The portion of DNA that contains the information of a functional biological product.
What are the monomers of a DNA polymer called?
Nucleotides.
What are the 3 components of a DNA monomer?
1. Nitrogenous Base.
2. Pentose (b-D-ribofuranose derivative).
3. Phosphate.
Is this a purine or a pyrimadine?
Does it belong to RNA or DNA?
What is it's name?
Purine,
DNA,
Deoxyadenosine
Is this a purine or a pyrimadine?Does it belong to RNA or DNA?What is it's name?
Purine,
DNA,
Deoxyguanosine
Is this a purine or a pyrimadine?Does it belong to RNA or DNA?What is it's name?
Pyrimidine,
DNA,
Deoxycytidine
Is this a purine or a pyrimadine?Does it belong to RNA or DNA?What is it's name?
Pyrimidine,
DNA,
Deoxythymidine
Is this a purine or a pyrimadine?Does it belong to RNA or DNA?What is it's name?
Purine,
RNA,
Adenosine
Is this a purine or a pyrimidine? Does it belong to RNA or DNA? What is it's name?
Pyrimidine,
RNA,
Cytidine.
Is the following a purine or a pyrimidine? Does it belong to RNA or DNA? What is it's name?
Pyrimidine,
RNA,
Uridine.
What is the correct numbering for this ring?
What type of ring is it?
Pyrimidine.
What is the correct numbering for this ring?
What type of ring is it?
Purine.
What is one mechanism by which DNA would be kept in the nucleus whereas RNA could leave?
The deoxythymidine has a methyl group on it's pyrimidine ring at position 5 that is not present in uridine. This could potentially end up keeping the DNA in the nucleus whereas RNA would be free to pass through.
What is the major structural difference between DNA and RNA nucleotides?
2' hydroxyl.
Which side of the DNA molecule is hydrophylic, and why is that?
The sugar-phosphate backbone. The oxygen in the phosphate and in the sugar are able to form hydrogen bonds.
What is the Directionality of a DNA molecule?
5'-3'.
The phosphate is at the 5' and the sugar's 3' carbon sits at the 3' end.
What is the type of lincage that hold the backbone together, and what is it's defining characteristic?
Phosphodiester linkage.
P-O-C(5')-C(4')-C(3')-O-P
Protein will absorb what wavelength of light?
280nm. This is because of the aromatic amino acids Trp, Phe, Tyr.
DNA will absorb what wavelength of light?
260nm.
Is there evidence to suggest that DNA has pi stacking interactions?
Yes, the Purines and Pyrimidines as well as the sugars are turned so that their rings lie over each other like stacks of coins. This allows for extensive pi stacking between polymers.
Describe intercalation:
Intercalators have aromatic structures that fit in between the stacked sugars and nitrogenous bases to induce variations in the energy associated with the placement of each monomer. They can be carcinogenic when it becomes more energetically favorable for a different base to replace the original base.
What form of DNA is this?
What is significant about it's structure?
B form.
You can clearly see the major groove and the minor groove.
What is significant about the A form and the Z form of DNA?
They are not especially relevant as biological molecules, but they are possible structures, and may have applications in nano-technology.
How is RNA more "flexible" than DNA?
1.It can have both information storing properties, and enzymatic properties.
2. Its single stranded nature allows for structural flexibility and altering base pairing interactions.
What 3 types of RNA are common?
1. mRNA
2. tRNA
3. rRNA
Given normal Guanine, what would oxidatively damaged guanine look like, and what would it be named?
What is the danger of uv radiation?
It can dimerize adjacent pyrimidine pairs through a forbidden Diels-Alder reaction.
Why is S-Adenosymethionine important?
It is involved in regulatory methylation at nucleophylic positions.
Describe the process of SAM Methylation:
What is the full name and structure of ATP?
Adenosine 5'-Triphosphate.
Why is Water important for ATP Hydrolysis?
Energy doesn't come from breaking ATP, The energy payoff comes from ADP being stabilized by water.
Describe cAMP:
Briefly describe genetic engineering:
Target genes are excised with a restriction enzyme.
The same restriction enzyme also cuts a bacterial plasmid.
The target gene and the plasmid join with sticky ends, and then a 2nd type of enzyme seals the two together.
Bacteria can now express the target gene.
What is the name of the "non-critical" bacterial DNA that is manipulated through genetic engineering?
Plasmid.
Could you go out an buy a certain type of pre-modified plasmid?
Yes.
What are 3 common features of cloning vector plasmids?
Origin of replication
antibiotic resistance
restriction sites.
