Topic IA-IC Flashcards
what’s collagen
3 α-helices woven together to form a 2˚ helix; makes up most of the extracellular matrix of connective tissue; strength & flexibility
what’s elastin
component of the extracellular matrix of connective tissue; stretches and recoils, restoring original tissue shape
what’s keratin
intermediate filament proteins in epithelial cells; contributes to mechanical integrity, acts as regulatory proteins
what’s actin
makes up microfilaments & thin filaments in myofibrils; most abundant protein in eukaryotic cells; has polarity
what’s tubulin
makes up microtubules
antibodies
immunoglobulins, Ig = proteins that function in the adaptive immune system. The antigen is the antibody’s ligand.
transport proteins
responsible for binding small molecules and transporting them to other locations in the organism.
The proteins must have a high affinity for the ligand when the ligand is present in high concentration [low concentration = low affinity]
motor proteins
generates force through conformational changes; myosin, kinesin, dynein; crucial for cellular motility. Uses ATP hydrolysis.
myosin
protein for contracting muscles
kinesin
intracellular transport; moves in anterograde (center to periphery)
dynein
intracellular transport also motility of cilia; retrograde
Cell adhesion molecules = CAM
Binds cells to other cells or surfaces. Includes cadherins, integrins, and selectins. All are integral membrane proteins.
Cadherins
glycoproteins; mediates calcium-dependent cell adhesion
Integrins
has 2 membrane-spanning chains & permits cells to adhere to proteins in the extracellular matrix; signaling capabilities; α and β binding
Selectins
allows cells to adhere to carbohydrates; most commonly used in the immune system; weakest bonds; expressed on WBCs & endothelial cells
Receptors
proteins that bind to signaling molecules, ie insulin
Ion channels
passageway for membrane bilayer; aqueous pores which are accessible after conformational change that causes the ion channel to open
-Binds a specific substrate, either to sequester it or hold its concentration at a steady state
Ligand
ion or molecule attached to a metal atom by coordinate bonding
Ungated channels
always open
Voltage-gated channels
open within a range of membrane potentials; no ligand involved
Ligand-gated channels
open in the presence of a specific binding substance, usually a hormone or a neurotransmitter; binds to allosteric site, rapid
enzyme-linked receptors
Uses extracellular ligand binding and second messenger cascades Cell-surface receptors; normally have large extracellular & intracellular domains; membrane-spanning region has a single α-helical region of the peptide strand
G protein coupled receptors
G protein coupled receptors are the largest class of membrane receptors and are found in eukaryotes only. Acts as molecular switches.
G proteins can bind GTP (activated) or GDP (inactive). Also initiate second messenger systems: ligand binding engages the G-protein. GDP is replaced w/ GTP; α subunit dissociates from the β and γ subunits. The activated α subunit alters the activity of adenylate cyclase or phospholipase C. GTP is dephosphorylated to GDP and the α-subunit rebinds to the β and γ subunits.
Cell-surface receptors
Cell-surface receptors = integral proteins that bind to external ligand molecules; performs signal transduction. Cell-surface receptor components: an extracellular domain, hydrophobic membrane-spanning region, and an intracellular domain inside the cell.
Lyases
- XABY↔ AB + XY / A = + XY
* Catalyzes cleavage without H₂O or the transfer of electrons
Synthases
the synthesis of 2 molecules →1 molecule; may also be catalyzed by a lyase
- Only requires 1 substrate when cleaving, but 2 for the reverse
- Generates either a double bond or ring structure
- Hydrolysis of fats
Isomerases
- AB ↔ BA
- oxidoreductases, transferases, lyases
- interconversion of isomers, constitutional & stereoisomers
- named as [substrate]isomerase or [substrate]ase
Ligases
- X+Y + ATP ↔ XY + ADP + Pi
- Nucleic acid synthesis
- Joining two large biomolecules, often of the same type
- Addition/synthesis reactions; requires ATP
- Named as [substrate]synthase or [substrate]synthetase
- Ligase is used to create bonds b/t Okazaki fragments
- Remember: synthase could also be a lyase but synthetase must be a ligase.
Hydrolases
- AB + H₂O ↔ AOH + BH
- Phosphatases, peptidases, nucleases, lipases
- Cleavage with H₂O
- Named as [substrate]hydrolase or [substrate]ase
Oxidoreductases
- A + H ↔ AH (reduction)
- A + D ↔ AD (oxidation)
- Dehydrogenase, oxidase, peroxidase
- Catalyzes oxidation-reduction reactions involving electron transfer
Transferases
- AB + C ↔ A + BC
- Moves a functional group from one molecule to another
- Aminotransferase
- Transaminase
- Methyltransferases
Kinases
• Kinases = phosphotransferases, usually with ATP as donor.
