Biochemistry Flashcards
1
Q
Describe enzymes and the unique catalytic features of enzymes.
A
- biological catalysts
- increase the rate of reaction
- do not effect equilibrium constant
- does not effect delta G
- results in higher reaction rates
- 10^6 to 10^12 times greater than non-catalyzed reactions
- milder reaction conditions
- can occur at atmospheric pressure, neutral pH, and temperatures less than 100C
- specificity - very rarely occuring side products
- can be regulated
- enzyme activity can vary in response to biological molecules other than the enzymes substrate or products
2
Q
Describe the affects of pH and temperature on enzymes.
A
- enzymes have an optimum temperature
- most human enzymes have a temp optimum of about 37C
- pH optimum
- pepsin in the stomach at pH 2
- trypsin in the small intestine at pH 8
3
Q
Classify and describe 6 types of enzymes.
A
- oxidoreductases
- catalyze oxidation-reduction reactions
- dehydrogenases, oxidases, reductases, peroxidases, catalase, oxygenases, hydroxylases
- transferases
- catalyze transfer of a group such as glycosyl, methyl, or phosphoryl
- transaldolase, transketolase, acyl, methyl, glucosyl and phosphoryl transferases, kinases, phosphomutases
- hydrolases
- catalyze hydrolytic cleavage of C-C, C-O, C-N and other bonds
- esterases, glycosidases, peptidases, phosphatases, etc.
- lyases
- catalyze cleavage of C-C, C-o, C-n and other bonds by atom elimination leaving double bonds
- decarboxylases, aldolases, hydratases, dehydratases, synthases
- isomerases
- catalyze geometric or stuctural changes within a molecule
- racemases, epimerases, isomerases, some mutases
- ligases
- catalyze the joining together of two molecules coupled to the hydrolysis of ATP
- synthetases and carboxylases
4
Q
What are the molecular mechanisms of enzyme activity? (5)
A
- the “active site”
- where catalysis occurs
- substrate binds to AS with highly specific manner to promote reaction being catalyzed
- structure of enzyme is critical for the appropriate structure of the AS
- specific residues can play important role
- changes in the enzyme structure may or may not have major effects on the activity of the enzyme
- catalysis by proximity
- for molecules to react, they must come within bond-forming distance
- AS of enzyme binds substrate creating a high local concentration of substrate
- substrates are bound in specific orientation conducive to reaction
- acid-base catalysis
- ionizable functional groups of amino acyl side chains may participate in the catalysis by acting as acids or bases
- catalysis by strain
- for the enzymes catalyzing bond breakage, they may bind in such a way to destabilize the bond to be broken (just snap off)
- covalent catalysis
- covalent bond between substrate and enzyme is formed
- covalently modified enzyme then becomes a substrate for a subsequent reaction (to make ultimate product)
- rxn of covalently modified enzyme to product is energetically more favorable than the rxn of substrate to product
5
Q
Describe the role of cofactors, coenzymes, and prosthetic groups.
A
- small, non-protein molecules and metal ions with participate directly
- many derived from vitamins
- prosthetic groups
- tightly and stably incorporated
- sometimes by covalent bonds
- metal ions most common
- “metalloenzymes”
- other examples: B6, B1, biotin, lipoic acid, FMN, FAD
- tightly and stably incorporated
- cofactors
- bind only transiently (reversible)
- Mg2+ is required for enzymes involving ATP
- coenzymes
- serves as “shuttle” or “transfer agents
- act more like a substrate and product of an enzyme catalyzed reaction
- product of one reaction becomes a substrate for another, regenerating the coenzyme
- examples: FADH2, NADH, CoA
6
Q
Describe isozymes.
A
- distinct enzymes (with different sequences) which catalyze the same reaction
- subtle differences
- kinetic differences
- regulatory differences
- same Keq
- Tissue differences
- isozymes may be differentially expressed in different tissues
- ex: LDH and CK isozymes
- LDH - 4 subunits with heart and muscle types; 5 isozymes
- CK contains 2 subunits, with muscle and brain types; 3 isozymes
7
Q
First Order
A
- v = k [A]1 = k [A]
- occurs with low concentration of substrate, so S<<km>
<li>v = Vm/Km x S</li>
</km>
8
Q
Zero Order
A
- v = k[A]0 = k
- occurs with very high concentration of substrate, so S >> Km
- v = Vmax
9
Q
Second Order
A
- v = k[A]2
- when substrate concentration equals the Km, so [S] = Km
- v = 1/2Vmax
10
Q
Turnover Number
A
- k1, k2, k3 are rate constants
- k3 is the rate limiting constant and is called kcat or turnover number
- number of product released per unit of time per enzyme at Vmax
- Vmax= kcat [Etotal]
- km = (k2 + k3)/ k1
11
Q
Write both the Lineweaver Burk equation and Michaelis Menten equation.
What is Kcat/Km?
A
- 1/v = Km/Vm x 1/[S] + 1/Vm
- v = Vmax [S]/Km + [S]
- catalytic efficiency, turnover numvber over binding constant, the higher the better
12
Q
Competitive Inhibition
A
- I competes with S for AS of enzyme
- I is structurally similar to S
- I binds only to free enzyme
- Km increases
- Vmax does not change
- S can be increased to overcome inhibition
- Kmapp = Km (1 + [I]/Ki)
- ex: statins
13
Q
Noncompetitive Inhibition
A
- I binds to E or ES; no need for similarity in structure
- does not bind to AS, so does not compete with S
- effect is similar to removing E from system
- Km does not change
- Vmax decreases
- increasing S does not overcome inhibition
- Vmapp = Vm/ (1+ [I]/Ki)
- ex: heavy metals - Hg, Pb
14
Q
Uncompetitive Inhibition
A
- I binds only to ES
- S binding alters E, allowing I to bind
- Increase in S will not overcome inhibition
- rare in single S enzyme
- Km decreases
- Vmax decreases
- increase S is proportional to increase in I
- Kmapp= Km/ 1+ ([I]/Ki)
15
Q
Allosteric Enzymes - describe cooperativity, Hill number and K0.5
A
- usually display cooperativity with their S
- binding of one S facilitates binding of subsequent S
- allosteric enzyme has more than one S binding, AS, and usually more than one subunit
- Hill equation kinetics
- v = Vm[S]n/K0.5 + [S]n
- n = the Hill number
- measure of degree of cooperativity
- larger n, larger cooperativity, larger sigmoidicity of curve
- no cooperativity, n =1, is MM equation
- K0.5 is not the same as Km
- does represent [S] at 1/2 Vmax
- rate constant is different for allosteric enzymes and MM enzymes
16
Q
Discuss Tense and Relaxed state in allosteric enzymes along with homotropic and heterotropic regulation
A
- conformational change of an allosteric enzyme from Tense (inactive) to Relaxed (active) conformations
- At low S, enzyme is in T
- as S binds, enzyme changes to R
- various inhibitors and activators of allosteric enzymes may stabilize the T or R state
- homotropic regulation ex: binding S, increasing affinity of the other catalytic sites for S
- almost always positive
- heterotropic regulation - regulatory molecule other than the S which binds to allosteric site and modulates activity
- can be either positive activators or negative inhibitors
17
Q
Distinguish between V-system effectors and K-system effectors
A
- V-system effectors affect the catalytic rate, influencing Vmax
- can be positive or negative
- K-system effectors affect the binding of S, influencing K0.5
- can be positive or negative
- does not affect Vmax
18
Q
Overview of Regulation of Enzymes
A
- Synthesis and Degradation
- change [Etotal]
- Compartmentation
- physically separates conflicting pathways of enzymes, cofactors, and substrates
- Allosteric Regulation
- non-covalent mod of enzyme activity through the binding of effector molecules to the enzyme
- second messangers, cAMP, hormones increasing Ca2+
- non-covalent mod of enzyme activity through the binding of effector molecules to the enzyme
- Covalent Regulation
- phosphorylation (reversible)
- uses protein kinase to phosphorylate
- uses protein phosphatase to dephosphorylate
- proteolytic activation
- synthesized in inactive form as zymogen or proenzyme
- ex: trypsinogen, chymotrypsinogen
- ex: fibrinogen, prothrombin
- phosphorylation (reversible)
- Some enzymes are subject to regulation by multiple means, eg glycogen phosphorylase
19
Q
Michaelis-Menten Equation
A
v = Vmax[S]/Km+[S]
20
Q
Km
A
Equals the [S] at which the velocity is one-half Vmax
21
Q
Vmax
A
Vmax = k3[Etotal]
22
Q
heptahelical or serpentine receptors
A
- heterotrimeric G protein-coupled receptors (characterized to date)
- span cell membrane 7 times
23
Q
function of heterotrimeric G protein
A
- Receptor binds hormone
- G protein exchanges GTP for GDP and a-subunit dissociates
- Target protein binds GTP-G a-subunit
- GTP is hydrolyzed by intrinsice GTPase and ATP signals cAMP (second messenger)
- a-subunit with GDP now dissociates
- a-subunit reassociates with b and y-subunits and receptor (starts cycle over)
24
Q
G protein a-subunit categories of family > effector
A
- Gs - adenylyl cyclase stimulated
- Gi- adenylyl cyclase inhibited
- Gq - phosphplipase C stimulated
- G12 - various ion channels (depending on source)
25
Second Messengers
* G protein coupled receptors generate intracellular molecules (signals) that are termed second messengers
* ex: cAMP, cGMP, Ca++, diacylglycerol, and phosphatidylinositides
26
intracellular receptors
* gene-specific transcription factors
* proteins that bind to DNA and regulate the transcription of certain genes
* messengers using intracellular receptors must by hydrophobic molecules that are able to diffuse through the plasma membrane
* Lipophilic hormones that use this are considered in the Steroid hormone/thyroid hormone superfamily of receptors
* steroid hormones
* thyroid hormones
* retinoic acid
* Vitamin D
27
What are some nuclear receptors that have been identified to play an important role in intermediary metabolism and are the target of lipid-lowering drugs?
* peroxisome proliferator activated receptors (PPAR a, b, and g)
* the liver X-activated receptor (LXR)
* the farnesoid X-activated receptor (FXR)
* the pregnane X receptor (PXR)
28
What are the two subclasses of Lipophilic hormone receptors?
* cytosolic (subclass-I) receptors
* in cytosol bound to heat shock proteins (hsps)
* hormone binding displaces hsps
* hormone-receptor complex migrates to nucleus
* binds to a hormone response element (HRE)
* interaction of hormone-receptor complex and HRE affects the transcription of gene either positively or ngatively
* sex steroids fall into this category
* nuclear (subclass-II) receptors
* found within the nucleus and are not bound to hsps
* binding of the hormones induces a conformational change (activation)
* activated hormone-receptor complex binds to a HRE and affects the transcription of genes either positively or negatively
* thyroid hormone, retinoids, Vit-D fall into this category
29
Signal termination
quickly?
slowly?
in contrast?
disease?
* quick termination to modify metabolic responses of cells or that transmit neural impulses - **diffusion/degradation**
* slow termination for signals like stimulating proliferation - **desensitization, down regulation, protein phosphatases**
* signals regulating differentiation may persist throughout the lifetime
* many chronic diseases are caused by failure to terminate a response at the appropriate time - cancer
30
down regulation
hormone or neurotransmitter is present in excess, the number of active receptors decreases
31
up regulation
when there is a deficiency of the chemical messenger, there is an increase in the number of active receptors
32
internalization
ligand-receptor complexes taken into the cell by endocytosis after going through the membrane to coated pits
33
desensitization
type of down regulation where receptors are chemically modified in ways that make them less responsive
34
ligands
substances that bind witha high degree of specificity to a receptor
35
agonists
ligands that bind to receptors and cause a maximal response
36
partial agonist
ligands that evoke a submaximal response, even when filling all the receptors
37
antagonist
some ligands bind to receptors with high affinity yet elicit no response
38
Graves Disease
* due to the production of antibodies against thyroid stimulating hormone (TSH) receptor.
* TSH is involved in the stimulation of thyroxine hormone
* antibodies act as an agonist and bind to TSH receptor and stimulate thyroxine production
* results in hyperthyroidism and Grave's disease with characteristic bulging of the eyes
39
myasthenia gravis cause
* antibodies against nicotinic acetylcholine receptors
40
What are 3 main signaling systems and what kind of messenger do they use?
* nervous system - neurotransmitter
* endocrine system - hormones
* paracrine - eicosanoids
* derived from arachidonic acid, prostaglandins, thromboxanes and leukotrienes
* autocrine - growth factors
* polypeptides that function through stimulation of cellular proliferation (eg EGF and FGF)
* immune system - cytokines
41
What is signal transduction?
* conversion of the signal of a chemical messenger to an intracellular response after it has bound to a receptor
* receptors have binding site for single chemical messenger and another site involved in transmitting the message
* receptors may be either plasma membrane receptors or intracellular receptors
42
What are plasma membrane receptors and give a list of examples of them
* span plasma membrane, contain extracellular binding domain for the messenger
* EC binding domain is where it binds
* transmembrane domain is helical in nature
* intracellular domain has an enzyme within it but is not active until signal or ligand binds to make the enzyme active
* examples include
* ion channel receptors
* tyrosine kinase receptors
* tyrosine kinase associated receptors (JAK STAT)
* serine-threonine kinase receptors
* G protein coupled receptors
43
ion channel receptor: nictinic acteylcholine
* ACh used in neuromuscular junction
* diffuses across cleft
* binds to nicotinic ACh receptors
* has 3 subunits (a,y,a) which go across membrane with passage through the membrane
* receptor acts like gate, allows Na+ to go through or not depending on whether ACh is bound
* as ACh binds to a receptor, conformational change opens the gate, allowing Na+ to diffuse in and K+ to diffuse out
* change in ion concentration activates sequence of events, eventually triggering cellular response - contraction of muscle fiber
44
Tyrosine kinase receptors (enzyme linked)
* exisit in membrane as monomers with single membrane spanning helix
* one molecule of GF binds to 2 molecules of receptor, promoting dimerization
* receptor dimer formed, intracellular tyrosine kinase domains of receptor phosphorylate each other on certain tyrosine residues (autophosphorylation)
* phosphotyrosine residues form specific binding sites for signal transducer proteins
45
JAK-STAT receptors and process
* tyrosine kinase associated receptors
* used by cytokines to regulate the proliferation of certain cells
* receptor itself has no intrinsic kinase axtivity but binds with the tyrosine kinase JAK (janus kinase)
* various cytostolic proteins such as STAT (signal transducer and activators of transcription) are phosphorylated by JAKs
1. binds messenger
2. receptor dimerizes
3. conformational change
4. enzyme activated
5. JAK phosphorylates STAT
6. STAT-Ps dimerized enter the nucleus and activate transcription by further binding
46
Serine-Threonine Kinase receptors
* used by proteins in the transforming growth factor superfamily, also associates with Smad Family (gene specific transcription factors)
* family includes TGF-b, a-cytokine, hormone involved in tissue repair, immune regulation and cell proliferation
* phosphorylates serine or threonine only
* recruits Smad
47
G-protein coupled receptor
* translate signal to biological effect by way of nucleotide regulatory proteins (G-proteins) that bind GTP
* GTP is a quanosine analong of ATP
* small G proteins related to ras oncogene are involved in
* vesicle transport
* interactions between cytoskeleton and cell membrane
* cell growth
* heterotrimeric G proteins coupled cell surface receptors to catalytic units
* these units catalyze the intracellular formation of second messenger or couple receptros to ion channels directly
* have 3 subunits (a,b,y)
48
function of membranes
* create a cellular boundary, a barrier, and intercellular compartments
* contain components that accomplish transfer and components for cell communication
49
Basic components of membrane
* lipids, proteins, carbohydrates
* carbohydrates are covalently bound to either proteins or lipids (glycoprotein or glycolipid)
* compounds that are soluble in organic solvent are called lipids
* major lipids here are phospholipids, glycosphingolipids and cholesterol
50
membrane structure
* has amphipathic lipids
* part hydrophilic - phosphate, amino acid derivatives, and carbohydrates
* part hydrophobic - fatty acids
* fatty acid chains are
* saturated - single bonds
* unsaturated - at least 1 double bond
* spontaneous formation of lipid bilaryer in aqueous environment or formation of liposome (has hollow core)
51
Name the common phospholipids and where they are found in membranes
* phosphatidylcholine (PC) -
* phosphatidylserine (PS) -
* phosphatidylethanolamine (PE) -
* phosphatidylinositol (PI) -
all are major bulk lipid molecules for
* mitochondria
* microsomes
* lysosomes
* plasma membrane
* Golgi membrane
* plasma membrane
52
Where is cardiolipin mostly found?
Where is sphingomyelin mostly found?
Where is cholesterol mostly found?
* cardiolipin - mitochondria
* sphingomyelin - lysosomes and plasma membrane
* cholesterol - plasma membrane
53
What is unique to DNA/RNA?
* Both composed of nucleotides
* Components of nucleotide
* Nitrogenous Base (Purine or Pyrimidine)
* Sugar Moiety (Ribose or Deoxyribose)
* Phosphate
* Rare or Minor Bases
* Bases can be modified via methylation (control gene expression), acetylation or hydroxymethylation
* tRNA contains a high % of **_minor bases_**
* rRNA contains a high % of methylated
* Most common modified base in DNA: 5-Methylcytosine
* Most often CpG sites are modified
* DNA Contains:
* Nitrogenous Base: A, T, G, C
* Sugar Moiety: 2-deoxyribose
* Phosphate
* RNA Contains:
* Nitrogenous Base: A, U, G, C
* Sugar Moiety: Ribose
* Phosphate
54
How are nucleotides linked in DNA/RNA?
* Mononucleotides are joined together by **_phosphodiester_** bridges between 5'-hydroxyl group of one nucleotide and the 3'-hydroxyl group of the adjacent nucleotide
* _Bases are not directly attached covalently to one another_
* Sequences are always written **_Left → Right: 5' to 3' direction_**, unless otherwise noted
55
Products of strong acid hydrolysis of DNA/RNA?
* Products:
* Purine Bases
* **_Pyrimidine Nucleosides_**
* Deoxyribose/Ribose
* Phosphate
* Pyrimidine Nucleosides are stable in acid
* Purine Nucleosides are labile (liable to change) in acid
56
Products of base hydrolysis of DNA/RNA?
* Important method for separating DNA from RNA
* DNA is stable in base (Does NOT contain 2'-hydroxy group)
* RNA is NOT stable in base
* Base hydrolysis of RNA proceeds through a 2', 3' cyclic phosphate intermediate
* Products: 2' and 3' nucleoside monophosphates
57
Peridocities found in DNA?
* 2 Periodicities
* Distance b/t stacked nitrogenous pairs: 0.34 nm or 3.4 Å
* Distance for one complete turn of double helix: 3.4 nm or 34 Å
* Meaning there are 10 base pairs for each complete turn
* AKA the B form of DNA which is the most abundant
* Major and Minor grooves in DNA (particularly major) provide surfaces to which regulatory proteins can bind
* Width of Major Groove = 22 Å
* Width of Minor Groove = 12 Å
58
Reasons for DNA stability?
1. Hydrophobic and electronic interactions b/t stacked bases of each strand
2. Hydrogen bonds that are formed b/t the complementary bases of 2 strands
3. Hydrophobic nitrogenous bases are shielded from aqueous environment
4. Negatively charged phosphate groups and polar sugar moieties are exposed to the aqueous environment
59
Details of DNA melting and denaturation?