What type of restriction enzymes cut DNA at specific sites?
Type 2
What are restriction endonucleases?
Restriction enzymes that cut DNA as specific sites.
About how long generally are the restriction sites for restriction endonucleases?
About 4-6bp
True or false, restriction sites are often complements of each other?
True.
What does DNA Ligase do?
It reseals sticky ends by reforming the phosphodiester bonds.
What are sticky ends?
After a restriction cut, they are the few base pairs that protrude. They are complementary to any other cut made by that enzyme.
Define Transformation:
Insertion of the modified plasmid into bacteria.
How is the protein product purified from the bacteria?
The bacteria overproduce the product.
Inclusion bodies are formed.
The celles lyse.
product is purified through affinity collumn.
If you found that you have 10% adenine residues by weight, what are the ratios of the other nucleotides?
A=0.1
T=0.1
G=0.4
C=0.4
Given 2 light absorbant values, A260 & A280, Describe the process of finding the protein concentration, and how would you find the DNA concentration?
Take 280/260, and compare this value to a chart.
This value will correspond to a F value, and to a proportion of nucleic acid at that value.
Multiply the A280 by the F value to find protein concentration in mg/ml.
The percentage of DNA at that reading is available on the chart.
What would account for a difference in melting point for two fatty acids with the same length carbon chain?
The number of cis-double bonds. The more it has, the lower it's melting point.
Would branched chain fatty acids increase or decrease membrane fluidity?
Increase fluidity.
(less side chain interactions)
What characteristic of phosphitydalcholine would allow it to stabilize butter and water?
It is amphipathic, and helps butter be more soluble.
Compositional analysis of a certain lipid shows that it has exactly 1 mole of fatty acid to 1 mole of inorganic phosphate. What type of fatty acid could this be?
Sphingolipid
What would a name of
18:1(9) cis-9-Octadecanoic acid mean?
18 Carbons
cis double bond from C9-10
What would it mean to say that a molecule is an 3 fatty acid?
That counting backwards the first carbon that has a double bond is carbon 3.
Describe a tryacylglycerol:
This type of lipid has 3 fatty acid chains all held together with an 3ester (acyl) lincages to a common 3 carbon backbone.
Describe the saponification of a triacyglyceride:
A base (like NaOH) would have to be used to cleave the 3 ester bonds:
What type of fat is not usually found in nature, and presents a problem for human metabolism?
The trans-fats are not easily metabolized. This is why they have such long shelf lives. The bacteria that would spoil them cannot metabolize the trans double bond quickly enough and they die.
In hydrating a cis-fat, and then dehydrating the saturated fat, why do trans-fats occur more often than cis-fat?
The trans-fats represent the low energy product of double bond formation.
Describe a glycerophospholipid:
2 fatty acid tails are ester linked to a 3C backbone. The 3rd carbon is attached to a phosphate, and then to a head group substituent.
What do plant cells use to replace the phosphate group in their version of glycerophosphopholipids?
They can replace the charged phosphate group with galactose or sulfate groups.
What do we mean when we say "phosphotidyl-x?
it is a glycerophospholipid with an x group coming off of the phosphate.
Describe a sphingolipid:
1 single fatty acid attached to a Sphingosine backbone with an amide bond. The sphingosine has 1 single polar OH group, and a head group substituent (X group).
What is significant about the amide bond in a sphingolipid?
The amide bond can come on and off much more easily than the traditional lipid acyl linkage.
What type of fats are associated with blood types and antigens?
sphingolipids.
Describe a sterol:
They are steroid alcohols with alkyl side chains.
One one side the polar OH group.
The 4 fused ring steroid nucleus.
The alkyl side chain.
What do sterols do for the cell membrane?
They decrease its fluidity, giving it more strength.
Describe an "active" lipid:
arachidonic acid derived eicosanoids are hormones that act locally and induce a specific response in tissues. They're a cause inflamation and clotting.
are steroid hormones biologically active?
Yes.
Name the fat soluble vitamins:
A
D
E
K
What evidence is there for lateral membrane diffusion?
Laser bleaching experiments show the return of fluorescent coated phospholipids, indicating that they have fluidly filled the gap.
What shape is important for a bilayer to form?
Cylindrical diacyl groups provide the correct shape for the bilayer. The head and tail diameters are the same.
About how thick across is a typical bilayer?
30 angstroms.
Are bilayers primarily lipid, or primarily protein?
It depends. There is an exceptionally high amount of protein in the bilayer, and in some cases it can be more present (by mass) than the lipid.
What physical state best describes a cell mambrane?