◦ Adds a phosphate group
Phosphatases
• Phosphatases = removes a phosphate group
Phosphorylases
Phosphorylases = introduces a phosphate group into an organic molecule, notably glucose
Polymerases
Polymerases = nucleotidyltransferases
Reduction of activation energy
Reactions must move through a high energy transition state before becoming products. In stabilizing the transition state, an enzyme ↓ activation energy and ↑ reaction rate.
Temperature changes
Doubles in velocity for every 10˚C ↑until the optimum temp is reached, then activity falls sharply (denatures)
Enzymes
stabilizes the transition state. They only affect the rate at which a reaction occurs; how quickly it gets to equilibrium, but doesn’t affect equilibrium itself.
Active site
the location within the enzyme where the substrate is held during the chemical reaction; defined spatial arrangement; site of catalysis
Substrate:
the molecule that the enzyme acts on
Lock & key theory:
enzyme & substrate are exactly complementary
Active site model:
location on the enzyme where it reacts w/ its substrate. Shape/characteristics (functional groups) of an active site are responsible for the specificity of the enzyme
Induced fit model:
initial binding = when the substrate first binds to the enzymes (not perfect). Enzyme & substrate mold their shape to bind together super tightly. Binding b/t the reactant & enzyme is strongest @ the transition state.
Orthosteric
regulatory elements interacting with enzyme @ it’s active site
Allosteric
those that bind @ a site other than the active site
Ligand
substrate + regulatory element
Cofactors
Inorganic ions and metal cation; like Fe²⁺ and Mg²⁺; directly involved in the enzyme’s catalytic mechanism
Apoenzymes
without cofactor
Holoenzyme
with cofactor
Prosthetic groups
tightly bound cofactors or coenzymes that are necessary for enzyme function
Approximation
brings reactants together in proximity and proper orientation
Mechanism catalysis
how a reaction is assisted in proceeding
Coenzyme
Organic molecule; like heme, NAD⁺ & Coenzyme A. Many are derived from vitamins.
vitamin precursor for NAD
Vitamin B3
vitamin precursor for CoA
vitamin B5
water soluble vitamins
B complex and ascorbic acid (C)
fat soluble vitamins
DAKE
Enzyme kinetics
characterizes the catalytic behavior of enzymes, specifically focusing on reaction rates.
Saturation kinetics
[substrate] ↑ = reaction rate ↑ until a max value is reached
v =
Km:
Km: The [S] at which an enzyme runs at half its Vmax. Km is a constant. Enzyme-catalyzed reactions w/ high enzyme-substrate affinity will reach the ½Vmax benchmark at a lower [substrate] aka have a lower Km; lower enzyme-substrate affinities → needing a higher [substrate] to reach ½Vmax (have a higher Km).
Km can be used to measure the affinity that an enzyme has for its substrate
Michaelis-Menten (not equation)
Hyperbolic curve. Calculates the rate of reaction (vₒ) using Vmax, the substrate concentration ([S]), and the Michaelis constant (Km).
Michaelis menten equation
Lineweaver-Burk
- X-intercept is -1/Km
- Y intercept is 1/Vmax
- If Vmax ↓ = y-intercept ↑ (moves further from origin)
- If Km ↓ = x-intercept ↓ (moves further from origin)
Irreversible inhibition: alters the ____ so …
alters the enzyme so that the active site is unavailable for a prolonged duration or permanently; new enzymes molecules must be synthesized for the rxn to occur again
Suicide inhibitor
A substrate analogue that binds irreversibly to the active site via a covalent bond.
Allosteric effector
Binds at the allosteric site and induces a change in the conformation of the enzyme so the substrate can no longer bind to the active site. Displays cooperativity, so it does not obey MichaelisMenten kinetics.
◦ Positive effectors: exert a positive effect, ↑ activity
◦ Negative effectors: negative effect ↓ activity
Homotrophic effector
An allosteric regulator that is also the substrate.
Heterotrophic effector
An allosteric regulator molecule that is different from the substrate.
Lineweaver-burk plot: y, m, x, bm 1/vo, slope =
Feedback inhibition
regulatory mechanism where the enzyme’s catalytic activity is inhibited but the presence of high levels of product later in the same pathway.
Reversible Inhibition
ability to either replace the inhibitor w/ a compound that has greater affinity or remove it with a lab tx. 4 main types of reversible inhibition.
Competitive inhibition: Binding site- Impact on KM- impact on Vmax- effect on X-intercept of LWBP- effect on Y-intercept of LWBP-
inhibitor is similar to the substrate and binds @ the active site
◦ Inhibitor binds in place of substrate in enzyme’s active site
Binding site- active site Impact on KM- increases impact on Vmax- no change effect on X-intercept of LWBP- ↑ (moves toward origin) effect on Y-intercept of LWBP- no change
Noncompetitive inhibition
Binding site- Impact on KM- impact on Vmax- effect on X-intercept of LWBP- effect on Y-intercept of LWBP-
Noncompetitive inhibition: inhibitor binds with equal affinity to the enzyme and the enzyme-substrate complex
◦ Inhibitor can bind to allosteric site either before or after the substrate has bound
Binding site- allosteric site Impact on KM- no change impact on Vmax- decreases effect on X-intercept of LWBP- no change effect on Y-intercept of LWBP- ↑ (moves away from origin)
Uncompetitive inhibition Binding site- Impact on KM- impact on Vmax- effect on X-intercept of LWBP- effect on Y-intercept of LWBP-
Uncompetitive inhibition: the inhibitor binds only with the enzyme-substrate complex.
◦ Inhibitor can only bind after substrate has bound and opened up the allosteric site
Binding site- enzyme-substrate complex
Impact on KM- decreases
impact on Vmax- decreases
effect on X-intercept of LWBP- ↓ (moves away from origin)
effect on Y-intercept of LWBP- ↑ (moves away from origin)
Mixed inhibition Binding site- Impact on KM- impact on Vmax- effect on X-intercept of LWBP- effect on Y-intercept of LWBP-
Mixed inhibition: the inhibitor binds w/ unequal affinity to the enzyme and the enzyme-substrate complex
◦ Inhibitor can bind to allosteric site either before or after the substrate has bound, but has a higher affinity for one state over the other
◦ If the mixed inhibitor ends up binding more readily to the enzyme Km is higher
◦ If the mixed inhibitor ends up binding more readily to the enzyme-substrate complex Km is lower
Binding site- allosteric site
Impact on KM- increases or decreases
impact on Vmax- decreases
effect on X-intercept of LWBP- ↓ or ↑ (moves towards or away from origin)
effect on Y-intercept of LWBP- moves away from origin
competitive inhibition
noncompetitive inhibition
uncompetitive inhibition
cooperativity
Cooperativity: substrate binding ∆ substrate affinity
Hill coefficient
measure of cooperatively (ie Hb, where you see sigmoidal kinetic curves that don’t follow Michaelis-Menten)
hill coefficient n < 1
Substrate binding decreases affinity for subsequent substrate
↑ in KM, parameter value _____; intercept ______; moves ____ origin
↑ in KM, parameter value doubles; intercept ↑ (becomes less -) ; moves towards origin
↑ Vmax, parameter value _____; intercept ______; moves ____ origin
↑ Vmax, parameter value halves; intercept increases (becomes more +); moves away from origin
enzyme turnover equation
enzyme turnover # = Vmax / total concentration of available enzymes
catalytic efficiency equation
(enzyme turnover #)/ Km
catabolism vs anabolism phosphorylation
catabolism = active; anabolism = inactive phosphorylation
Dephosphorylation
removing a phosphate group = catalyzed by phosphatases
Glycosylation
covalent modification w/ carbohydrate (adding sugar)
Zymogen
enzyme precursors; secreted in an inactive for and are activated by cleavage (ie trypsinogen)
Nucleosides
= 5-carbon sugar pentose bonded to a nitrogenous base; formed by covalently linking the base to C-1’ of the sugar
Nucleotides
phosphate group attached to C-5’ of a nucleoside; named according to the # of phosphates present; are the building blocks of DNA
Chargaff’s rule
dsDNA: %A = %T & %C = %G
Sugar-phosphate backbone — describe
covalent phosphodiester bonds; phosphate group forms an ester bond to the 3’ carbon of one sugar molecule and the 5’ carbon of another
B, A, Z, DNA
B-DNA = double helix; right handed; 1 turn every 3.4 nm; 10.5 bp A-DNA = tighter; 2.3 nm; 11 bp Z-DNA = zig-zag; left handed; 3.8 nm; 12 bp
DNA gyrase (topoisomerase II)
alleviates supercoiling, working ahead of helicase, nicking the strand(s) relieving the torsional pressure
DNA ligase
fuses the DNA strands together to create one complete molecule
DNA polymerase
has proofreading activity, corrects any mistakes (mutations) it makes
explain the differences in DNA replication between Prokaryotes and eukaryotes
State the differences in steps of replication between prokaryotic and eukaryotic cells
Helicase
uses hydrolysis of ATP to “unzip” or unwind DNA helix at replication fork to allow resulting single strands to be copied
Primase
polymerizes nucleotide triphosphates in a 5’ to 3’ direction. Synthesizes RNA primers to act as a template for future Okazaki fragments to build on to.
DNA Polymerase III
synthesizes nucleotides onto leading end in classic 5’ to 3’ direction.(adds nucleotides to growing daughter strand)
DNA Polymerase I
synthesizes nucleotides onto primers on lagging strand, forming Okazaki fragments. This enzyme cannot completely synthesize all the nucleotides.
Ligase
glues together Okazaki fragments, an area DNA Pol I is unable to synthesize
Telomerase
catalyzes lengthening of telomeres; enzyme includes molecule of RNA that serves as template for new telomere segments
Nuclease
excises or cuts out unwanted or defective segments of nucleotides in DNA sequence
Topoisomerase
introduced single-strand nick in the DNA, enabling it to swivel and thereby relieve the accumulated winding strain generated during unwinding of double helix
Single Strand Binding Proteins
holds the replication fork of DNA open while polymerases read the templates and prepare for synthesis (prevents reannealing)
Telomerase
lengthens telomeres with repetitive sequences proteins the tellers from loss during replication
oncogenes
promotes cell cycling = can lead to cancer; Oncogenes = stepping on gas pedal
Tumor suppressor genes
Code for proteins that reduce cell cycling or promote DNA repair = cutting the breaks.
Explain Mismatch repair, nucleotide excision repair, and base excision repair
Mismatch repair = occurs during the G2 phase using MSH2 and MLH1. It cuts the strand that doesn’t have methylation
- Nucleotide excision repair: Fixes helix-deforming lesions of DNA like thiamine dimers. A cut-and-patch endonuclease; (requires an excision endonuclease)
- Base excision repair: Fixes non-deforming lesions of the DNA helix such as cytosine deamination by removing the base, leaving apurinic/apyrimidinic (AP) sites. AP endonuclease removes the damaged sequence which can be filled in with the correct bases.
Describe the phase(s) of the cell cycle and the key enzymes/genes that occurs in each of the following: DNA polymerase, Mismatch repair, nucleotide excision repair, base excision repair
Describe the Meselson-Stahl experiment
It explained that DNA replication is semi-conservative. they grew E. coli in a medium containing 15N (heavy nitrogen) which made the bacteria’s DNA heavy. Using centrifugation, it separated things according to their weight. Initially all the DNA in the cell was all heavy and at the bottom of the tube. They then grew these cells in the absence of the heavy nitrogen, so all of the new DNA made in subsequent cell divisions would be lighter. After one cell division, the DNA was half as heavy (half of the DNA molecule had heavy nitrogen and the other didn’t. This ruled out the conservative method, which if were true, would’ve produced one molecule that was all light and the other all heavy. After 2 cell divisions, the DNA molecule was now either half heavy and half light or all light. This ruled out the dispersive method, which if were true, would’ve produced a mixture of heavy and light DNA molecules after two cell divisions. The result of the experiment was in line with the semi-conservative postulate by producing a situation where every cell gets one old DNA strand and one new one. So they discovered that the two strands of DNA can separate and this provides the template for the production of two NEW STRANDS
Describe the Hershey-Chase experiment
- Question: Is protein or DNA the genetic material of the cell?
- Background: It was known that some viruses consisted solely of DNA and a protein coat and could transfer their genetic material into hosts
- Hypothesis: Hershey & Chase conducted a series of experiments to prove that DNA was the genetic material
- Test: T2 bacteriophages (viruses) were grown in one of two isotopic mediums:
◦ Protein radiolabeled viruses = grown with ³⁵S (b/c sulfur is present in the protein but not the DNA component of the virus)
◦ DNA radiolabeled viruses = grown with ³²P (phosphorus is present in DNA but not proteins)
◦ The viruses were allowed to infect E. coli and then the virus and bacteria were centrifuged = separated
- Results: The larger bacteria formed a solid pellet while the smaller viruses remained in the supernatant
- Analysis: The bacterial pellet was found to be radioactive when infected by the ³²P–viruses (DNA) but not the ³⁵S–viruses (protein)
- Conclusion: DNA was the genetic material b/c DNA was transferred to the bacteria
Genes
codes for making polypep3des
Genetic code
sequence of bases on mRNA which tells the ribosome which amino acids to use.
what is the central dogma
DNA - transcription in the nucleus > RNA - translation in the cytoplasm > Protein
Codon
mRNA = sequence of nucleo3des, but it CODES for a sequence of amino acids. Every 3 nucleo3des = 1 amino acids. They are con3nuous, non-overlapping, and degenerate.
anticodon
is the 3 bases on the 3p of the tRNA. A single tRNA contains a single an3codon at the “3p” and the corresponding amino acid at the “tail.” An3codons are complementary to their corresponding codon.
Misssense codon
mutated codon → different amino acid
nonsense codon
a stop codon (UAG, UAA, UGA)
Initiation codon
starts translation = AUG; lies just downstream of the Shine Dalgarno sequence (Kozak sequence for eukaryotes)
Termination codon
(UAG, UGA, UAA) = ends translation; no tNA is involved; protein “release factor” comes along and terminates translation
Describe what degenerate and wobble means
degnerate = multiple codons can code for the same amino acid and wobble means that the 3rd base in the sequence could be mutated and still code for the same amino acid
Describe eukaryotic mRNA
it’s the product of transcription and the template for translation; carries the message from DNA in the nucleus via transcription of the gene; has a 5’ cap and a 3’ poly A tail to protect it from exonuclease degeneration
describe tRNA
hairpin structure responsible for translating b/t codons & anticodons (brings int eh correct amino acids during translation and recognizes the codon on the mRNA using its anticodon aminoacyl-tRNA synthetases changes tRNA molecules by attaching the C-terminus of the amino acid to the 3’ end of the tRNA molecule; requires APT; mature tRNA is found in cytoplasm
describe rRNA
it’s synthesized in the nucleolus; made of nucleotides. makes up ribosome, enzyme responsible for translation. contains the active site for catalysis. used for protein assembly in the cytoplasm.
large subunit: rRNA catalyzes the formation of the peptide chain
prokaryotes = 50S; eukaryotes = 60S
small subunit rRNA: reads the RNA
prokaryotes = 30S; eukaryotes = 40S
overall: prokaryotes = 70S; eukaryotes = 80S
is also important in splicing out its own introns w/in the nucleus
describe transcription & translation, short & sweet summary
transcription = initiation → elongation → termination
describe the 6 steps of transcription
- Helicase unwinds DNA helix; RNA polymerase II binds to TATA box
- hnRNA = synthesized from DNA template strand (an3sense)
- New strand undergoes post-transcrip3onal modifica3ons:
- 7-methylguanylate triphosphate cap = added to 5’ end; poly-A tail added to 3’ end
- snRNA & snRNPs in the spliceosome remove introns in the lariat structured
- Exons are ligated together
describe the 3 steps of translation
- Ini3a3on in prokaryotes— 30S ribosome adaches to the Shine-Dalgarno sequence; lays down N-formylmethionine in P site of ribosomes; in eukaryotes= 40S ribosome adaches to 5’ cap; lays down methionine
- Elonga3on— addi3on of new aminoacyl-tRNA to A site; growing polypep3de chain is transferred from the P site tRNA → A site tRNA and then to the E site then exits the ribosome
- Termina3on— The codon in the A site = a stop codon; release factor puts H₂O molecule on the polypep3de chain releasing the protein. Postransla3onal modifica3ons can occur
Prokaryotes increase gene variability through ____ while eukaryotes accomplish it through ____
Prokaryotes ↑ gene variability through polycistronic genes Eukaryotes ↑ it through alternative splicing.
DNA gyrase (DNA topoisomerase II)
introduces negative supercoils; alleviates the torsional stress and reduce risk of strand breakage
Tandem repeats vs trinucleotide repeats
• Tandem repeats (repeats right next to each other, eg:
CAGCAGCAG)
• Trinucleotide repeats: too much can cause diseases. Eg:
Huntington disease and fragile X syndrome
single copy DNA
a long unique sequence of nucleotides in the DNA; mainly found in the axons; are the coding regions; the sites for transcription to make mRNA; present tin the euchromatin part as they are coding regions; similar in many individuals;
DNA sequence that doesn’t repeat; has a low mutation rate
Repetitive DNA
DNA sequence that does repeat; nucleotides don’t code for proteins; found near the centromeres; in the introns and other noncoding parts of the DNA; present in heterochromatin; have a higher mutation rate; unique in different individuals
Heterochromatin
dense, transcriptionally silent; tightly coiled regions off chromatin, not available for transcription; often consists of DNA w/ highly repetitive sequences
euchromatin
majority of DNA is in the form; only prokaryotes have euchromatin; it’s the less dense and transcriptionally active DNA; appears light under light microscopy; portions are uncoiled and available to be read by transcription machinery
telomeres
capping regions that exist on the ends of chromosomes; high GC content that protects the chromosomes from degradation during replication
centromeres
single point region located in the middle of the chromosome that connects the two sister chromatids (original chromosome and its replicated partner until they’re separated)
The Operon Concept
describes genetic regulation that is the hallmark of the Jacob-Monod Model. This identifies and conceptually organizes the parts of prokaryotic gene expression as an operon; Operon = gene expression; they allow a bacterium to respond to changes in its environment by increasing or decreasing the expression of certain genes as appropriate.
positive control- operon
activator stimulates transcription
negative control- operon
repressor prevents transcription by binding to the operator
Negative inducible operon
repressor is normally present and the genes aren’t expressed except under specific conditions
Negative repressible operon
genes are usually transcribed, but transcription can be halted by binding the repressor in appropriate conditions
Promotor
regulatory DNA sequence where the RNA polymerase can attach
Promotor + multiple genes [sequences of DNA] = operon
Jacob-Monod model
Jacob-Monod model of repressors & activators— operons = inducible or repressible clusters of genes transcribed as a single mRNA and responsible for the expression and regulation of the specific mRNA transcript.
Regulatory gene
produces repressor protein that binds to operator to block RNA polymerase
Structural gene
codes for the protein of interest
Operator site
non-transcribable region of DNA capable of binding a repressor protein
Promoter site
provides a place for RNA polymerase to bind
Inducible systems
Bonded to a repressor under normal conditions; they can be turned on by an inducer pulling the repressor form the operator site. Example: Lac operon.
Repressible systems
Transcribed under normal conditions; they can be turned off by a corepressor coupling with the repressor and the binding of this complex to the operator site. Example: Trp operon
explain the main differences b/t inducible and repressible systems (8)
describe the differences between the absence and presence of lactose in E. coli (lac operon gives E. Coli the ability to metabolize lactose if it’s present (energetically expensive)).
Lactose is absent = repressor is bound to the operator and prevents RNA polymerase from transcribing the structural genes
- Lactose is present = allolactose (isomer) binds w/ the repressor; it dissociates from the operator which allows RNA polymerase to transcribe the structural genes. E coli now has the ability to metabolize lactose
- Doesn’t metabolize lactose if ample glucose is present
When tryptophan is absent or at low levels, the repressor protein is ____. the operator is _____.
When tryptophan is absent or at low levels, the repressor protein is inactive. The Operator (the gate) is opened the genes to make tryptophan are transcribed to make W.
Describe the Trp operon
Trp operon = essential for the production of tryptophan
The trp operon allows the expression of genes in the absence of tryptophan (W), but not when tryptophan is present because this would be energetically unfavorable.
In the absence of W, the repressor doesn’t bind to the operator and W synthesis proceeds.
• In the presence of W, it binds to the repressor protein and causes it to bind to the operator, thus inhibiting synthesis.
The trp operon = a repressible negative operon b/c it can be induced by environmental conditions, but transcription is not repressed by default.
- W present = attaches to the repressor molecules and ∆ their shape → activated → inhibits W synthesis
- W absent = trp repressor = inactive (b/c no tryptophan is available to bind to and activate it). It doesn’t attach to the DNA or block transcription, and this allows the trp operon to be transcribed by RNA polymerase
repressible vs inducible operons
- Repressible operons = default is on, can be turned off
* Inducible operons = default is off, can be turned on
Operator
the sequence of DNA where a transcription factor protein can bind
Promoter
the sequence of DNA where RNA polymerase binds to start transcription
Operator
the sequence of DNA where a transcription factor protein can bind
Promoter
the sequence of DNA where RNA polymerase binds to start transcription
Enhancers
sites on the DNA that are bound to by activators in order to loop the DNA in such a way that brings a specific promoter to the initiation complex which enhances transcription of the genes in a particular gene cluster.
- Enhancers are usually cis-acting (acting on the same chromosome) but they don’t necessarily need to be particularly close to the gene that it acts on.
- Enhancers don’t act on the promoter region itself, but they’re bound by activator proteins which can interact w/ the mediator complex I.
Repressors
proteins that bind to the operator and impedes RNA polymerase progress on the strand thus inhibiting expression of the gene.
Silencers
regions of DNA that are bound by repressor proteins in order to silence gene expression.
Transcriptional regulation in prokaryotes vs eukaryotes:
Prokaryotes:
‣ Transcription regulation is necessary for the cell to adapt to its environment ‣ The presence, quantity, and type of nutrients determines which genes are expressed ‣ A combination of activators, repressors, and rarely enhancers determines whether a gene is transcribed ‣ Regulates for response ‣ Simple & short ‣ Trp operon attenuation & antitermination
◦ Eukaryotes ‣ Transcriptional regulation tends to involve a combo of interactions b/t several transcription factors = more sophisticated response ‣ Since eukaryotes have a nuclear envelope which prevents transcription and translation from occurring simultaneously = adds an extra special and temporal control of gene expression ‣ Regulates for maintenance ‣ Long & complex ‣ Chromatin structure & gene expression
Transcription factors
search for promoter (w/in 25 bp of transcription start site) and enhancer regions (> 25 bp) in the DNA
are DNA binding proteins that bind to specific regions of the DNA (DNA-binding domain) to influence transcription:
◦ Activators bind to activate transcription & repressors bind to repress transcription
Gene amplification
a structural change to DNA that gives way to duplicate copies a gene being present, which in turn can lead to an increase in transcription and protein product for that gene.