* When DNA is heated hydrogen bonds are disrupted thus separating the two strands
* Segments rich in A-T melt at lower temperatures than those rich in C-G
* Can be followed by an increase in UV light absorbance
* Stacked bases in double-stranded DNA decrease UV absorption relative to free nucleotides in solution
* Known as **_hypochromic effect_**
* Annealing
* Opposite of melting
* Slow cooling allows the two complementary strands to reassociate
60
Conformations of DNA?
* DNA can assume different conformations under different physical conditions
* May function in regulation of gene expression
* DNA B, A, C, Z
* DNA B
* Most common conformation
* Right-handed helix
* DNA A
* 11 base pairs per complete turn
* Distance for one complete turn = 2.8 nm
* Nitrogenous bases are not perpendicular to the long axis of the double helix but have a tilt of about 20º
* Forms under low hydration conditions
* Probably occurs in very short stretches in DNA
* Sequence Dependent
* DNA C
* 9 base pairs per complete turn
* Distance for one complete turn = 3.3 nm
* Not thought to occur *in vivo*
* DNA Z
* 12 base pairs per complete turn
* Observed in crystals of synthetic hexanucleotide d (CpG)3 and in fibers of the alternating d(GC)n polymers
* Left-handed helix
* May exist *in vivo*
* Regions rich in G-C base pairs
* Methylation of deoxycytidine groups in these regions may favor Z-DNA formation
* Possible for B-Z transition to occur
* Details on how Z-DNA functions in gene regulation are emerging
* Proteins have been purified that bind specifically to Z-DNA
* Not a separate conformation, but adenine residues can cause bending of the helix
* 6 in a row can cause bend of 18º
* May be feature recognized by DNA binding proteins
* Expression of genes is generally blocked in these regions
61
Coding Strand
* DNA is composed 2 strands one is the coding strand the other is the template strand
* Nucleotide sequence in the coding strand is the same as that of the RNA made from the DNA w/ uracil replacing thymine
62
Template Strand
* Noncoding strand
* Is complementary to the coding strand and also to the RNA which will be synthesized
* If the two strands of DNA are separated, the newly synthesized RNA would bind (hybridize) to the template strand
63
Factors that can cause denaturation?
1. High Temperature
2. High pH
3. Low ionic strength\*
1. Not sufficient to cause complete denaturation, but in combination w/ the two above is experimentally used to denature DNA
64
Southern Blot
1. DNA sample electrophoresed
2. Denatured
3. Transferred to nitrocellulose and hybridized w/ radiolabeled probe
Reason for Use: Often used to see gene structure or complexity
65
Northern Blot
1. RNA sample is electrophoresed
2. Transferred to a membrane and immobilized
3. Hybridized w/ DNA probe
Reason for use: Used to measure RNA message levels for genes
66
List 3 passive forms of selective permeability and transport
* simple diffusion - non mediated
* restricted to hydrophobic molecules or small uncharged polar molecules
* H2O, O2, CO2, urea
* simple diffusion - mediated by channel proteins
* channel protein forms a water-filled pore through the membrane
* channel proteins still display selectivity in the molecule passing through
* facilitated diffusion
* transport or carrier protein is required
* interact with transported molecule and due to some change in the molecule-protein complex, release the molecule on the opposite side of the membrane
FOR ALL - THE NET FLOW IS HIGH CONCENTRATION TO LOW CONCENTRATION
67
What is the plasma membrane characterized by?
* greatest amount of cholesterol
* major components PE, PS, PC, PI, and sphingomyelin
68
Discuss lipid asymmetry.
* outer leaflet of the plasma membrane contains PC and sphingomyelin
* inner leaflet of the plasma membrane contains PS and PE
* lateral movement of phospholipids is common, but movement from one leaflet to another leaflet is rare
* translocases or flipases catalyze the movement from one leaflet to another
* the enzymes are responsible for maintaining the asymmetry
* carbohydrate portion of glycolipids extends into the extracellular space
69
Discuss protein asymmetry.
* orientation of protein is associated with function
* orientation is a function of synethesis (inherent part of proteins aa sequence)
* may be anchored with other cellular elements either outside (extracellular matrix) or inside (cytoskeleton) of the cell
* carbohydrate protion of glycoproteins extend only into the extracellular space.
70
Discuss membrane fluidity and its phases.
* exisit in either liquid-crystalline phase (fluid-like) or gel phase (solid-like)
* gel phase is less permeable to small molecules
* for any given composition of lipids, there is a temperature at which this phase transition occurs, called the transition temperature, Tm
* So, temperature affects membrane fluidity
* protein activity is altered when in different phases
71
What affects membrane fluidity?
* fatty acid composition
* unsaturated = lower melting temp (double bond dramatically decreases melting temp)
* an increase in unsaturated fatty acids causes an increase in membrane fluidity
* cholesterol
* at temps below Tm, it increases fluidity
* at temps above Tm, it decreases fluidity
72
Discuss active transport
* requires specific transport protein (pumps)
* requires input of energy
* ATP hydrolysis to ADP and Pi
* an ion concentration gradient (to go against)
73
Discuss classifications of transport
* uniport - only one solute transported
* symport - two or more different solutes transported in the SAME direction
* antiport - two or more different solutes transported in the OPPOSITE direction
* mediated transport can be classified as:
* electroneutral - transport results in no difference in charge across the membrane
* electrogenic - transport results in a charge difference across the membrane
74
Classify the kind of transport protein:
Na+, K+ ATPase is responsible for transporting 3Na+ out of the cell as 2K+ into the cell at the expense of 1 ATP
electrogenic antiport active transport protein
75
Western Blot
1. Protein sample is electrophoresed
2. Transferred to a membrane
3. Visualized by an immunological procedure
Reason for use: not given in handout, but essentially to isolate detectable proteins
76
RNases
Hydrolyze RNA
77
DNases
Hydrolyze DNA
78
Exonucleases
* Enzymes which degrade DNA or RNA one base at a time starting from either the 5' or 3' end of the molecule
* 5' exonucleases
* 3' exonucleases
79
Endonucleases
* Enzymes which hydrolyze in the interior of a polynucleotide
* Some are specific to 5' and some for 3' side
* DNase I cuts double stranded DNA in a nonspecific fashion
80
Restriction Enzymes
* Endonucleases that recognize specific base sequences in double-helical DNA
* Cleave both strands of the duplex DNA
* Found in a wide variety of bacteria
* Restrict the growth of bacterial viruses (bacteriophages)
* Valuable tools for:
* Analyzing chromosome structure
* Creating recombinant DNA molecules
* Isolating genes
* Sequencing very long DNA molecules
* Often recognize palindromic sequences
81
How are restriction enzymes used to create a DNA library?
* Recombinant DNA molecule created by linking of restriction enzymes of DNA from one organism w/ another
* DNA fragments are linked to a vector (bacterial or viral fragment of DNA) which can be propagated in the appropriate host
82
Cloning DNA Procedure
_Cloning into Bacteria_
1. DNA fragment from a genomic or cDNA library is introduced into a plasmid
2. Plasmid is reintroduced into a bacteria (transformation)
3. Bacteria possessing the "engineered plasmid" are identified and isolated
_Selection of Vector_
* pBR322 is an example of a plasmid vector
* Desirable Characteristics
* possesses a number of sites which are cleaved by different restriction enzymes
* Bears genes which code for proteins that impart **resistance** to antiobiotics (permits selection of bacteria into which the construct has been inserted)
* Possesses internal signals which permit the insert to be transcribed and subsequently translated into the eukaryotic protein of interest
_Formation of Cohesive Ends_
* **Sticky ends**
* Through hydrogen bonding complementary extensions from enzyme digestion facilitate joining of DNA fragments by **ligation**, an enzymatic joining of DNA by phosphodiester bonds
_Isolation of the DNA to be Cloned_
* If cloning experiment begins with "pure fragment" of DNA to be cloned, then all of the transformed *E. coli* cells should contain the DNA of interest
* In many cases, gene isolation is wanted which requires:
1. Screen DNA library, a group of genesj
2. Have a method for selecting the clone that possesses the gene of interest
83
What are the two basic types of gene libraries and how are they constructed?
What size of genomic DNA can be cloned in a lambda vector? What is a COSmid?
1. Genomic Library
* a digest of the genomic DNA
* created by cloning all the DNA from the genome of an organism, use lambda phage to keep important sequences within the same clone or few clones
2. cDNA Library
* DNA prepared from the total mRNA of the cell
Ultimately, a library consists of a population of transformed cells bearing different fragments of DNA or cDNA
Lambda phage vectors can accomodate 20kb.
COSmids are plasmids which contain DNA sequences (called cos sites) that allow phage DNA to be packaged into a bacteriophage protein coat allowing efficient expression. DNA inserts can be up to 50kb in size for COSmids
84
How is insert DNA prepared?
* restriction endonuclease is introduced to genomic DNA
* ex: EcoRI
* creates sticky ends
* fragments are seperated by size via electrophoresis and appropriate size range is removed from the gel for cloning
85
Discuss DNA sequence frequencies, specifically moderately repetitive DNA and a nonrepetitive fraction.
* in eukaryotic DNA, sequences can be highly repetitive (many copies in the genome), moderately repetitive, or nonrepetitive (single copy)
* **moderately repetitive DNA**
* both transcribed and untranscribed sequences
* transcribed, moderately repetitive examples: genes that code for 5.8S, 18S, and 28S ribosomal RNAs (rRNA), tRNA, amd 5 classes of histone proteins
* they have an untranscribed spacer in between them
* # in each genome go from several hundred to several thousand
* need these multiple copies to meet needs at certain times for the RNAs or proteins for each they code
* requirement for the synthesis of large candidates of histone proteins during cell division.
* highly repetitive - known as satellite DNA, found in centromere region of chromosome, not transcribed
* ex: Alu I, may have role in recombination of genes
86
Differentiate between histones and nonhistones.
* histones - major quantitative proteins responsible for packaging the DNA
* 5 classes of histone proteins: H1, H2A, H2B, H3 and H4
* all are very basic due to presence of arginine and lysine
* have a large net positive charge and can bind to negatively charged molecules (like phosphates in DNA)
* nonhistones- regulatory proteins, enzymes, amd other structural proteins
* heterogeneous class of proteins
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Chromatin structure
DNA organization - haploid cells contain how many base pairs of DNA? How many genes exist for humans?
* combination of DNA with the histones form chromatin
* highly condensed is heterochromatin
* less condensed is euchromatin (active transcription taking place)
* haploid human cells contain approx. 3.5x109 base pairs of DNA
* about 25,000 genes are estimated to exist for humans
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Nucleosome structure with specificity about H1
* nucleosome is a repeating unit within chromatin
* each nucleosome contains about 200 base pairs of DNA, two molecules of histones H2A, H2B, H3, and H4 and one molecul of H1
* H1 is bound to the linker DNA
* core forms an octomer sphere
* DNA wraps around core, using sphere for most surface area in small space
* H1 proteins interact to form a solenoid structure and binds to both the DNA of the core particle and the linker DNA
* takes on an important role in this order of packaging
* width of nucleosome is 11nm (100A)
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Why does acetylation occur?
* disrupts the binding of histones to DNA in order for the DNA to be replicated and transcribed
* both acetylation and phosphorylation of the N-terminal aa in histones changes their positive charge to negative, separating the histones from the negatively charged DNA wrapped around them
* acetylation of histones occurs strongly in expresseed regions of chromatin
* gives rise to R-banding of chromosomes in histology
* high mobility group proteins (HMG) are needed for unwinding of nucleosomes to produce regions of DNA that can be transcribed