Liquid-ordered state.
It cant be too disordered: para-crystalline (gel)
Or too fluid.
Describe membrane symmetry:
Membranes are asymmetrical, they also catalyze trans-bilayer diffusion.
What is the difference between latteral diffusion and trans-bilayer diffusion?
Lateral diffusion is side to side and very fast: microseconds.
Transbilayer is flipflop and very slow: days.
Describe Flippase:
Flips bound substrates (lipids etc) from outside to inside.
ATP catalyzed movement.
Describe Floppase:
Flops bound substrates (lipids etc) from inside to outside.
ATP catalyzed movement.
Describe scramblase:
Moves bound substrates (lipids etc) from out to in (flip) or in to out (flop).
Not ATP catalyzed, just runs on equilibrium constants.
What is the "cell death" phospholipid that should remain on the inside of a healthy cell?
Phosphatidylserine.
What is an IMP?
Integral membrane protein.
They are classified by type.
The non-polar residues are the transmembrane portions,
The polar residues are the external portions.
Describe signal specificity:
Only the signal molecule fits in it's complementary binding on the complementary receptor. Other signals do not fit.
Describe signal amplification:
Enzymes activate enzymes and the number of molecules involved at each step increases geometrically.
Describe signal desensitization and adaption:
Receptor activation triggers a feedback mechanism that either deactivates the receptor, or removes it from cell surface.
Describe signal integration:
When 2 signals have opposite effects, the regulatory outcome is the integrated input from both receptors.
What is one of the most widely used signaling methods that cells use?
G protein coupled receptors (GPCR)
True or false, GPCR's involve a secondary messanger?
True.
What is the end result of the adrenal/epinephrine pathway?
Release of glucose frome glycogen.
What would it mean to say that the receptor protein is heptahelical serpentine?
That the receptor protein make 7 transmembrane passes before coupling with the different G subunits.
Describe the basic action of a GPCR
Signal binds to receptor.
G subunits attached to receptor separate.
1 subunit moves & is activated by GTP replacing GDP
Active G subunit attaches to Adenylyl Cyclase
AC catalyzes formation of cAMP.
How is the alpha subunit held to the plasma membrane?
Palmitoyl subunit.
What is the 2nd messenger in the epinephrine response pathway?
cAMP
What key feature of GPCR's does Ras demonstrate?
The gamma phosphate in the new GTP causes a conformational shift that activates the Gprotein. (adducts the 2 side subunits towards the center)
How do G proteins keep from being constantly activated?
They have intrinsic GTPase properties, continually cleaving the activating phosphate.
In the case of Ras, what would happen if it were to lose its GTPase capability?
It would remain constantly activated.
Because it is proto-onco, if it is constantly on cancer would result.
After adenylyl cyclase forms cAMP, where does the 2ndary messenger go, and what does it do?
cAMP binds to protein kinase A, releasing the catalytic subunits fromt the inhibitory subunits.
What is the eventual outcome of the cAMP?
Eventually it is hydrolyzed to 5'-AMP.
For each signal molecule in the epinephrine mechanism, around how many blood glucose molecules are released?
1:10,000
How is phospholipase C activated by GPCR?
Hormone H binds to receptor.
Occupied receptor Causes GDP/GTP exchange with attached Gq.
Active Gq moves to PLC and activates it.
How are PIP2, DAG, and IP3 related?
PIP2 spits to form DAG and IP3.
PIP2 loses its phosphate and P-attached sugar.
DAG is glycolipid backbone
IP3 is P-attached sugar.
What is the function of Phospholipase C?
It cleaves PIP2 at the phosphate to form PIP and IP3.
Describe the action and biproducts of PLC:
cuts right at attached phosphate.
How many 2nd messengers are produced from PIP2 from Phospholipase C activity?
2. DAG and IP3
Describe an adapter protein:
Lack Catalytic function
Bring various protein together.
Grb2 is an example.
What is the similarity between lipid rafts and Adapter proteins?
Both lack catalytic function,
Both bring various proteins together.
Describe a lipid raft:
It is a Liquid-ordered region inside a sea of liquid dissordered phospholipid.
It can attribute its stability to increased sphingolipid and cholesterol concentration in the region.
Where on the cell membrane are GPCRs concentrated?
Lipid rafts.
what is a protein "gone bad?"
An oncogene.
What is a typical membrane potential?
Between 50-70mV
Describe the 2 gradients in the cell:
The Electrochemical gradient: The sum of + or - on both sides of the cell membrane.
The concentration gradient: how much of a certain molecule is on one side vs. the other.
