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SSN SBPMD Block 1
Katie Lemberg ([email protected])
Ankoor Shah ([email protected])
Introduction to the Cell
• Plasma Membrane
• Integral and peripheral membrane proteins embedded within a lipid bilayer
o Membrane phospholipids are amphipathic
o Precise composition of phospholipids varies from membrane to membrane
depending on function.
• Selective barrier: proteins in membrane determine what goes in and out.
o DNA => RNA => Proteins
o Proteins in membrane determine specific function of the cell.
Prokaryotic vs. Eucaryotic Cells
• Prokaryotic Cells: No nucleus; No cytoplasmic compartments; Ribosomes; Mesosomes =
invagination of plasma membrane; Circular, double stranded DNA; Limited size (usually
small)
• Eucaryotic Cell:Vacuolar (Cisternal) Compartment = Endoplasmic Reticulum (E.R.);
Nucleus = storage site for DNA; Membrane Bound Organelles; Cytoskeleton
Overview of Organelles:
• Cytoplasm: contains cytosol and organelles
o Cytosol contains solutes including: Carbohydrates, Proteins, Fatty acids, and Ions
• Nucleus: DNA Storage, DNA transcription, Ribosome assembly (Nucleolus)
o Membrane is contiguous with E.R.
• Golgi Apparatus: Modifies and distributes proteins
o Distributes Proteins and lipids from the rough E.R. to the plasma membrane,
lysosomes, and secretory vesicles.
o Contains three compartments each containing specific enzymes: Cis, Medial,
Trans
• Peroxisomes: metabolism of hydrogen peroxide; detoxification catalysis of FAs;
breakdown of acetyl-CoA
• Lysosomes: contains digestive enzymes, for NAs, proteins, lipids, carbohydrates, and
phospholipids
o Autolysis of dead cells
o Lower pH than remainder of the cell, prevent accidental autolysis
o *Lysosomal enzymes are tagged in ER with mannose-6-phosphate
• Endoplasmic Reticulum
o Smooth ER: synthesis of membrane lipids; steroid and triglyceride biosynthesis
o Rough ER: synthesis and secretion of membrane proteins (visibly lined with
ribosomes)
• Mitochondria: Takes up Oxygen to produce ATP
o Endosymbionic Hypothesis
Has its own genome which encodes for 13 proteins (E.H.)
Has its own ribosomes
Similar in size to bacteria
Genes may have moved from mitochondrion to host cell.
Protein Movement
• Gated Transport: ligand gated (binding of ligand opens or closes gate)
o Examples: transfer of proteins from the cytosol to nucleus
• Trans-membrane Transport: requires trans-membrane protein; channels can be
opened or closed
o Example: cytosol to mitochondria, ER, plastids, or peroxisomes
• Vesicular transport: movement within vesicles, allows for protein secretion
o Example: ER Golgi Secretory Vessicle Cell Surface
Secretion can be regulated – e.g. cell in pancreas hold enzymes until hormones signal
release
• *Signal Sequence (linear sequence) and Signal Patches (conformation of protein) direct
protein distribution
Chemical Components of the Cell: Lipids, Sugars, Proteins, Nucleic Acids
Lipids: function as barriers and prohomones (many are cleaved with fragments acting to
transduce signals
• Fatty Acids = (CH2)n COOH; Tri-acyl-glycerides = 3 fatty acids linked by glycerol
• Types of lipids differ by: polar head groups, chain length, double bonds (kinked w/
double bond)
Sugars: (CH2 O)n define carbohydrates
• Differentiating sugars:
o Number of carbons
o Position on OH groups (can create many isomers)
o Many modifications can occur: COOH, NH2, N-acetyl
• Post-translational modification can include glycosylation.
o Sugars are added to proteins via fatty acid dolichol.
o Sugars are then matured
• Maturation of Sugars
o Sugars added in the E.R. lumen are high mannose sugars with glucose
o Enzymes in the Golgi modify the polysaccharides by adding other sugar units
(vertebrates remove many mannoses, and replace with other sugar)
• Functions: 1. species defining tags; 2. define blood groups; 3. receptors for matrix
proteins & growth factor
• Other: 1. bacteria attach to cell surface sugars; 2. defective glycosylation of proteins is
linked to disease.
Protein Structure and Function—From amino acids to biological machines!
• 1o structure—sequence of amino acid residues
(We will discuss important groups of amino acids in lecture. You probably don’t need to
memorize all 20 structures, but know the breakdown by groups: acidic, basic,
hydrophobic, small&polar.)
• The peptide bond—partial double bond character, no rotation.
• 2o structure—local H-bonding patterns among backbone atoms result in a-helices and bsheets.
• --These are the structures formed using rotations about the other two bonds (,) within
the backbone. Only certain values of these dihedral angles are allowed to prevent steric
clashes (Ramachandran plot.)
• 3o structure—folding into larger globular structure (non-local, mostly non-covalent
interactions)
• --Types of interactions: salt bridges (electrostatics), hydrogen bonds, hydrophobic,
disulfide bonds (Cys-Cys)
• --Unfolded Native transition is entropically unfavorable (forcing “order.”)
• --Driving force for folding = hydrophobic collapse to put greasy residues in center, away
from water.
• Domains—“independent” tertiary structures within a larger polypeptide chain. Can be
separated from each other at low protease concentrations.
• Subunits—many proteins require multiple folded polypeptide chains interacting in order
to be functional. We will discuss one of these in a few minutes!
Enzyme Kinetics and Mechanisms
•
A General Description of Enzymes: bind substrate and lower activation energy; do NOT alter
equilibrium
• *Almost all enzymes are protein – exception --> ribozymes = RNA
• 6 Classification of enzymes
o Oxidoreductases – add/remove H atoms in redox reactions
o Transferases – transfer a chemical group
o Hydrolases – cleavage by water
o Lysases – cleavage without water
o Isomerases – change of molecular configuration
o Ligases – joining of two compounds
• Simple and Sigmoidal Types
• Hill coefficients: characterizes degree of cooperativity: higher hill coefficient less
likely binding sites are partially filled
Michaelis-Menten Plots (see curved fit graph )
• v=Vmax [S]/(km + [S])
• km = [S](1/2 Vmax)
Linewaever-Burk Plots (see linear fit graph )
• 1/v = (km/Vmax)(1/[S]) + 1/Vmax
Control of Enzyme Activity:
• Changes in substrate or product amounts
o Stoiciometric control: changes
in amount of substrate
o Some enzymes are product inhibited: accumulation of product
• Changes in amount of enzyme
• Changes in activity of enzyme, or rapid preduction of enzyme precursors
o Zymogen activation: from off to on ONLY
o Covalent modification: switch on or off
o Enzyme inhibitors: non-covalent binding
Zymogens: Inactive precursor form of some enzymes; irreversible, need inhibitor to inactivate
• Proteolytic cleavage activates zymogens
• Example: conversion of fibrogen to fibrin in blood clotting (prothrombin cleaved to
thrombin allows conversion
o conversion of trypsinogen to trypsin in digestion.
Inhibitors
• Competitive Inhibition: Reversible binding at active site (Vmax no change; Km increased)
• Non-competitive Inhibition: Reversible binding to alternate site (Km no change; Vmax
reduced)
• Uncompetitive Inhibition: Reversible binding with ES complex (Km and Vmax change,
but slope of plot remains constant (parallel lines)
• Irreversible Inhibition: Irreversible covalent bonding to enzyme, MM kinetics not
applicable
• Feedback Inhibition: downstream product of pathway inhibits enzymes earlier in the
pathway
Hemoglobin—a teaching model for cooperativity AND a protein of great clinical relevance
Structural characteristics:
• Adult hemoglobin (Hb)—4 subunits. 2, 2. Both types have globin-type fold.
• Strongest (most noncovalent) interactions between 1, 1 and 2, 2 pairs.
• Heme moiety bound within each subunit. Fe2+ in center.
• Fe2+ prefers six ligands. 4—from porphyrin ring, 1—from a proximal His. In oxyHb, the
final ligand is the O2 molecule.
Conformational change between deoxy and oxy Hb:
1. General conformational change (globally):
T
R
strong
a1
b2
b1
a2
O2 binding
Rotation and sliding of a1,b2 and a2, b1
relative to each other because of
changes in non-covalent interactions.
2.
•
•
•
Molecular level reason for change:
Heme moiety “puckered” towards proximal His in deoxy state because only 5 ligands.
O2 binds to Fe2+ and has one H bond to a distal His. Relieves heme “pucker.”
Proximal His shifts postion propagated to a global change in subunit interactions.
Example from class of change in interfacial non-covalent interactions:
Asp94 from a1 and relationship to Asn and Trp in b2 chain.
Cooperativity of O2 Binding: The Hb Dissociation Curve!
100%
Percent
saturated
pO2
Cases to Discuss:
1. Normal Hb oxygen binding:
At low pO2 _______________________________________________
After one O2 binds ____________________________________________
At high pO2 _________________________________________________
• The curve is sigmoidal. Means that protein has increased sensitivity for binding
ligand. We can think of it as if the subunits were “talking” to each other so each
is aware of the other’s state.
• P50=the partial pressure at which 50% of the binding sites are full.
2. Myoglobin and HbF
Have higher affinity for O2 than Hb does. Draw in the curves.
3. Allosteric effectors
-Small molecules that bind at distant sites from oxygen binding site, yet they affect the
protein’s conformation and oxygen affinity.
-One allosteric effector of Hb is O2. (Binding at one site affects binding at a distant
heme.)
-Other effectors:
1. H+ ( pH)—this is the Bohr effect
--Mechanism: H+ binds to any of several residues on Hb and leads to a distant
change (decrease) in O2 affinity.
2. CO2 —binds to N-terminal end of each subunit
--Contributes to pH in blood
--By binding, carbon dioxide further stabilizes T state
3. 2,3-BPG—binds between b-subunits in T state
--Positively charged residues lining this cavity interact with phosphate groups of
2,3-BPG.
--Decrease in affinity for O2.
• All three effector molecules above decrease the affinity of Hb for O2 . What will the
curve look like?
• P50 increases. You need to be at a higher pO2 to have 50% of the sites saturated.
Hemoglobin in Disease:
1. Sickle cell anemia—point mutation in DNA coding for beta chain
Autosomal recessive disease—must be homozygous for mutation to see symptoms.
Glu6 Val6 (Charged Hydrophobic on exposed surface)
Hb molecules can aggregate by hydrophobic interactions into fibers in RBCs.
This changes the shape of RBCs (sickling), influences oxygen-carrying capacity of blood,
RBCs are more fragile, etc.
2. Beta thalassemia—defect in synthesis of one or both b-globin chains of Hb.
DNA SYNTHESIS AND MANIPULATION
Max Merkow: [email protected]
SSN Workshop 9/14/05
NUCLEIC ACIDS
- Comprise nitrogenous bases, ribose sugars and phosphate backbone
Nucleoside, e.g. Adenosine
Sugar and Base
Nucleotide, e.g. Adenosine monophospate
Nucleoside and Phosphate group
-
Functions: Cell signaling (e.g. AMP is a starvation signal); Energy intermediate
(e.g. ATP); Transfer of complex groups; Coenzyme formation (Coenzyme A)
-
Polymerization
o End of growing chain, i.e. 3’-OH of ribose sugar, attacks alpha phosphate
of incoming nucleoside triphosphate.
o Displacement of pyrophosphate molecule is highly energetically favorable
DNA and CHROMOSMES
-
2 Major Points:
o 1. DNA is the language that directs cells and
o
-
2. Protein molecules package and unpackage DNA
Decoding of DNA assembles proteins to carry out cell function (e.g. bone cell),
differentiation (stem cell to mature epithelium), and death (webs of fetus).
-
**Central analogy: DNA is language in which bases are letters, words are 3 bases
long, and genes are complete instructions
-
DNA is packaged to store and separate and unwound to read and replicate.
States of Chromatin
Euchromatin
Heterochromatin
Light, unraveled, diffuse
Dark, condensed
Predominant in Interphase
10% of genome during interphase
Replication, gene expression
Trascriptionally inactive
- Chromosomal DNA is wrapped around histones, which form the basic packaging unit:
nucleosomes. Analogies: beads on a string and telephone cord
- Chromatin structure is dynamic: chromatin remodeling complexes change structure for
changing needs for gene expression, DNA replication and Chromosome separation.
- Facts to Know: fundamental principles from in Griffith and Hershey & Chase
experiments; DNA has 10 bases/ turn; .34nm/base; 3.4 nm/ turn. Each turn comprises 1
minor and 1 major groove.
REPLICATION, REPAIR, AND RECOMBINATION
-
Major Points:
1. DNA carries redundant information and thus
a. Can separate and replicate and
b. Has backup information for repair if damaged.
2. Asymmetry of DNA replication -- Figures 6-12, 6-17
3. DNA repair adds to fidelity and consists of
a. Nuclease excision of strand
b. Repair DNA Polymerase synthesis of new DNA and
c. DNA Ligase seal of nick
4. Genetic recombination is a means of genetic variation.
a. Homologous recombination (e.g. mitosis)
b. Transposons
- 1 DNA polymerase mistake/ 10^7 bp * 1 missed repair/ 100 bp = 1 mistake/10^9 bp
- DNA polymerase proofreads as it replicates; it can excise and replace mispaired
nucleotides
- Common mutations include spontaneous depurination and deamination events, UV
induced thymine dimers, and DNA polymerase errors- ultimately forming dysfunctional
proteins- BAD!
- Homologous recombination occurs between 2 double-stranded DNA molecules
anywhere there is homology in the sequence (typically two copies of same chromosome).
Rotation of Holliday junction leads to more genetic exchange.
- Transposons- only in bacteria- contrasted to homologous recombination b/c they can
move anywhere in genome.- e.g. greater expression of gene, multiantibiotic resistant
strains.
Homologous recombination
+
vs.
+
A
A
C
B
B
C
Transposition
SSN #1 – Transcription, Translation and Gene Expression
Melissa Ruiz
[email protected]
BIG Overview:
Eukaryotes AND Prokaryotes
_____________
DNA
_____________
RNA
Protein
- Transcription = _____________ synthesis
= _____________ synthesis
- Translation
** Regulation is most commonly at the level of ______________________.
RNA
DNA
Deoxyribose
Uracil
More labile (i.e. more unstable)
Base pairs _________molecularly
therefore folds into defined
structures
Eukaryotes:
_________________
Less labile
Base pairs _________molecularly
therefore double-stranded
__________________
1 RNA
Transcript
DNA
mRNA
export mRNA from ______________ to ________________ . . .
_________________
mRNA
Transcription: General overview
Protein
gene X
Strand A
Strand B
Basic Steps:
1. RNA polymerase II binds promoter
2. RNA pol II reads template 3’ > 5’, synthesizing RNA ____’ >_____’
3. RNA pol II reaches termination sequence and release transcript
Prokaryotes
RNA pol II binds DNA directly
Primary RNA transcript =
mRNA
- no introns
i.e. non-coding regions of DNA w/in
exons
No compartmentalization
Eukaryotes
RNA pol II binds DNA via __________
mRNA processing
1.
2.
3.
mRNA exported out of nucleus to
cytoplasm
- Promoter: initial binding site for RNA polymerase (e.g. TATA)
- Enhancer: short region of DNA upstream or downstream from gene that when bound
by an activator protein boosts the level of transcription of gene
* Can be several hundred thousand base pairs away
- Activator: proteins that bind enhancer sequences
- Repressors: various actions, such as . . .
• interfering with TFs binding to DNA
• interfering with activators binding regulatory regions of DNA
- Bacterial operons:
Set of genes encoding various proteins involved in a specific process; transcribed as one
unit
(i.e. via one promoter therefore at similar levels)
- Trp: fig. 8-6 ECB
- Lac: fig. 8-9 ECB
- Transcription factor: protein that binds a specific regulatory sequence thereby
facilitating
initiation of transcription
- For 3 important classes see fig. 8-5 ECB
Translation: Basic Steps
1. Initiation
2. Elongation
a. “Charged” tRNA enters A site (A = amino acid)
b. Peptide bonds forms & peptide shifts from last tRNA to “new” tRNA
c. “new” tRNA moves into P site (P=peptide)
“old” tRNA moves into E site (E=exit)
3. Termination
Regulation within transcription
1. Transcription factors
a. specific combination to transcribe a specific gene
b. anterior-posterior polarity during development (fig. 8-16, 8-17)
c. cell fate (e.g. transcription factor myoD converting epithelial cell to
muscle cell)
2. Enhancers/Activators; Repressors
3. Histone Modification fig.8-14 ECB
Human Genetic Variation
Gene Structure: Introns, exons, promoters, enhancers & splice sites are all potential
places for gene mutations
Mutations at the Gene level: changes in nucleotide sequence or arrangement of DNA,
EFFECT ON PROTEIN TRANSCRIBED IS CRITICAL
Category
Mutation
Effect on Amino
Overall effect
Acid Sequence
Point Mutations –
Silent
single base pair
change
Missense
Nonsense
Insertions/Deletions
Frameshift
Mutation: Insertion
or deletion of base
pair(s) not in
multiples of 3
Expanded Repeats
Homologous
Recombinations
Unequal crossing
over of repeats
Splicing Mutations
Destruction or
creation of a splice
site
Polymorphism: Alternate genotype expressed by at least 1% of the population, it is seen
at a frequency greater than that which can be maintained by mutation alone; e.g. blood
type. Polymorphisms at splice sites are often used to identify mutations
1. SNPs: Single Nucleotide Polymorphisms substitute a single nucleotide
2. STRs: Short tandem repeats;
3. RFLPs: Restriction fragment length Polymorphisms are mutations that alter the
site recognized by a restriction enzyme and thereby alter the fragment lengths
seen on gels. This is very useful for determining _____________________
KNOW how to calculate frequency of a mutation and carrier frequency
p2 + 2pq + q2 = 1
p = frequency of normal allele
q = frequency of mutant allele
q2 = ________________________
2pq = carrier frequency
Sample Problem: 1 in every 2000 children in the US has cystic fibrosis, what is the likelihood
that a child carries a single mutant CF allele?
KNOW the principles of the Hardy Weinberg Equilibrium: Allele frequencies are the same from one
generation to the next if…
1. the population is large
2. mating is random
3. there is no preference for or against a particular phenotype
4. mutation rate is constant
5. there is no immigration or emigration
ALSO BE SURE TO KNOW how mutations arise and what the frequencies of mutations depend on.