golgi
N-linked glycosylation (book)
dolichol phosphate inserted into er membr - core glycosylation
carb. groups added to po4 group of dolichol po4.
first 2 groups added are N-acetylglucosamine (GlcNAc)
Next, 5 mannose groups added.
Translocation of growing core oligosaccharide from cytosol to er lumen by flippase.
inside er lumen, dolichol po4 carriers bring mannose and glu units that are added to specific positions
completed core oligosaccharide is then transferred from dolichol to asparagine residue of recipient prot - catalyzed by oliosaccharyl transferase.
3 glu units and on mannose removed by glucosidases and mannosidases.
usually core oligosacch is added to prot as polypeptide is being synth by ribosome bound to er membr. - cotranslational glycosylation
calnexin and calreticulin promote disulfide bond formation forming ERp57.
further processing in golgi - prot move from cis thru medial to trans face of golgi stack - terminal glycosylations.
terminal glycosylation includes removal of a few carb units of core oligosacch.
initial steps of n glycosylation take place where
cytosolic side of er membr.
glycosylation (book 12-6)
biosynth of core oligosacch for n linked glycosylation of certain asparagine residues
initial processing of core oligosacch.
identification and removal of misfolded proteins.
attachment of n-acetylgalactosamine to serine or threonine.
1st step of phosphorylationo f lysosomal prot's
removal of mannose
2nd step of phosphorylation of lysosomal prot's
removal of mannose and attachment of n-acetylglucosamine
addition of galactose and sialic acid
addition of sialic acid and attachment of sulfate to tyrosine
protein trafficking (p.p)
prot's from er must go to correct site, stay there or be excreted - targeting tags on prot's, tags on lipids of vesicle membr's
sorting - early in er, cgn, med. cisternae, later sorting in tgn (packaging)
prot's impt to remain in er
prot's impt to remain in golgi may have tags, may form large complexes (can't escape)
protein trafficking (book)
once a prot reaches an organelles where it is to remain, must be mechanism to prevent it from leaving.
each prot contains a specific tag - a.a sequence, oligosacch. side chain, hydrophobic domain
memb lipids may also be tagged to help vesicles reach proper destination - attach po4 groups to 3, 4, or 5 of memb phosphatidylinositol (PI) mol by specific kinase
Overview of protein trafficking
Prot's synth by ribosomes attached to cytosolic surface of rough er - initial glycosylation occur in er lumen
transition vesicles carry newly synth lipids and glycosylated prot's to CGN
Lipids and prot's move thru cisternae to TGN
At TGN, ves. bud of to form secretory ves or endosomes
secretory ves. move to plasma memb
prot's taken into cell by endocytosis form endocytic ves that fuse w/ early endosomes.
components not destined for digestion after endocytosis are recycles to plasma memb.
endosomes w/ material for digestion mature to form late endosomes then lysosomes
retrograde traffic returns prot's to earlier compartments
Protein glycosylation
addition of CH side chains to specific a.a. residues of prot's

strict dependence on each step in previous modification
N-linked glycosylation
via asparagine
O-linked glycosylation
via serine, threonine
oligosaccharyl transferase
catalyzes the transfer of a complete core oligosaccharide to asparagine of recipient prot.
catalyzes the transfer of a complete core oligosaccharide to asparagine of recipient prot
oligosaccharyl transferase
how many glu. and how many mannose removed when core oligo is being trimmed and modified? what enzymes perform this func?
3 glu
1 mannose
glucosidases and mannosidases
when is core oligosaccharide added to a prot?
when the polypeptide is being synthesized by a ribosome bound to the er membr.

cotranslational glycosylation
cotranslational glycosylation
core oligo usually added to prot as the polypeptide is being synthesized by a ribosome bound to the er membr.

promotes proper prot folding
what does cotranslational glycosylation help do
promote proper prot folding
proper folding of a newly synth glycoprot is sensed by what enzyme?
er glucosyl transferase
ERAD
er-associated degradation - if a improperly folded glycoprot is not corrected
if glycoprot not properly folded, what can happen to correct it?
add glu
promotes disulfide bond formation
calnexin
calreticulin
catalyzes disulfide bond formation
ERp57
ERp57
catalyzes disulfide bond formation
terminal glycosylations occur where?
golgi
microtubules (MT)
largest structures
2 types: cytoplasmic, axonemal
2 types of microtubules
cytoplasmic
axonemal
cytoplasmic microtubules
maintain polarized shape
orientation of cellulose microfibrils in plants
form mitotic and meiotic spindles
axonemal microtubules
highly organized
found in cilia, flagella - struct's impt to cell movement
microtubule structure
straight hollow cylinders
protofilaments organized longitudinally.
13 around lumen is most common
basic subunit of protofilaments
heterodimer of tubulin prot
alpha/beta tubulins
have GTP binding site at N-terminus
domain at C-terminus which interacts with MAP's
all tubulin dimers oriented in same direction - gives polarity
why are tubulin dimers oriented in the same direction
gives polarity
how are microtubules formed?
reversible polymerization of tubulin dimers
microtubule formation
reversible polymerization of tubulin dimers
requires GTP, Mg++
aggregation of tubulin dimers into clusters = oligomers
oligomers serve as nuclei from which new MT's can grow = nucleation
then elongation - addition of subunits at either end.
What is needed for microtubule formation?
GTP, Mg++
importance of GTP in microtubule formation
each tubulin heterodimer binds 2 GTP mol's.
forms GTP cap - protects MT from subunits coming off plus end, provides a tip to which further dimers can be added.
hydrolysis of GTP by beta tubulin results in unstable tip
in microtubule formation, what hydrolysis of GTP result in?
unstable tip which can lead to depolymerization

GTP - GDP - unstable tip - depolymerization
what hydrolyzes GTP in MT formation?
beta tubulin
microtubule originate from where?
microtubule-organizing center (MTOC)

regulated, ordered construction
microtubule organizing center (MTOC)
MT's originate from here
anchors one end of MT

centrosome acts as MTOC during cell division (interphase) - near nucleus w/ 2 centrioles associated.
centrioles
symmetrical - walls formed by 9 pairs of triplet MT's
Impt to formation of basal bodies
basal bodies impt for formation of cilia, flagella
NOT impt in plant cells
most impt role of MTOC
its ability to nucleate and anchor MT's
how does MTOC limit number of MT"s
it has a limited number of nucleation and anchorage sites
microtubule associated prot's (MAP's)
bind at regular intervals along MT wall - allows for interaction w/ filaments and other cellular struct.
incr MT stability and affect MT bundle density
impt in brain - MT bundels denser in axons
neurofibrillary tangles
neurofibrillary tangles
from dysfunction of MAP's
dense tangles of neurites
can lead to alzheimers, palsy.
microfilaments (MF's)
smallest of cytoskeletal filaments
impt to movement - cell migration, amoeboid movement
produce cleavage furrows that divides the cytoplasm of animal cells
help maintain cell shape and development
cell cortex
structural core of microvilli
cell cortex
dense network of MF's
form structural core of microvilli
microfilaments
actin
once synthesized, folds into U shaped mol w/ central cavity that binds ATP or ADP.
individual actin mol's
G-actin (globular actin)
G-actin (globular actin)
individual actin mol's

G-actin polymerized to form MF's - now F-actin
G-actin polymerizes into what?
microfilaments of F-actin
G-actin monomers polymerize reversibly
why is polarity impt in MF's
allows for independent regulation of actin assembly and disassembly at either end of the filament.
microfilament structure
ends of growing MF have ATP-F-actin
but bulk of MF has ADP-F-actin
receptor
cellular struct, usually a prot, that receives a signal from another cell
embedded in cell membr
cytosolic or nuclear
ligand
mol that binds cellular receptor
signal transduction
cell's translation of receptor-ligand interaction
changes in cell behavior or gene expression
second messenger
cellular mol's produced when receptor/ligand bind
01: In what step does most eukaryotic regulation take place?
TRANSCRIPTION
cell growth is generally accompanied by:
cell division
02: If both an activator and repressor are working, which usually wins out?
the repressor
M phase
Point in cell cycle when the cell actually divides

involves 2 overlapping events in which the nucleus divides 1st and the cytoplasm 2nd
03: Where does RNA Pol bind?
the promoter
Phase in cell cycle when the cell actually divides.
M phase
04: How is the eukaryotic regulatory region different from prokaryotes?
it is often very far (thousands of BP) away from the promoter
growth phase b/t divisions
interphase
05: How does a regulatory specify which gene it works on?
BOUNDARY ELEMENTS (aka INSULATORS): regions that define where a gene is. will have proteins bound to them
interphase
growth phase b/t cell divisions

most cellular contents are synthesized cont. during interphase = incr in cell mass
06: Does transcription in eukaryotes usually require one regulatory element?
NO: usually needs lots to initiate transcription
S phase
specific portion of interphase

new nuclear DNA is synthesized
07: What helps us learn about gene eukaryotic regulation? One example
REPORTER GENE:
a gene whose activity is easily measured.

ie β Galactosidase:
can add a protein which will change color depending on how much of it is available.

if you put in a regulatory region of another gene that will instead act on your reporter, you can see where those genes act.
seperates S phase from preceding M phase
G1 phase
08: What is the structure of eukaryotic regulatory proteins?
often DIMERS:

have separate BINDING and ACTIVATION domains
seperates the end of S phase from the onset of the next M phase.
G2 phase
09: How can we have variation in eukaryotic regulatory proteins?
HOMODIMERS: same dimers.
HETERODIMERS: different dimers together, gives larger possibilities for variation
overall length of cell cycle
generation time
10: Where do eukaryotic regulatory proteins bind?
ACTIVATING SEQUENCES: can be upstream or downstream. can have multiple binding sites
nuclear DNA doubles in what phase?
S phase of interphase
11: what did we learn from the DOMAIN SWAP EXPERIMENT?
It showed us that both the activating and binding regions of the regulatory protein are needed.

Activating regions can bind to other binding regions to activate the binding region's gene.
phase in which a "decision" is made as to whether the cell is to divide again
G1 phase
12: What are some binding motifs?
HOMEODOMAIN: single subunit...esp helix-turn-helix

ZINC FINGERS: charged, will interact w/ negative DNA backbone

LEUCINE ZIPPER: dimer. each part could have different binding domains.

HELIX-LOOP-HELIX
cells that become arrested in G1 phase, awaiting a signal that will trigger reentry into the cell cyle
G0 phase
13: What is a common mechanism for activating proteins? What is the significance?
activator brings in MEDIATOR: holding onto RNA Pol via many NON-SPECIFIC SEQUENCES...means that it can work with several different activators.

mediators are often positively charged
G0 phase
cells that become arrested in G1 phase, awaiting a signal that will trigger reentry into the cell cycle and a committment to divide.
14: Is there a holoenzyme for eukaryotic activation?
it is debatable, we think it only forms in vitro.
terminal differentiation
cells exit from cell cycle entirely, destined to never divide again.

most nerve cells are in this state.
15: What is one way the mediator can act?
can push the LexA protein (bound to Rna Pol) onto lexA site on DNA
semiconservative replication
half of parent mol is retained by each daughter mol
16: What is a very important overall eukaryotic activation/repression mechanism? How does it act?
NUCLEOSOME REMODELING:

activator protein binds, brings in acetyl transferase, expands chromatin to 10nm fiber

OR:
activator recruits chromatin remodeling complex, moves DNA around histones
replication of circular DNA
begins at a single origin
proceeds bidirectionally around the circle w/ 2 replication forks moving in opp. directions.
process generates intermediates that resemble theta = theta replication
membr growth b/t attachment sites of 2 replicating copies move daughter chromosomes toward opp sides of cell.
17: How do boundary elements work?
(aka insulator region)
they act like a FENCE: stops enhancer from turning on a promoter of the wrong gene.

it can also act as a boundary for CHROMATIN MODIFYING: will stop it from going further down the chromatin
replicons
multiple replication units
DNA of eukaryotic chromosomes is initiated at these multiple sites.
maybe thousands
18: what is an LCR?
LOCUS CONTROL REGION: lets you turn on access to a number of genes.

ie β Globulin: you have several different types. LCR turns on access to all the genes, which can then be individually activated.

like turning on the power to the entire building, then flipping switches in individual rooms.
origin of replication
special DNA sequence at the center of each replicon.

where DNA synthesis is initiated by a mechanism involving several groups of initiator prot's.
19: Are genes usually on or off?
OFF: most need an activator to turn them on since they have a poor promoter
ensures only one copy of each chromosome
licensing
20: what is the importance of SIGNAL INTEGRATION?
there are many different activators and repressors all acting at once.

the summation of their effects together is often much more powerful than summing them individually.

you can also have one protein act as both a positive and negative signal, depending on concentration gradient.
cyclin-dependent kinase (Cdk)
functions both in activating DNA synth. at licensed origins and in ensuring these same origins can't become licensed again.
21: basic mechanisms of cooperative binding(4)
1: A+B need each other to work as activators

2: A+B need to bind to a third molecule in between them to act as activators

3: A works, blocking B, but then brings in a chromatin modifier so that B can bind.

4: A binds, unwinding DNA so that B can bind.
geminin
prot that inhibits relicensing.

blocks binding of MCM prot's to DNA.
22: how is the HO gene controlled?
three molecules of SWI5 bind to sites

allows CHROMATIN REMODELERS to bind

makes the SBF site available, 3 molecules SBF bind to activate gene
Catalyzed DNA elongation
DNA polymerase
23: How is the human interferon protein regulated?
HMG proteins bend the DNA in a "U" shape so that other proteins can bind.

ENCHANCEOSOME complex forms with several proteins, all of which are needed to turn on the gene.

this complex forms in many organisms
joins DNA fragments
DNA ligase
24: What is COMBINATORIAL CONTROL?
one protein regulating many genes in combination with other proteins (ie CAP)

ie one gene needs activators 1, 2, 3, 4

another gene needs 3, 5, 6
strand synthesized as a cont. chain in the 5'-3' direction
leading strand
25: are most activators specific?
some are TISSUE SPECIFIC,

most are NOT gene specific
lagging strand
must grow in the 3'-5' direction
formed as a series of short, discontinuous Okazaki fragments that are synth. 5'-3' b/c DNA polymerase can't add nucleotides in 3'-5' direction.
fragments joined by DNA ligase to make a cont. new 3'-5' strand.
26: What is an example of gene control in S. Cervisae (yeast)
mostly haploid, but when stressed will become diploid

MCM1 protein acts as an activator in certain cells, repressor in others, depending on the cell protein that it binds to.
Primase
synth. RNA fragments a/b 10 bases long using DNA as a template.

can initiate RNA synth from scratch by joining 2 nucleotides together.
27: ways repressors work (4)
COMPETITION: both trying to bind to overlapping sites

INHIBITION: both binds, but repressor occludes the activator's activating site

DIRECT REPRESSION: repressor interacts w/ mediator to stop transcription

INDIRECT REPRESSION: interacts w/ deacetylase to transform chromatin to 30nm fiber
Primers
RNA primers needed to initiate DNA synth.
synth'd by primase
28: What is an example of yeast genetic repression?
Gal1 gene:

Mig1 binds to Mig1 site.
Tup1 binds to Mig1 protein, turns off gene.

overrides UAS which promotes gene
DNA helicase
prot responsible for unwinding DNA.
uses E derived from ATP hydrolysis
29: What is a second Yeast repressor?
Gal80, occludes Gal4 activator dimer.
binds to activator when no galactose.
topoisomerases
relieves supercoiling
DNA gyrase=type II topoisomerase (cuts both DNA strands)
30: How can we silence genes at the chromatin level?
esp a TELOMERE:

RAP protein binds to telomere
SIR2 binds to it, then SIR 3, 4, which bind to chromatin across it

keeps telomeres condensed always.

it becomes HETEROCHROMATIN
single-stranded DNA binding prots (SSB)
keep DNA strand separated
31: What is a long term way to silence genes?
DNA METHYLATION: blocks activation in the long term.

proteins bind methylated DNA which blocks transcription oven more.
Central Dogma of Molecular Biology
the concept of directional flow of genetic information

Francis Crick
32: What is another way DNA methylation could act?
could block a BOUNDARY ELEMENT SITE: then an enhancer will act across it and turn on the wrong gene.
Transcription
synth. of an RNA mol using DNA as a template
33: when do you use the CHIP ASSAY?
to amplify sections of DNA that a certain protein binds to

can do it without knowing the sequences
mRNA
RNA that is translated into a prot
34: How does the CHIP assay work?
purify DNA (or chromatin) and shear it into fragments. can use entire chromosome

then add a binding protein, and use an antibody against it.

antibody will make a large chain from the protein, which will precipitate in solution. skim rest off, centrifuge to form a pellet.

you can remove the proteins and antibodies, and clone or PCR the DNA fragments to have pieces of all the genes that the binding protein binds to.
RNA that is translated into a protein:
mRNA
Exceptions to Central Dogma
Retroviruses - "backward" flow of genetic info, Ex. HIV

RNA viruses - RNA genome is template for RNA synth., Ex. influenza, hepatitis C
The genetic code is dependant upon:
the relationship b/t:

DNA nucleotide sequence.
linear order of a.a.'s in prot. mol.
triplet code
3 base pairs in double-stranded DNA required to specify each a.a. in a polypeptide.
gene amplification
multiple copies of same gene.

by repl. of DNA in specific chromosomal region
Genomic control mechanisms
gene amplification
gene deletion
gene rearrangement
DNA methylation
5 main levels gene expression is regulated:
the genome
transcription
RNA processing and export from nucleus to cytoplasm.
translation
post-translational events
why can gene amplification be regarded as an example of genomic control?
it is a regulatory change in the make-up of structural organization of the genome.
genomic control by gene amplification
creates mult. copies of same gene.
gene deletion
a means of genomic control

some cells delete genes whose products are not required.
occurs in RBC's - discard entire nuclei after sufficient hemoglobin mRNA has been made.
DNA rearrangement
movement of DNA segments from one location to another w/in the genome.
used by lymphocytes to prod. antibody mol's
antibodies
prot's composed of 2 polypeptide subunits - heavy chain and light chain
lymphocyte DNA rearrangement
lymphocytes use small # of DNA segments and rearrange them in various combinations

V, J, D, C DNA segments
4 DNA sequences used in lymphocyte rearrangement
V, J, D, C segments
DNase 1
an endonuclease from pancreas.

degrades transcriptionally active DNA in chromatin - evidence that DNA is uncoiled.
DNA methylation
genomic control
addition of methyl groups to selected cytosine bases in DNA
usually at promoter region (5').
DNA methylation at promoter regions can have what effects?
can block acces of prot's required for transcription activation.
can serve as binding sites for prot's that condense chromatin to an inactive configuration - no gene expression
histones H3, H4
acetylation of these histones is associated w/ gene activation
Transcriptionally active chromatin often lacks what histone and why:
histone H1 - keeps chromosome uncoiled.
Flow of genetic information
The nucleus
Location of
Chromosomes
Replication
Transcription
Double-membrane structure
Nuclear envelope
Perinuclear space between inner, outer membranes
outer nuclear membrane
Continuous w/ ER
Perinuclear space continuous w/ ER lumen
Ribosomes
Cytoskeleton anchoring proteins bind actin, intermediate filaments
Movement
Nucleoplasmic reticulum – projections
Contacts inner membrane
nuclear pores
Connect cytosol w/ nucleoplasm
Specialized channels
3000-4000
Fuse inner/outer membranes
nuclear pore complex
Impt to transport
Need enzymes, proteins, precursors for replication/ transcription
Move mRNA, ribosomal subunits to cytosol
nucleoporin protein complexes
Octagonal symmetry
Protrude into cytosol and nucleoplasm
Wheel-like structure
<30,000 MW mol’s move between “spokes of wheel”
Center = “transporter”
transport through nuclear pores
Active
Energy
Specific binding to pore complex proteins
Nuclear localization signals (NLS) on protein moving into nucleus
8-10 aa sequences (incl proline, lysine, arginine)
Recognition
Nuclear export signals (NES) on mol’s moving out of nucleus
Aa sequences
importin
binds NLS

Shuttles/chaperones protein through center of NPC
Releases protein as assoc’s w/ GTP-binding protein (Ran-GTP) in nucleoplasm
Importin-GTP-binding prot complex shuttled back through NPC  cytosol
Recycled
exportin
similar protein needed to export mol’s out of nucleus
Impt mostly for RNA movement
Also shuttles through center of NPC
Uses same GTP-binding protein (Ran-GTP)
import/export cycle (image)
Role of Ran-GTP in import/export cycle
Ran-GTP concent gradient impt: [Ran-GTP] higher inside nucleus than in cytosol
Guanine nucleotide Exchange Factor in nucleus
Encourages incr’d Ran-GTP over Ran-GDP
GTPase Activated Protein in cytosol
Encourages hydrolysis Ran-GTP  Ran-GDP
Equilibrium w/ high [Ran-GTP] in nucleus
Promotes release of Importin from imported prot’s inside nucleus
Promotes binding of Exportin to RNA to be exported from nucleus
nuclear matrix
Protein network shapes nucleus
Organizing skeleton for nucleic acids (?)
May anchor DNA/RNA during synthesis
nuclear lamina
Fibers lining inner membrane
Intermediate filaments (lamins)
Mutations give rise to diseases
Nuclear shape distortions
nucleolus
Ribosome factory
One or several  1000’s
Varied sizes, shapes; usually rel large
Size dependent on activity prot synthesis
Contains DNA w/ genes coding for rRNA
DNA Nucleolus Organizer Regions
Higher organisms have 100s-1000s NOR copies
Contains fibriles
DNA being transcribed  rRNA
Contains granules
rRNA packaging w/ proteins  ribosomal subunits
Many in cells w/ high prot synth activity
Some additional activities
Nuclear transport
Chem mod’n RNA
Cell division control
DNA replication
Accuracy  duplicate copies to daughter cells

Purine/pyrimidine pairing fits physical dimensions
Strands held by H-bonds
Allows separation
Complementarity allows strand sequence determined by opposite strand
Template
Right handed helix (B-DNA)
Left-handed helix can form (Z-DNA)
Antiparallel strands
double helix model
Watson/Crick, 1952
Repeating phosphate/deoxyribose units
Nitrogenous base attached to each sugar
A/T, G/C could hydrogen bond
Franklin’s X-ray diffraction showed repeating structures
“Circular staircase”
major groove
exposes H, O, N (all can form H bonds) and T’s methyl
Regulatory proteins can bind
supercoiling
Studied in closed, circular DNA
Bacteria, viruses, mitochondria, chloroplasts
Linear DNA when anchored, cannot freely rotate
Compacted chromosomes
During replication, transcription
Impt to interaction w/ other molecules
topisomerase
Topoisomerases – enzymes catalyzes supercoiled  relaxed DNA forms
Type I introduce ss breaks, Type II ds breaks
DNA strands pass through break  unwinding
Type II requires ATP
Resealing also catalyzed by topoisomerase
DNA gyrase
DNA gyrase – bacterial
Can relieve supercoiling AND induce supercoiling
Impt to replication
DNA denaturation
Strand separation
H-bonds relatively weak, rel easily broken w/ incr’d heat, pH
What holds each strand together??
Use UV absorbance to monitor strand sep’n
Ss DNA has higher absorbance
Tm = DNA melting temp = temp @ which ½ abs change found
DNA melting temperature
Tm depends on base composition
3 H-bonds bind GC base pairs
2 H-bonds bind AT base pairs
Which base pair requires more heat to separate?
Incr directly proportional to # GC bp’s
Proper base pairing also  incr’d Tm
DNA renaturation
Renaturation w/ lowering temp
Use UV abs to determine hybridization ability
Tells complementarity of strands
Can use probes to locate genes
DNA-DNA
DNA-RNA
RNA-RNA
genome
DNA w/ one complete copy of all genetic info of that organism
Prokaryotes, viruses – one or few linear or circular DNA mol(s)
Eukaryotes – nuclear, mitochondrial and chloroplast genomes
Mitoch, chloroplast – single, circular (usually)
Nuclear – multiple DNA mol’s
bioinformatics
Impt to locating genes w/in genome, determining which proteins genes code for
Much info needs computer analyses
Book: <2% human genome codes for proteins; unclear function of rest
transcriptomes
Transcriptomes – entire set of RNA mol’s prod’d by genome
Products of transcription
DNA microarrays use cDNA from RNA, then DNA-DNA hybridization
proteomes
Proteomes – all proteins produced by a genome
More complex than genome
Book: human 25,000 genes  100,000’s proteins
Alternative splicing
Biochem modifications
Protein microarrays used to study chem properties of proteins
human genome
Human genome project studied genomes of 10 people
All humans’ genomes 99.7% identical
0.3% variable ~ 3.2 Gb
SNPs
CNVs
single nucleotide polymorphisms
Single Nucleotide Polymorphisms (SNPs) – single base changes w/in genome
~1x107 SNPs explain variability
Some understood due to known diseases/ dysfunctions
Most not in coding regions
Many related to each other
When on same chromosome, inherited in sections (haplotypes)
So easier to locate several at once
copy number variations
Copy Number Variations (CNV’s)
Also impt to variability in genome
DNA rearrangements, deletion, duplications
Involve 100’s of Gb’s as segments of Kb’s

Useful medical info, but difficult ethically
tandem repeated DNA
Multiple copies arranged next to each other
10-15% mammalian genome
Varies in length of repeat, # times repeated in succession
Length 1-2000 bp; usually simple sequence (<10 bp)
“Satellite” DNA

Much of centromeres, telomeres
Book: human telomeres 250-1500 copies TTAGGG; highly conserved among vertebrates
Small satellite DNA used for DNA fingerprinting
Excessive repeats found in diseases
interspersed repeated DNA
Scattered throughout genome
100’s-1000’s bps length per unit x 100’s-1000’s of copies
Transposons (transposable elements) move around
Some w/ genes coding for enzymes that copying, insert these sequences elsewhere
Believed impt for evolutionary adaptation
May be impt in gene regulation
bacterial chromosome
Commonly single, circular molecule
Nucleoid region
Anchored to RNA, protein
DNA wrapped around basic proteins  bead-like packets
Packets arranged in loops held by proteins
Loops supercoiled in nucleoid
bacterial plasmids
Small, circular DNA
Mostly nonessential genes + genes for plasmid repl’n
Conjugation
Resistance to antibiotics
Secr’n antibiotics
Secr’n toxic proteins
Metabolic rxns
Supercoiled
linker DNA
~146 bp
Histone H1 assoc’d w/ linker
looped domains
30-nm fibers folded
Protein scaffold assists
Active DNA less tightly packed, more accessible in loops
euchromatin
transcriptionally active DNA (less packed)
Includes most DNA in metabolically active cells
Compaction ~750x
heterochromatin
tightly packed (~15,000x compaction)
Not active transcriptionally
All chromatin in dividing cells is heterochromatin
organellar DNA
Mitochondria, chloroplasts
Also have enzymes, proteins for repl’n, transcription, translation
Semiautonomous (nuclear genome needed)
Symbiotic origin?
No histones
Circular, relatively small
>90% apparently noncoding
cell growth
Macromolecule synthesis
Increased cell volume  decr’d surface/vol ratio  ineffective exchange ability
Cell division accompanies cell growth
Increases cell # in organism OR
Replaces dead/injured cells
Daughter cells are genetic duplicates of parent
REMEMBER: somatic cells
M phase of cell growth
Mitosis – nuclear division
Cytokinesis – cytoplasmic division
Chromatin condensed  chromosomes
DNA has replicated, so 2 duplicate chromosomes (sister chromatids)
Stay together til cell divides
Nuclear envelope dissolves
Mitotic spindles guide chromosomes to poles
What type of structure are mitotic spindles??
interphase
Rest of cell cycle; cell growth
Continuous synthesis of macromolecules
S phase – nuclear DNA doubles
6-8 hours
G1, G2 phases – time gaps
G1 8-10 hours
G2 4-6 hours
G0 – temporary arrest from cell growth
Await signals to reenter cell cycle
(M phase 30-45 mins)
eukaryotic replication
more complex
Larger genome; takes longer
Nucleosomes must be unwound
Multiple origin sites  multiple replicons
Center = origin of replication
AT rich (why??)
Initiator proteins bind DNA, unwind helix
Prereplication complex  “licensing” repl’n
Two replication forks synth DNA in opp directions  replication bubble
Bubbles merge
licensing
ensures only one copy of each chromosome
Occurs through initiator proteins (particularly MCM proteins)
Requires all proteins be present
MCM proteins prevented from re-binding same origination site
Cyclin-dependent kinase (Cdk) prod’d at beginning of S phase
Phosphorylates intiator proteins  inhib’n of those proteins from re-binding
Geminin made during S-phase
Blocks binding of MCM prot’s to DNA
DNA polymerase
Catalyzes DNA elongation
Energy released as NTP’s inc’d (dNMPn + dNTP  dNMPn+1 + PPi)
Nucleotides added to 3’ –OH end of chain as phosphodiester bond between PO4 at 5’ C of dNTP and 3’ –OH of chain
Growth is 5’ 3’
primers
RNA found to be needed to initiate DNA synthesis
DNA polymerase can only add dNTP’s to established chain (can’t start new chain)
Short RNA segments found @ initiation site
Primase synthesizes
Uses DNA as template
Primosome in E. coli – primase + 6 proteins
Recognize origination site, unwind helix
primase
Primase in eukaryotes closely assoc’d w/ DNA polymerase
Leading strand needs 1 primer
Lagging strand needs 1 primer for each Okazaki fragment
Polymerase adds nucleotides til reaches next primer
Primers later cleaved, dNTP’s fill in
E. coli – DNA polymerase does both
Ligase connects sections
Best method for minimizing errors
“Accessory” Enzymes Needed for Replication
DNA helicases
Unwinding; H-bonds broken
Where are the H-bonds??
Unwinding  supercoiling
ATP nec
E. coli – part of primosome
Topoisomerases
Relieves supercoils
DNA gyrase – impt in E. coli; Type II
Single-strand Binding Protein (SSB)
Keep DNA strands separated
DNA helicase
Unwinding; H-bonds broken
Where are the H-bonds??
Unwinding  supercoiling
ATP nec
E. coli – part of primosome
single stranded binding prot
Keep DNA strands separated
telomeres
Short repeating units at ends of each linear chromosome
TTAGGG
Humans 100-1500 copies
Noncoding
No coding info lost w/ repl’n

Telomerase catalyzes add’l copies of telomeres

Telomeres protected by telomere capping proteins
Bind 3’ end of DNA
Loops back, base pairs w/ opp DNA strand
telomerase
Telomerase found in germ cells, actively proliferating cells
Other cells don’t have telomerase
Telomere shortening  DNA coding region shortening
Signals apoptosis
May be impt to degenerative diseases w/ aging
Telomerase dysfunction leads to similar diseases, premature aging
Telomerase found in cancer cells
Highly proliferative
cell cycle regulation
General cell cycle progression over ~24 h
G1, S, G2, M
Varied overall length, stage length, mitosis/cytokinesis pairing
Regulated to meet cell, organism needs
Cancer cell research
cell cycle length
Stem cells, sperm cells constantly divide
~8 hours
Where are adult stem cells??
Some cells never divide (example??)
Liver cells, lymphocytes divide only when stimulated
Most time variation is in G1
Non-dividing cells  G0
Rapidly dividing may skip G1
speed G1 phase
Increase number of replicons  shortened time for each synthesis event
What is a replicon??
Large eggs w/ much cytoplasm  little time needed to replicate cytoplasm contents
TOR
prot kinase
decision points in cell cycle
Cell must check for
Appropriate sequence, time for each mechanism
Completion of phase before next is started
Environmental conditions or signals
Cell uses biochemicals at particular stages
Late G1, late G2, during M (metaphase/ anaphase)
decision points in cell cycle graph
late G1 control point
Time varies among cell types/species
G1 inhibited to  S w/ decr’d nutrients, space or w/ inhibitors
Restriction point
Presence of growth factors impt
 S phase (committed), OR
 G0
Need signal to  G1
late G2 control point
Arrest here possible for long periods
Less common than at G1
Important factors
Cell size
DNA viability
Sufficient DNA
metaphase/anaphase control point
Movement of 2 chromosome sets  2 poles
Spindle attachment crucial
May lag for “catch-up”
cyclin-dependent kinsases (Cdk's)
Cytoplasmic molecules
Kinases commonly regulate protein activity
Phosphatases must also be present (why??)
Cyclins
Proteins that must be present
Activate kinases that regulate other proteins impt to cell cycle progression
So kinases are Cdk’s
Get Cdk-cyclin complexes
Cyclin” refers to “cycle”
Concentration in cell incr’s, decr’s at different stages of cell cycle
G1 cyclins bind G1 Cdks
Mitotic cyclins bind mitotic Cdks
S cyclins impt for DNA repl’n
Mitotic cyclin/Cdk complex
mitotic cyclin/Cdk complex
Maturation (Mitosis) Promoting Factor (MPF)
Triggers onset of mitosis
Cyclin/Cdk’s must themselves be regulated to function at appropriate time

Mitotic Cdk always present during all cycle stages
BUT kinase activity only when bound to mitotic cyclin
Mitotic cyclin concentration low, increasing over G1, S, G2 until critical concentration at end G2
Now Mitotic Cyclin/Cdk complex  mitosis
activation of Cdk
For activity, Cdk must be specifically phosphorylated
Inhibiting kinases add 2 PO4 grps
Activating kinase adds 1 PO4 grp
Phosphatase removes 2 inhibiting PO4 grps
Now get positive feedback loop
Activated mitotic Cdk stimulates phosphatase
Get more PO4 removal  more activated mitotic Cdk  more stimulated phosphatase
Overall, more rapid activation
Now protein kinase subunit active to trigger mitosis
Nuclear envelope effect
Phosph’n lamin proteins of nuclear lamina, and other prot’s assoc’d w/ envelope
 depolymerization of lamins
 breakdown of nuclear lamina
 nuclear envelope destabilized
condensation of chromatin fibers
Activated by phosphorylation of condensin
 condensin binding to DNA
 supercoiling
 compact chromosomes
Assembly of mitotic spindle
Phosphorylation of microtubule-assoc’d proteins
active mitotic-cyclin-Cdk activates:
nuclear envelope breakdown, chromosome condensation, mitotic spindle formation, targeted protein degradation
G1 Cyclin/Cdks
Regulates progression to S phase
Nutrients
Growth factors
Cell size
Progression  commitment to cell division
Signals  activation G1 Cdk/Cyclin  phosph’n/activation proteins

Rb protein binds, inhibits E2F transcription factor
 E2F inactivated
 Genes not transcribed
Genes code for proteins needed for DNA replication initiation
Cells don’t replicate DNA, don’t divide
Growth factor signal  2nd messenger pathway  G1 Cdk/Cyclin activation
 Phosph’n Rb protein
 Releases E2F
 Genes transcribed
 DNA repl’n, cell division
mitotic spindle checkpoint
Can halt cell cycle if improper conditions
Mitotic spindle checkpoint
Check kinetochore attachment to microtubules
If not attached, signal inhibits anaphase-promoting complex
 Cohesion proteins not degraded
 Sister chromatids held together
DNA replication checkpoint
Checks for complete DNA synthesis before mitosis
Proteins impt to repl’n inhibit phosphatase
 no final dephosphorylation Cdk-Cyclin
 Cdk-Cyclin not activated
 No progression beyond G2 phase
DNA damage checkpoints
Checks for viability of DNA before cell division
Altered DNA  activation ATM protein kinase
 Phosphorylation p53 protein
 Protection from degradation  p53 builds up in cell
 Activation two pathways
Pathway 1 codes for p21 protein
Inhibits Cdk-Cyclin activity
 Halts cell cycle
Pathway 2 codes for Puma protein
Binds apoptosis-inhibiting proteins
 Apoptosis signalled
Cell cycle control
Autonomous clock
Fixed cycle repeats
Due to rhythmic synthesis, degradation of cyclins that bind Cdks
Cyclin/Cdk complex activate cell cycle progression
External, internal cues interrupt, regulate cycle when needed
Additional proteins influence Cdk’s, cyclins
Phosphatases, kinases
stimulatory growth factors
Some very specific, some signal many cell types
Only cells w/ receptors for particular factor will respond
Impt during embryo development, tissue damage/wound repair
Platelet-Derived Growth Factor (PDGF), Epidermal Growth Factor (EGF)
growth factors commonly bind what type of receptor?
Growth factors commonly bind tyrosine kinase receptors
Binding  receptor autophosphorylation  phosphorylation of second messengers  signal transduction cascade
signal transdution cascade
Signal transduction cascade: phosphorylation events include
Raf
MEK
MAPK’s
Mitogen Activated Protein Kinases
inhibitory growth factors
Ex: Transforming Growth Factor b (TGFb)
May sometimes be stimulatory (cell dependent)
Antagonizes EGF, PDGF stimulating effects
Work through yet another cascade
Smad proteins  nucleus  regulation of gene expression
Activates gene expression coding for proteins that inhibit cell division
the genetic code
Set of rules governing gene expression
Dependent upon relationship between
DNA nucleotide sequence and
Linear order of amino acids in protein molecule
Genetic Code: A Triplet Code

Deciphering this “code”-- major scientific breakthrough
When synthesizing the polypeptide chain, mRNA is read as 3 base units—codons
Each amino acid (aa) coded by one or more triplet codes
properties of the genetic code
Unambiguous
Degenerate
Nonoverlapping
Almost universal
code vs codon
CODE = genetic code expressed in DNA language
Bases A, T, C, G
CODES are written 3' to 5' as they appear in the DNA template strand
CODON = genetic code expressed in mRNA language
Bases A, U, C, G
CODONS are complementary and antiparallel to genetic code of DNA from the template strand
Actual coding units read by protein synthesis mechanism
bacterial RNA polymerase
Very well characterized (480,000 Da)
Multiple subunits:
Core enzyme (2α + 1β + 1β’ subunits) + 1 sigma factor
“Core" + sigma = holoenzyme
Sigma ensures that RNA synthesis initiates at right place on DNA (determines correct reading frame; core only will start at random)
Sigma factor readily dissociates
Transcription Factor Binding of RNA Pol II
6 general transcription factors (TF) required for RNA Polymerase II to bind
(Nomenclature: (e.g TFIID)
Roman numeral – Polymerase type (II)
Letter – specific TF protein (D))

Before Pol II can initiate transcription,
(1) ATP-dependent phosphorylation of Pol II
(2) Pre-initiation complex needs to be released
For intact cellular DNA: requires participation of other regulatory proteins that open chromatin for initiation complex to bind
code vs codon
CODE = genetic code expressed in DNA language
Bases A, T, C, G
CODES are written 3' to 5' as they appear in the DNA template strand
CODON = genetic code expressed in mRNA language
Bases A, U, C, G
CODONS are complementary and antiparallel to genetic code of DNA from the template strand
Actual coding units read by protein synthesis mechanism
bacterial RNA polymerase
Very well characterized (480,000 Da)
Multiple subunits:
Core enzyme (2α + 1β + 1β’ subunits) + 1 sigma factor
“Core" + sigma = holoenzyme
Sigma ensures that RNA synthesis initiates at right place on DNA (determines correct reading frame; core only will start at random)
Sigma factor readily dissociates
Transcription Factor Binding of RNA Pol II
6 general transcription factors (TF) required for RNA Polymerase II to bind
(Nomenclature: (e.g TFIID)
Roman numeral – Polymerase type (II)
Letter – specific TF protein (D))

Before Pol II can initiate transcription,
(1) ATP-dependent phosphorylation of Pol II
(2) Pre-initiation complex needs to be released
For intact cellular DNA: requires participation of other regulatory proteins that open chromatin for initiation complex to bind