Describe Na+ K+ ATPase:
3 Na from Inside bind.
ATP phosphorylates, opening up to outside
3 Na release to outside.
2 K from outside bind.
P dephosphorylates, opening up to inside.
2 K release to inside.
In ATPase Na is moved _____ the ____ gradient:
Against Electrochemical gradient.
AND: against the concentration gradient.
In ATPase K is moved ____ the ____ gradient:
Against the Concentration gradient.
AND: With the electrochemical gradient.
What is the general electrochemical environment just inside and just outside the cell membrane?
Inside is generally negative.
Outside is generally positive.
What establishes the membrane potential?
The electrogenic ATPase action moving 3 Na out and 2 K in for every ATP hydrolysed.
How do Na, Ca, or Cl depolarize a cell?
They have high concentrations outside of the cell. As they move across the membrane, they decrease the differing charges across the membrane, lowering the membrane potential.
How does K hyperpolarize the cell?
K has a (+) charge and is plentiful inside the (-) cell. As it moves from high concentration to low concentration out of the cell, it removes its portion of (+) charge with it. Leaving a more polarized cell, with a greater membrane potential.
What does IP3 signal?
One function of IP3 is to tell the endoplasmic reticulum to release Ca2+.
Describe the MAPK/ERK (insulin receptor) pathway Just after the GEF (sos) stage:
Ras->Raf->Mek->Erk which is synonymus with
G-Protein->MapKKK->MapKK->MapK
How is Ras activated?
Sos binds to Ras, causing Ras to release its GDP and bind a new GTP.
How is Raf activated?
Activated Ras binds to Raf (MapKKK). This is the first kinase.
Describe the MapK/Erk (insulin receptor) pathway from insulin to the Gef (sos) stage:
Ins + InsReceptor=Active Kinase.
Active InsR phosphorylates Irs-1 at 3 tyr residues.
Irs-1 moves to adapter proteing Grb2 which simultaniously binds sos and Ras.
Describe the role of adapter protein Grb2:
Grb 2 is activated by Irs-1 phosphate group. It has binding sites for sos and Ras. (Gef and G-protein)
What would MapK (Erk) need to do to stimulate cell division?
It moves into the nucleus and phosphorylates transcription factors. The transcription factors do the rest.
Describe a complexity associated with the insulin and epinephrine pathways:
Insulin can actually hijack the epinephrine pathway by shutting down the GPCR through multible phosphorylations.
Describe
At equalibrium, what is Delta G?
Why is this?
0.
This is because the Concentration gradient and Electrical potential cancel each other out.
What is the Nernst Equation, and what does it solve for?
It solves for membrane potential at equalibrium.
How can you check to make sure that your energy equations are correct?
Before the calculation, you should be able to say if delta G is going to be + or -, then adjust the in/out to out/in depending on the situation.
What does R equal?
8.315 J/mol•K
How do you know the correct tempurature to use in the Nernst equation?
Kelvin
273+Celcius
Norm Body temp: 310K
What number does Z stand for?
1
What number F equal?
96500 J/V•mol
Where can we find voltage gated ion channels?
Nerve Cells:
Describe a synaptic wave of depolarization up to the synaptic cleft:
Voltage Gated Na+ channels allow rapid intake of Na moving down to the synapse,
At the synapse voltage gated Ca2+ take over.
Acetylcholine vesicles are induced to exocytose at the secretory synaptic cleft.
With the rapid influx of + charge, the voltage gated K+ channel releases rapid amounts of K+ from the cell, causing a wave of hyperpolarization.
Describe the effect of acetylcholine:
At the synaptic cleft acetylcholine is released in vesicles. These travel to the post synaptic neuron and activate Na+/Ca2+ ion channels.
This propogates the wave to the next neuron.
Describe a Voltage Gated Ion channel:
The activation gate is held closed by the voltage sensors which are sensitive to the polarized sides of the membrane.
The channel is opened when the (+) voltage sensors are pushed open by an influx of + charges on the cytosolic side of the membrane (membrane depolarization).
At a certain point an inactivation gate responds to the changing polarity and shuts down the channel (think of the flushing mechanism of a toilet).
What would be the effect of small amounts of Aspartame being injected into the body?
Nerves ion channels would be left open, and overload would occur.
Describe how Sight, Smell, and Taste work:
Signal protein binding releases messengers to PIP2->DAG + IP3->ER to release Ca2+. Ca2+ also activates Na+ pump. Depolarizing cell membrane causes rapid exocytosis to afferent nerve fibers.
What 3 common features are needed for cancer to occur:
