Reading...
Play button
Play button
Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
262 Cards in this Set
- Front
- Back
|
Differences between animal and plant cells
|
1) Chloroplasts (p)
2) Cell Wall (p) 3) Vacuole (p) |
|
|
Conformation
|
the spatial arrangement of substituent groups that, w/o breaking any bonds, are free to assume different positions in space b/c of rotation around a bond
|
|
|
Configuration
|
fixed spatial arrangement of atoms that cannot change w/o breaking any bonds. i.e. stereoisomers
|
|
|
Chiral Center
|
4 different paths around one carbon center
|
|
|
Stereoisomers
|
same chemical bonds but different sterochemistry
|
|
|
Enantiomers
|
mirror images of one another
|
|
|
diastereomers
|
chiral molecules that are stereoisomers but not enantiomers
|
|
|
L/D systems
|
D molecules have the carbon next to the carbonyl with the hydroxyl on the same side as D-glucose
|
|
|
Enthalpy
|
H- the number and kinds of bonds
|
|
|
Free Energy
|
G=H-TS
DeltaG neg ==> exergonic and spontaneous DeltaG pos ==> endergonic and not spontaneous |
|
|
Entropy
|
S- the randomness of a chemical system
-S (order increases, randomness decreases) +S (order decreases, randomness increases) |
|
|
If a protein was broken down into amino acids, would deltaS be + or -
|
- because randomness is increasing. A protein is much more ordered than all of its respective amino acids in solution
|
|
|
Gibbs Free Energy Equation
|
deltaG=StandarddeltaG+RTln([C][D]/[A][B])
|
|
|
K(eq)
|
K(eq)=([C][D])/([A][B])
|
|
|
Catabolic Reaction
|
Breaks down something and is exergonic
|
|
|
Anabolic Reaction
|
Builds something up and is endergonic
|
|
|
Relation between DNA, RNA, and Proteins
|
DNA-->RNA-->Proteins
|
|
|
4 Weak (non-covalent) Interactions
|
1)H-bonding
2)Ionic 3)Hydrophobic 4)Van der Waals |
|
|
Which interactions are important in stabilizing non-polar lipids in water?
|
Hydrophobic
|
|
|
What type of bonds (and how many) hold water molecules together in ice?
|
H-bonds and 3.6 bonds/water
water can normal H-bond 3 times |
|
|
What makes the best H-bond?
|
CNOFS
When all the molecules involved are in a straight line |
|
|
What actually happens when a salt crystal dissolves in water?
|
delta S is increasing (+) and delta G is decreasing (-)
The H-bonds from water stabilize the ionic attractions of each of the molecules that were in salt |
|
|
What are the Colligative Properties?
|
Osmolarity, Vapor Pressure, boiling point, melting point
|
|
|
What is an Amphipathic molecule?
|
contain regions that are polar and regions that are non polar
|
|
|
What structures do amphipathic molecules form in water? and why?
|
micelles because the number of water molecules that are ordered around the hydrophobic portion is minimized when they are all in one group
|
|
|
What does a Hydronium ion look like and why do we care about it?
|
H3O+ and it carries a proton around. It is formed through H-bonding that eventually forms a covalent bond.
|
|
|
Keq of Water
|
=[H+][OH-]/[H2O]=1x10^-14
|
|
|
Henderson-Hasselbalch equation
|
pH=pKa+ log [A-]/[HA]
|
|
|
pH
|
is defined as the -log of the concentration of H+ present in an aqueous solution
|
|
|
Conjugate acid-base pair
|
A-=base
HA=acid |
|
|
Ka and pKa
|
Ka the equilibrium constant for the disassociation of a weak acid-base pair.
pKa is the pH at which [A-]=[HA] |
|
|
Buffer zones
|
pKa + or - 1 pH point
|
|
|
Acid Titration Curves
|
-Completely protonated on the right
-Completely deprotonated on the left -X-axis [OH] -Y-axis pH -Slope =0 at equivalence points -isoelectric point is when the molecule is neutral and the slope is undefined at this point |
|
|
Hydrolysis and Condensation Reactions
|
Hydrolysis adds water to a molecule and condensation removes a water molecule
|
|
|
Glycine
|
Non-polar
Gly G |
|
|
Alanine
|
Non-polar
Ala A |
|
|
Proline
|
Non-polar
Pro P |
|
|
Valine
|
Non-polar
Val V |
|
|
Leucine
|
Non-polar
Leu L |
|
|
Isoleucine
|
Non-polar
Ile I |
|
|
Methionine
|
Non-polar
Met M |
|
|
Phenyalanine
|
Aromatic
Phe F |
|
|
Tyrosine
|
Aromatic
Tyr Y |
|
|
Tryptophan
|
Aromatic
Trp W |
|
|
Serine
|
Polar, uncharged
Ser S |
|
|
Threonine
|
Polar, uncharged
Thr T |
|
|
Cysteine
|
Polar, uncharged
Cys C |
|
|
Asparagine
|
Polar, uncharged
Asn N |
|
|
Glutamine
|
Polar, uncharged
Gln Q |
|
|
Lysine
|
+ charged
Lys K |
|
|
Histidine
|
+ charged
His H |
|
|
Arginine
|
+ charged
Arg R |
|
|
Aspartate
|
- charged
Asp D |
|
|
Glutamate
|
- charged
Glu E |
|
|
What amino acid allows disulfide bonds?
|
Cysteine
|
|
|
What physical property of certain amino acids (and which ones) allows us to determine if a solution contains proteins?
|
The aromatic amino acids, Phe, Tyr, and Trp absorb UV light at 280nm allowing us to see if there are proteins in a solution.
|
|
|
How do you calculate Isoelectric Point?
|
Average the pKa values
|
|
|
What atoms make up a peptide bond?
|
O
ll R--C--NH--R |
|
|
What names are given to the ends of peptides?
|
Amino terminus is the N-side
Carboxyl terminus is the C-side Usually written from N-->C |
|
|
How is a peptide bond formed?
|
Condensation reaction so a water is made everytime
|
|
|
4 levels of protein structure
|
1)Primary-amino acids connected in the correct order
2)Secondary- some folding, disulfide bonds, starting of beta sheets and alpha helices 3)Tertiary- functionally folded protein 4)Quaternary- functional protein that consists of multiple subunits |
|
|
Conjugated Proteins
|
lipoproteins and glycoproteins that have other things attached to them
|
|
|
What 2 different methods could I use to learn the relative mw of an unknown protein?
|
Gel Electrophoresis or Size-exclusion chromatography
|
|
|
What is the most efficient order of column chromatography methods to purify a protein?
|
ion-exchange then size-exclusion then affinity chromotography
|
|
|
What is specific activity when referring to the purification of proteins?
|
The number of enzyme units per mg of total protein
|
|
|
What method of working with proteins requires ampholytes?
|
Isoelectric focusing
|
|
|
What does SDS actually do in gel electrophoresis?
|
It attaches to proteins to negate any charge by providing it with a large negative charge. It also binds relatively similar no matter the protein so relative mw are still present
|
|
|
What methods and in what order are they used in 2-D electrophoresis?
|
1st isoelectric focusing and then gel electrophoresis so things are separated by charge and then by mw.
|
|
|
How do you break disulfide bonds in a protein?
|
performic acid or DTT (dithiothreitol)
*KNOW THIS! |
|
|
Difference between Edman and Sanger procedures
|
Edman-labels and removes only the amino-terminus residue from a peptide while leaving the rest of the peptide bonds intact
Sanger-same thing but it destroys the protein after removing the amino-terminus amino acid Sanger- |
|
|
What is a Proteome? Genome? Metabolome?
|
Proteome- the entire protein complement encoded by an organism's DNA
Genome- the complete sequence of an organism's DNA Metabolome- the complete sequence of the cellular intermediary metabolism |
|
|
What do you learn about a protein using Mass Spectroscopy?
|
The mass of the protein along with sequences of short stretches of polypeptides
Sequence of peptide |
|
|
What is a protein's native conformation?
|
The Tertiary or Quaternary functional form of the protein
|
|
|
How is function changed when the structure of a protein is altered?
|
Shapes can change that means active sites can be changed or things won't fit where they need to so that biological processes can be carried out
|
|
|
What is a solvation layer?
|
A layer of water around non-polar molecules. Proteins get this when hydrophobic molecules are grouped together in one part of the molecule
|
|
|
What does a Ramachandran Plot tell you about protein structure?
|
It tells us which secondary structures are possible by comparing rotation around the alpha C-C bond and the alpha C-N bond of each amino acid
|
|
|
X-axis and Y-axis of a Ramachandran Plot
|
phi bonds are- AlphaC-N
psi bonds are- AlphaC-C psi vs. phi C-C vs. C-N |
|
|
What are the major Secondary protein structures?
|
alpha helix and B-sheet and B-turns
|
|
|
Alpha Helix
|
-R-groups are on the outside
-Can't have anything too big or small and NO PRO -H-bonds every 4 amino acids so a turn every 4 amino acids |
|
|
B-sheet
|
-antiparallel is more stable because there is a straight line between H-bonds
-small amino acids like Gly and Ala |
|
|
B-turns
|
-180 degree turn containing 4 amino acids
-carbonyl oxygen of 1st h-bonds with the amino group H of the 4th -Pro common b/c turns on its own |
|
|
Alpha vs. Beta
|
Alpha- tough and insoluble structures of varying hardness and inflexibility
Beta- Soft, flexible but non-stretching filaments |
|
|
Heme group structure
|
-Protoporphyrin ring bound to a Fe +2 molecule. 4 coordination bonds from the ring, 1 from His in protein, 1 for O2
|
|
|
Heme group placement w/n proteins
|
-Placed on the inside of the molecule so that only O2 binds with it
-Keeps CO2 from getting in |
|
|
NMR vs. X-ray diffraction
|
NMR-- solution of macromolecules determines structure
X-ray diffraction-- macromolecules must be in crystallized form to determine structure |
|
|
5 different graphic representations of proteins
|
1)polypeptide backbone- highlights regions of secondary structures
2)mesh- emphasizes the protein surface 3)surface contour image- shows pockets where other molecules can bind 4)ribbon representation-shows side chains 5)space-filling- show realistic shape of protein |
|
|
Domains and motifs
|
Domains- a functional part of one polypeptide
motifs-folds or supersecondary structures that are common in a lot of proteins |
|
|
Basic motifs
|
B-alpha-B loop
alpha-alpha corner B-barrel 1.Burial of hydrophobic residues requires at least 2 layers of secondary structures. 2. alpha-helix and B-sheets are found in different parts of a protein |
|
|
Rotational and Helical Symmetry
|
individual subunits can be superimposed on others by rotation about one or more rotational axes
|
|
|
How do you denature a protein?
|
Heat it up or change pH. It just has to be able to refold into it's natural conformation.
denaturing means taking it out of its natural, functional conformation |
|
|
Why are Chaperones needed?
|
Proteins have 1 natural conformation but they may be able to fold into different structures first, so these just make sure they fold into the right one first
|
|
|
How do the two main classes of Chaperones work?
|
HSP70- (heat shock protein 70) bind to regions of unfolded proteins that are hydrophobic
chaperonins- elaborate protein complexes required for the folding of proteins that don't fold spontaneously |
|
|
Define: Ligand
|
a molecule bound reversibly by a protein
substrate |
|
|
Define: binding site
|
where the ligand binds. it is complementary to the ligand in size, shape, charge, and hydro-character
active site |
|
|
Define: induced fit
|
structural adaptation that occurs between protein and ligand is called induced fit
|
|
|
Hemoglobin vs. Myoglobin
|
Hemoglobin- movement of Oxygen out of the lungs to the tissues. 4 subunits very similar to myoglobin. 4 binding sites for O2
Myoglobin- used for long term storage of oxygen. 1 binding site for O2 |
|
|
Ka and Kd
|
Ka=[PL]/[P][L]
Kd=[P][L]/[PL]=1/Ka Kd is used more often. Kd is smaller than affinity for substrate is higher. |
|
|
Ligand Binding Curves
|
-X-axis-[L]
-Y-axis-Theta -Theta=fraction of ligand binding sites that are occupied by ligand Kd=.5Theta |
|
|
Theta
|
=[L]/[L]+Kd
=Binding sites occupied/total binding sites |
|
|
What shape is the Ligand Binding Curve for Myoglobin? Hemoglobin?
|
myoglobin- hyperbolic
hemoglobin-sigmoidal |
|
|
T-State vs. R-State in hemoglobin
|
T-state- when O2 binds to it, it changes to R state. Low-affinity state
R-state-high affinity state |
|
|
Allosteric protein (ex. hemoglobin)
|
-As more O2 molecules bind to it it is easier to change from T to R state
-Sigmoidal binding curve -Changes shape |
|
|
2,3-BPG effects on hemoglobin binding sites
|
heterotropic allosteric moderation-- reduces affinity of hemoglobin for O2
Used to help O2 be released in the tissues more readily. The hemoglobin doesn't want O2 when BPG is bound to it |
|
|
CO effects on hemoglobin
|
when CO is present, the hemoglobin doesn't release O2 in the tissues
|
|
|
H+ effects on hemoglobin
|
When [O2] is high hemoglobin binds O2 and releases H+ in the lungs
When [O2] is low, H+ binds and the O2 is released in tissues |
|
|
Bohr Effect
|
The effect of pH and CO2 concentration on the binding and release of O2
|
|
|
Hill Coefficient
|
nh=1 the binding is not cooperative
nh>1 positive cooperation nh<1 negative cooperation |
|
|
Hill Plot
|
log(theta/1-theta)=nlog[L]-logKd
|
|
|
2 models for cooperativity
|
1)concerted model- assumes all subunits are functionally identical and all transition from one state to another simultaneously
2)sequential model- lingand binding can induce a change of conformation in an individual subunit-hemoglobin |
|
|
What is the major defect in sickle cell disease?
|
When hemoglobin is deoxyginated 1% of it is sickled, meaning it is changed to an dysfunctional form
|
|
|
Structure of Actin
|
globular domains put together into filamentous proteins
|
|
|
Structure of Myosin
|
filamentous body with a globular head that has an actin binding site
2 light chains are used to in the globular head 4 heavy chains |
|
|
Action of Actin and Myosin
|
1)ATP binds to myosin and a cleft in myosin opens so bound actin is released
2)ATP is hydrolyzed causing a high energy state to change orientation in relation to actin 3)Phosphate is released and the myosin rebinds to actin one spot up 4)power stroke where it returns to rest and it is one actin molecule head of where it was before |
|
|
How do enzymes decrease free energy changes?
|
They lower the activation free energy by hold molecules closer together and providing environments that encourage product formation
|
|
|
Define: Coenzyme
|
complex organic or metalloorganic molecule that binds to an enzyme to make it functional
|
|
|
Define: Cofactor
|
one or more inorganic ions that bind to an enzyme to make it functional
|
|
|
Define: Prosthetic group
|
a coenzyme or metal ion that is very tightly or covalently bound to an enzyme
|
|
|
Define: Holoenzyme
|
a catalytically active enzyme together with its bound coenzyme or metal ion
|
|
|
Define: Apoenzyme or Apoprotein
|
the protein part of the holoenzyme
|
|
|
Reaction Coordinate Diagrams
|
-Y-axis- Free energy, G
-X-axis- Reaction Coordinate -Transition state-where it is equally probable for the molecule to go to products and reactants |
|
|
How can you find the rate limiting step on a rxn coordinate diagram?
|
The step with the highest transition energy
|
|
|
How can you find activation energy on a rxn coordinate diagram?
|
The delta G from reactants to transition state
|
|
|
How can you find Transition state on a rxn coordinate diagram?
|
the highest energy state on the diagram is the highest transition state
|
|
|
What is a Rate Equation?
|
V=k[S]
where k is a constant. This shows how fast a rxn can overcome deltaG of the transition state |
|
|
What is Binding Energy?
|
the energy derived from enzyme-substrate interaction is called binding energy, delta Gb
|
|
|
What is Desolvation of the substrate? And when does this happen?
|
enzyme substrate interactions replace most or all of the hydrogen bonds between substrate and water. Happens with polar substrates
|
|
|
3 basic mechanisms for catalysis
|
1)Acid Base
2)Metal Ion 3) Covalent |
|
|
Acid Base catalysis
|
specific- uses only the H+ and OH- present in water
general- proton transfers mediated by other classes of molecules Charged intermediates can often be stabilized by the transfer of protons to or from the substrate to form a species that breaks down more readily |
|
|
Covalent Catalysis
|
a transient covalent bond is formed between the enzyme and the substrate
|
|
|
Metal Ion Catalysis
|
Ionic interactions between an enzyme-bound metal and a substrate can help orient the substrate for reaction or stabilize charged reaction transition states
|
|
|
Velocity plots for enzyme catalyzed reactions
|
Km=Michaelis Constant=[S]@1/2Vmax (mM/ml)
Vmax=theoretical speed= mM/s Km can be looked at as affinity of enzyme for it's substrate |
|
|
Michaelis-Menten Equation
|
Vo=Vmax[S]/Km+[S]
|
|
|
How can you tell which enzyme is most sensitive when looking at Initial Velocity plots?
|
The one with a lower Km. A smaller change in concentration of substrate causes a bigger change in initial velocity
|
|
|
What is the steady state of a rxn? How is this different from equilbrium?
|
Steady State-the rate of formation of ES is equal to the rate of it's breakdown.
Its Equilbrium for enzymes |
|
|
How do I find Vmax and Km on double-reciprocal plots?
|
Km is the x-intercept and Vmax is the y-intercept
|
|
|
How do you specify the mechanism of inhibition using a double-reciprocal plot?
|
With Competitive Inhibition Vmax will be the same but Km will be higher (1/Km will be smaller)
With Noncompetitive- Vmax will be lower and so will Km With Mixed Inhibition- change in both Vmax and Km |
|
|
What is Kd and Kcat?
|
They measure the affinity of enzyme for its substrate
|
|
|
What are suicide inactivators?
|
Pharmacy uses these to make drugs. They bind to the enzyme and start the reaction, but then cannot finish or be released from the enzyme, so they render it useless.
|
|
|
How do you tell the difference between reversible inhibitors and allosteric inhibitors?
|
reversible inhibitors will have a hyperbolic curve and Vmax will be lower
allosteric inhibitors will have a sigmoidal curve and Vmax should be the same as without inhibition. |
|
|
What is Feedback inhibition?
|
Where a molecule made at the end of the pathway regulates earlier steps in the pathway. This allows molecules to self-regulate and not create to much of one thing.
|
|
|
What things happen to enzymes at higher or lower pH values than the optima?
|
Because enzymes are proteins they can denature and become non-functional with a change in pH
|
|
|
What is a pro-enzyme?
|
a protein that has to be modified before it can be an enzyme. So it must be activated
|
|
|
Alpha vs. Beta in carbohydrates
|
If the hydroxyl group on the anomeric C is on the same side as the C6 group then it is Beta Conformation
Alpha conformation has the OH on opposite sides of C6 |
|
|
What does the arrow mean when naming carbohydrate linkages?
|
single arrow goes from anomeric C to another C
double arrow goes from one anomeric C to another anomeric C |
|
|
Draw the structure of Glucose
|
look it up
|
|
|
What are epimers?
|
They are carbohydrates with all the same number of carbons but with one chiral center with a different stereochemistry
|
|
|
Anomeric Carbons
|
Named as Carbon #1
-If it is an aldehyde then its an aldose -If it is a keytone then it is a ketose |
|
|
Hemiacetal vs. Hemiketal
|
Hemiacetal-formation of a ring by a reaction between alcohol and aldhyde
Hemiketal-formation of a ring by a rxn between alcohol and a keytone |
|
|
What is the reducing end of a sugar?
|
The end of a chain of carbohydrates with a free anomeric carbon
It can be oxidized in linear form and only a free anomeric carbon can switch back and forth between linear and cyclic |
|
|
Difference in structure and function for starch vs. cellulose
|
Starch-contains all alpha linkages and is stored as forms of fuel and reduced one sugar at a time
Cellulose-Beta linkages and tough, water insoluble |
|
|
How does H-bonding effect Cellulose and Chitin? And Starch and glycogen?
|
For cellulose and chitin all of the molecules are held in 180 degree turns from each other in their chair forms which makes it very tough
For Starch and glycogen inter chain H-bonding holds it in a helical form |
|
|
What are 3 basic conjugates? And what do they do?
|
Proteoglycans- macromolecules of the cell surface. Glycoaminoglycan chains joined covalently to a membrane protein
Glycoproteins-one or several oligosaccharides joined to a protein glycolipids- membrane lipids in which the hydrophilic head groups are oligosaccharides |
|
|
What makes up glycoaminoglycans?
|
Heteropolysaccharides of the extracellular matrix
|
|
|
What do Lectins bind to?
|
Proteins bind to carbohydrates with very high affinity
|
|
|
What do Lectins and Selectins do?
|
Lectins- cell to cell recognition, signaling, and adhesion processes and intracellular targeting of new proteins
Slectins-plasma membrane lectins that mediate cell to cell recognition and adhesion |
|
|
Define: gene
|
a segment of DNA molecule that contains the information required for the synthesis of a functional biological product
|
|
|
Define: Ribosomal (rRNA)
|
components of ribosomes, the complexes that carry out synthesis of proteins
|
|
|
Define: Messenger RNA (mRNA)
|
intermediaries, carrying genetic information from one or a few genes to a ribosome
|
|
|
Define: Transfer RNA (tRNA)
|
are adapter molecules that translate the info in mRNA into a specific sequence of amino acids
|
|
|
Difference between a triphosphate and a trisphosphate
|
Triphosphate has 3 phosphates on the same C
Trisphosphate has 3 phosphates on different C |
|
|
What is the name of the bond that joins nucleotides in DNA?
|
A phosphodiester bond between the 5' phosphate and the 3' hydroxyl
|
|
|
Three different forms of DNA
|
B-form- most stable structure for a random sequence of DNA under physiological conditions
A-form- favored in solutions devoid of H2O-wider helix Z-form- left-handed helix purines flip with pyrimidines |
|
|
Draw Deoxyadenylate
|
A, dA, dAMP, deoxyadenosine
|
|
|
Draw Deoxyguanylate
|
G, dGMP, dG, deoxyguanosine
|
|
|
Draw Deoxythymidylate
|
T, dT, dTMP, deoxythymidine
|
|
|
Draw Deoxycytidylate
|
C, dC, dCMP, deoxycytidine
|
|
|
Draw Adenylate
|
A, AMP, adenosine
|
|
|
Draw Guanylate
|
G, GMP, guanosine
|
|
|
Draw Uridylate
|
U, UMP, uridine
|
|
|
Draw Cytidylate
|
C, CMP, cytidine
|
|
|
What is Watson-Crick base pairing?
|
G (triple bond) C
A (double bond) T |
|
|
What holds a double stranded DNA helix together?
|
H-bonding between base pairs on complementary strands of DNA
|
|
|
What holds DNA in a Triplex form?
|
Hoogstein pairings that H-bond in the major groove. G (triple) C and a C+ and A=T*A
|
|
|
5' vs. 3' ends
|
5' end has a open phosphate group and the 3' end has a an open OH group on C3 of the sugar
5' is traditionally written on the left |
|
|
What did Hershey-Chase conclude?
|
they used radioactive viruses to conclude that DNA was what carried the genetic information of a cell not anything else
|
|
|
What did Avery-Macleod conclude?
|
they used heat killed diseases and rats to determine that DNA was able to be transform other DNA even after it had been denatured
|
|
|
What did Watson-Crick conclude?
|
Double helix and base pairing
|
|
|
What kinds of structures can DNA and RNA make as a result of base pairing and folding?
|
hairpin turn or cruciform develop because strands are self-complementary
|
|
|
Where are the Major and Minor grooves and what function do they have?
|
Major groove is in between the backbone turning around. It is a place for Hoogstein pairing to take place and for molecules such as initiators to bind
|
|
|
What is denaturing?
|
when the DNA helix unwinds a little bit or completely
|
|
|
What can you learn about DNA structure using 1)heating 2) UV light 3) hybridization?
|
1)heating you can see if you have more AT pairs or GC pairs because they denature at different temps
2)UV light you can see if you have completely denatured a protein because they absorb more light if they are in single strands 3) hybridization you can check for specific base-pair matching if you hybridize with known strands of DNA |
|
|
Can DNA and RNA absorb light?
|
Yes at a lower wavelength like 260nm
|
|
|
What can UV light do to DNA and RNA?
|
it induces the condensation of two ethylene groups and makes cyclobutane pyrimidine dimers
|
|
|
How does DNA sequencing work?
|
Altered bases are added to the end of a complementary sequence then ran through a gel. The size of the resultant bases tells us which nucleotide goes where
|
|
|
What are some other functions of nucleotides in the cell?
|
-energy carriers (ATP)
-NAD+ -FAD -cofactors -regulatory molecules |
|
|
What are blunt ends and sticky ends?
|
blunt ends are where restriction enzymes have cut both strands of DNA at the same nucleotide
sticky ends are where the restriction enzymes cut at different nucleotide numbers |
|
|
Which one, blunt or sticky ends, are preferred for cloning and why?
|
sticky ends because you can orientate which way the strand goes into the vector by cutting certain sites on the vector with the same restriction enzymes
|
|
|
What do I get using an expression vector that I usually do not get from a cloning vector?
|
You get the transcription and translation signals needed for regulated expression of the cloned gene
In other words, you get to regulate when that gene you cloned is expressed |
|
|
What is a Southern Blot?
|
It is a gel with radioactive DNA fragments in it that is used to match key areas of DNA to known sequences
|
|
|
How do microarrays work and what can I use them for?
|
You can see which genes are being expressed in a cell by seeing the florescent present on the chip.
|
|
|
What is the advantage of using a Bacteriophages?
|
You can get a lot of DNA into a cell relatively easily
|
|
|
What is the purpose of a two-hybrid system?
|
You can tell which genes are expressed together in a a cell
|
|
|
Define: transcription
|
the act of making a complementary RNA strand to the template DNA strand
|
|
|
Define: translation
|
taking the complementary RNA strand and attaching amino acids together according to the code on that piece of RNA
|
|
|
What enzymes make RNA from DNA?
|
RNA Polymerase, precursors of the binding unit and Mg+2
|
|
|
Promoters and Repressors?
|
they are areas of an DNA molecule that tell the RNA polymerase to transcribe or not to transcribe. They can also be in the form of molecules who bind to the promoter or repressor si
|
|
|
What are the main stages of RNA synthesis?
|
1)assembly of RNA Polymerase and transcription factors at the Promoter
2)RNA strand Initiation and Promoter Clearance 3)Elongation, Termination, Release |
|
|
What are transcription factors and what kind are there?
|
general transcription factors are the same in all eukaryotes and they bind to the promoter region so that the RNA polymerase can bind
|
|
|
What term refers to the non-coding portion of a primary RNA?
|
introns
|
|
|
What term refers to the coding portion of a primary RNA?
|
exons
|
|
|
What is splicing?
|
Splicing is removing the introns from a pre-RNA. This happens through spliceosomes (snRNPs)
|
|
|
What processing does pre-mRNA go through before it is translated?
|
-Introns are taked out by spliceosomes
-5' cap is put on the 5' end to protect from degradation -3' poly a tail is put on 3' end to protect from degradation |
|
|
Can there be more than one way to process mRNA?
|
yes, to make different proteins from the same strand of DNA you can splice-out different introns for different proteins
|
|
|
What is a codon and anticodon?
|
a codon is on the mRNA strand and it is a group of 3 nucleotides
an anticodon is on the tRNA and it is what is complementary to the mRNA to bring in the correct amino acids |
|
|
What is the Initiation codon?
|
AUG---Methionine
|
|
|
What are the termination codons?
|
UAA, UAG, UGA
|
|
|
What does Degeneracy mean with reference to the genetic code?
|
It means amino acids can be coded for by different codons but a codon will only code for 1 amino acid
|
|
|
What are the basic elements of the wobble hypothesis?
|
-1st 2 bp follow Watson Crick pairing very strongly
-3rd one does not bind as strongly, so it does not determine which amino acid is present as strongly as the 1st 2 |
|
|
What are the 5 stages of Protein Synthesis?
|
1) Activation of Amino Acids- the carboxyl group is activated to make peptide bond
2)Initiation- smaller of the two subunits of ribosome binds to mRNA with initiation tRNA 3)Elongation-lengthened by covalent attachment of each amino acid 4)Termination & Release- signaled by stop codon 5)Folding & Post-translational Processing- removal of amino acids |
|
|
Which enzyme attaches amino acids to the tRNA?
|
Aminoacyl-tRNA synthetases
|
|
|
What are tRNAs called after they have their corresponding amino acid attached to them?
|
Aminoacyl-tRNAs
|
|
|
What are the subunits of ribosomes? How are they described and structurally defined?
|
the 30s subunit binds first with initiation factors and the initiation aminoacyl-tRNA
70S subunit binds over that with 3 spaces- A, E, and P |
|
|
What are the parts of a tRNA?
|
1)codon arm
2)amino acid arm 3) 2 other arms that have no specific purpose yet |
|
|
What are the A, E, and P sites? What molecule are they on and what are they for?
|
They are located on the 70s ribosome. They are sites of elongation of the protein.
A-activation-where the carboxyl is activated to create peptide bonds P-peptidyl-where the covalent peptide bond is formed E-exit site-tRNA leaves without its amino acid |
|
|
What is a Shine-Dalgarno Sequence?
|
It is the initiation sequence that guides the initiating AUG into place and the 30s ribosome
|
|
|
What is significant about the first amino acid for all newly synthesized proteins? How does it differ between bacteria and mammals?
|
The first amino acid is always a form of Met.
In Bacteria it is f-met that is later processed In mammals it is met but there is a different aminoacyl-tRNA that supplies internal met |
|
|
What is the role of GTP in protein synthesis?
|
It is an elongation factor
|
|
|
What is Post-translational Processing? Name a few
|
Alterations to the protein after translation is complete
-Loss of signal sequences -Modification of individual amino acids -Attachment of carbohydrate side chains -Addition of prosthetic groups -Formation of Di-sulfide crosslinks |
|
|
What are storage lipids made of and what are they called?
|
-Triacylglycerides
Glycerol -Fatty Acid -Fatty Acid -Fatty Acid |
|
|
What are the 2 types of Phospholipids?
|
Glycerophospholipids and Sphingolipids
|
|
|
What does a glycerophospholipid look like?
|
Glycerol
-Fatty Acid -Fatty Acid -PO4-OH |
|
|
What does a sphingolipid look like?
|
Sphingosine
-Fatty Acid -PO4-Choline or -Monosaccharide |
|
|
What are the 2 types of glycolipids?
|
Spingosine and Galactolipids
|
|
|
What does a galactolipids?
|
Glycerol
-Fatty Acid -Fatty Acid -Monosaccharide |
|
|
How do you abbreviate a lipid with 24 carbons and one double bond between 9 and 10?
|
24:1(delta^9)
|
|
|
How does the degree of saturation of fatty acids effect melting point?
|
as the number of double bonds increase, the melting point goes down b/c the molecule is already disordered
|
|
|
What is a phosphatidylinositol and what does it do?
|
It is a lipid derivative that acts as an intercellular signaling molecule
|
|
|
What are the three main groups of eicosanoids and how do they differ?
|
Prostaglandins (PG)-5C ring
Thromboxanes-6C ring Leukotrienes-highly conjugated double bond |
|
|
What do eicosaniods do and what molecule gives rise to all 3 different categories?
|
They act on cells near the point of hormone synthesis and then carry signals to other cells. Arachidonic acid
|
|
|
What are waxes?
|
They are long carbohydrates attached to long chain alcohols. They are water proof
|
|
|
How are steriod hormones and cholesterol related?
|
Steriod hormones have the 4 ring structure without the long chain carbohydrate connected to the end as in cholesterol
|
|
|
Which vitamins are hormone precursors?
|
A&D
|
|
|
Which vitamins are cofactors or anti-oxidants
|
E&K
|
|
|
What are the main methods for working with lipids?
|
TLC, separating by polarity and mass spectroscopy for total structure
|
|
|
3 basic structures of lipids in aqueous environments
|
bilayer-all hydrophobic on the inside of the bilayer, outsides still exposed
micelle-hydrophobic core, hydrophillic circle around outside liposomes-no hydrophobic parts exposed to water |
|
|
Why are lipids asymmetrically distributed in membranes?
|
Some have attached proteins that need to be on the inside or outside of the cell.
|
|
|
What are Flippases?
|
They are the enzymes that flip a lipid through the hydrophobic region to the opposite side of the bilayer
|
|
|
How do temperature changes effect membrane properties?
|
Cold-Gel state-slow moving almost rigid
Warm-liquid-ordered state-moving enough to have Fluid Mosaic but not disordered Hot-liquid disordered state- moving too much around in the membrane and membrane can fall apart |
|
|
What are Microdomains and Membrane rafts?
|
Membrane rafts form transient clusters that largely exclude glycerophospholipids.
Microdomians of cholesterol and sphingolipids are thinker than phospholipids |
|
|
What does caveolin do?
|
It is an integral membrane protein w/ 2 globular domains that binds cholesterol into the membrane
|
|
|
Peripheral vs. Integral Proteins
|
Peripheral can move in and out of the membrane when they need too.
Integral are permanently attached to the membrane and usually go all the way through it. |
|
|
Define: transmembrane proteins
|
proteins that span the entire membrane
|
|
|
Define: lipid-linked membrane proteins
|
These are membrane proteins that are covalently attached to lipids that insert themselves into the membrane
|
|
|
What is a hydropathy plot used for? What does it tell us?
|
It is used to determine whether a protein will be a transmembrane protein or not. If it has enough hydrophobic amino acids it will be.
|
|
|
What is a Type I integral protein?
|
OOC- on the inside transmembrane to NH3+ on the outside
|
|
|
What is a Type II integral protein?
|
NH3+ on the inside transmembrane to OOC- on the outside
|
|
|
What is a Type III integral protein?
|
1 protein that spans the membrane several times
|
|
|
What is a Type IV integral protein?
|
multiple subunits that span the membrane several times
|
|
|
What is a Type V integral protein?
|
a lipid-linked membrane protein that is on the inside
|
|
|
What is a Type VI integral protein?
|
a transmembrane protein with a lipid linked portion
|
|
|
How are carbohydrates oriented in the membrane?
|
Always on the outside because they are hydrophobic
Opposite the cytosol always |
|
|
What general steps are required to transport solute into or out of a molecule by fusion?
|
Cell has to recognize the molecule
the lipid bilayer becomes disrupted resulting in fusion of outer leaflets |
|
|
Passive vs. Active Transport
|
Passive-with a concentration gradient
Active- against a concentration gradient |
|
|
Diffusion vs. Facilitated Transport
|
non-polar molecules can diffuse through the membrane but polar molecules need help from channels or other proteins that is called facilitated transport
|
|
|
What are the two components of an electrochemical gradient?
|
Electrical gradient and a concentration gradient
|
|
|
What constant is a measure of the distribution of charge across a membrane?
|
Vm
|
|
|
How is water transported across the membrane without the direct need for energy?
|
Aquaporins. They are channels that are small enough for just water and have h-bonding to stabilize the water as it goes through. It also has + charges placed so that H+ can not get through
|
|
|
How is glucose transported across the membrane without the direct need for energy?
|
The glucose transporter has several helices in a circle that provide many hydrophilic residues that H-bond with glucose specifically as it moves through the channel. Replaces h-bonding with water
|
|
|
Primary vs. Secondary Active Transport
|
Primary-directly paired with exergonic reaction
Secondary-indirectly paired with exergonic movement of something with the electrochemical gradient |
|
|
3 different Active Trasporters
|
Uniport- one thing moving one direction
Symport- two things moving in the same direction Antiport-two things moving in opposite directions-Chloride-Bicarbonate exchanger |
|
|
4 types of transport ATPases
|
P-cation transporters that are reversibly phosphorylated by ATP as part of transport cycle
F-moves protons against their concentration gradient by hydrolyzing ATP V-acidify intracellular compartments Multi-drug-ABC transporters |
|
|
Ion and Ligand Gated Channels
|
Ligand-wait for the ligand to bind before they open or vice versa
Ion-wait for the electrochemical gradient to reach a certain point then open or close |