Small interfering RNA (siRNA) and microRNA (miRNA)
Non-coding RNA sequences; ◦ Small interfering RNA (siRNA) = short, double-stranded; ◦ microRNA (miRNA) = single-nucleotide strands; characteristic hairpin loop; They differ in terms of structure but both are ∼ 22 nucleotides long. They both silence genes by interrupting expression between transcription and translation
Enhancer RNA
eRNA sequences are transcribed from enhancer regions of the DNA and appear to be associated with increased transcriptional activity.
compare acetylation to methylation (short and sweet)
- Acetylation = ↑ transcription = ↑ accessibility
- DNA methylation- a methyl group is added to cytosine or adenine.
- Methylation = deactivates genes (↓ accessibility)
compare acetylation to methylation (short and sweet)
- Acetylation = ↑ transcription = ↑ accessibility
- DNA methylation- a methyl group is added to cytosine or adenine.
- Methylation = deactivates genes (↓ accessibility)
Proto-oncogenes
genes that function as oncogenes after mutation or inappropriately elevated expression
Receptor tyrosine kinases
epidermal growth factor receptor [EGFR] & platelet-derived growth factor receptor [PDGFR]; transfusing signals involved in activating cell growth & differentiation
Cytoplasmic protein kinases
Src family & Raf family
Regulatory GTPases
Ras family — signaling in a major growth/differentiation pathway
Important oncogenes
◦ TP53 gene which is implicated in more than half of all human cancers
◦ Mutations in the BRCA genes = breast & ovarian cancer
Deacetylase (HDAC)
enzyme that removes the acetyl groups
Recombinant DNA
DNA composed of nucleotides from 2 different sources
DNA cloning
the process of making multiple, identical copies of a particular piece of DNA
the process of DNA cloning
Procedure:
◦ Gene or other DNA fragment is inserted into a DNA vector plasmid using restriction enzymes, enzymes that “cut”, and DNA ligase that “pastes”. This produces a molecule of recombinant DNA
◦ The recombinant plasmid is introduced into bacteria. Antibiotic selection identifies the bacteria that took up the plasmid. Then they use a reporter gene.
◦ The bacteria w/ the plasmid are grown and used as factories to make the protein. They reproduce they replicate the plasmid and pass it on to their offspring. Harvest the protein from the bacteria and purify it.
plasmid qualities
◦ Has a restriction site
◦ Has an origin of replication
◦ Has antibiotic resistant genes which let you kill the bacteria w/o the plasmid
◦ Replicates independently of the Genomic DNA of the bacteria
restriction enzymes
cut dsDNA @ palindrome sequences → restriction fragments
cleaves vertically across the recognition site → fragments = blunt ends
◦ Cleaves zig-zag fashion → sticky ends (EcoRI restriction enzyme)
◦ Sticky ends can hybridize; blunt ends can’t (Smal restriction enzyme)
• Restriction enzymes generate fragments that can be put back together by DNA ligase and doesn’t have to be in the same order as before
Process of recombination
- 2 different sources of DNA in a Petri dish
- Digest sequences w/ restriction enzyme
- Treat fragments w/ DNA ligase
Plasmids
short circular DNA; replicates independently in bacteria
1. Introduce the recombinant plasmids w/ the DNA into E. Coli
◦ 2. Plasmid & bacteria replicate
◦ 3. Plasmid DNA can be isolated making large amounts of recombinant molecules w/ the fragment of interest
◦ 4. The fragment can be isolated from the rest of the vector DNA by restriction endonuclease digestion & gel electrophoresis
cDNA libraries
made with cloned, reverse-transcribed mRNA. Lack DNA sequences that correspond to genomic regions that aren’t expressed. They generally have much smaller fragments than genomic DNA libraries and are usually cloned into plasmid vectors.
To generate recombinant DNA plasmids
- human DNA & plasmid DNA are digested w/ restriction enzymes
◦ 2. DNA ligase reseals
◦ 3. E coli cells are treated w/ the plasmid/DNA mixture → diverse set of bacteria
◦ How to distinguish b/t the bacteria that haven’t taken up any plasmids, those that took up the nonrecombinant plasmids & those with recombinant plasmids? = 4. use of antibiotics
◦ 5. Then to distinguish b/t non-recombinant & recombinant plasmids = use a reporter gene (codes for a product leading to an obvious phenotypic change & contains sites for the restriction enzyme that’s used in the restriction)
Hybridization
a technique that harnesses the base-pairing of complimentary strands to ascertain the presence of particular mRNA transcripts in a sample by exposing the sample to known complimentary mRNA and measuring the amount of binding.
Southern blotting
DNA probes hybridize onto the DNA fragments that have the target sequence
Gene cloning
hybridization is the process where sticky ends from a restriction fragment of a gene base pairs with the same sticky ends on a plasmid.
RNA sequence cloning
- RNA sequence cloning = accomplished through the generation of cDNA (complementary DNA)
- Step 1. Synthesize a DNA copy of RNA using reverse transcriptase. This makes cDNA
- cDNA ligated to vector DNA
- cDNA cloning allows the mRNA corresponding to a single gene to be isolated as a molecular clone.
PCR
technique used to make many copies of a particular region of DNA in vitro. It uses Taq polymerase (the DNA polymerase enzyme used in PCR) and DNA primers. The rxn is repeatedly cycled through a series of temp changes which allows many copies of the target region to be produced. DNA amplified by PCR may be sent for sequencing, visualized by gel electrophoresis, or cloned into a plasmid for further experiments.
PCR Basic steps
- Denaturation (96˚C) — heat the reaction to separate the DNA strands → makes single-stranded template.
- Annealing (55-65˚C) — cool the rxn so the primers can bind to their complementary sequences on the ss-template DNA
- Extension (72˚C) — raise the rxn temp so Taq polymerase extends the primers, synthesizing new strands of DNA
- Cycle repeats 25-35 in a typical PCR reaction; generally takes 2-4 hours
- Results of PCR are visualized using gel electrophoresis
Southern blot
Southern blot- used to detect the presence & quantity of various DNA strands in a sample
• Steps:
◦ 1. DNA is cleaved by transcription enzymes
◦ 2. The DNA fragments are run through gel electrophoresis, separating them based on size
◦ 3. DNA fragments are transferred to a filter membrane, still separated
◦ 4. The filter membrane is exposed to radiolabeled DNA probe. The radio-labeled DNA will be complement to the gene of interest. This will anneal the gene of interest.
‣ So far we have a (1) radio labeled piece of DNA stuffed to a (2) DNA fragment that’s complement to the gene of interest
◦ 5. We need to be able to visualize the DNA so we expose the filter to an X-ray film. B/c the piece of DNA is radiolabeled, it pops up on the x-ray film.
DNA sequencing and process
Used to determine the sequence of nucleotides in a strand of DNA; uses ddNTPS; These are missing the OH group on the 3’ carbon so they’re unable to create a new 5’ → 3’ phosphodiester bond putting us in control of terminating the replication
◦ 1. DNA strand of interest is denatured using an NaOH solution to create a ssDNA strand that we can use for replication
◦ 2. ssDNA strand of interest is added to a solution containing:
‣ A radiolabeled DNA primer that is complementary to the gene of interest ‣ DNA polymerase ‣ All four dNTPs (dATP, dTTP, dCTP, dGTP) ‣ A very small quantity of a single ddNTP (e.g., ddATP) ‣ This is done once for each of the four nucleotides in separate solutions
◦ 3. Each solution containing a specific dNTP and ddNTP are placed in their own lane of a gel and ran under gel electrophoresis. The gel is transferred to a polymer sheet and autoradiography is used to identify the strands in the gel.
◦ For each respective nucleotide, the insertion of a ddNTP will terminate replication and create various strands of different length that correspond to that specific nucleotide. Therefore, the gel can be read from bottom-to-top to determine the nucleotide sequence. The smaller the fragment, the further it travels in the gel.
Gene expression
a process where a gene is used to synthesize some sort of product
Northern blot
detects a specific RNA in a mixture of RNAs.
Uses:
Electrophoresis- separates RNA molecules by size
◦ Blotting- transfer molecules from one membrane to another
◦ probing (hybridization)- label the target molecule with a radioactive or fluorescent tag
RT-qPCR
RT-qPCR: used when the starting material is RNA.
- Total RNA or mRNA is transcribed into cDNA by reverse transcriptase
- cDNA is used as the template for the qPCR rxn
- One-step assays: combines reverse transcription and PCR in a single tube and buffer, using a reverse transcriptase along with a DNA polymerase. Only utilizes sequence-specific primers.
- Two-step assays: the reverse transcription & PCR steps are performed in separate tubes w/ different optimized buffers, rxn conditions, and priming strategies.
Western blot
detects a specific protein in a sample
◦ 1. Proteins from a sample are loaded into an SDS-PAGE gel and separated based on size
◦ 2. They’re transferred to a polymer sheet and exposed to a radiolabeled antibody (sometimes using two antibodies; one specific to protein of interest and another radiolabeled antibody that binds to first antibody) that is specific to protein of interest
◦ 3. The polymer sheet is viewed using autoradiography. The protein of interest that is bound to the radiolabeled antibody will be visible.
In-Situ Hybridization
studies gene expression in tissue or embryo.
• 1. A thin slice of tissue is fixed to a slide and permeabilized to open the cell membrane
- A labeled probe is added to the selection of tissue and it binds to the transcript of interest
- An enzyme-linked antibody is added to the tissue selection and it binds to the probe
- An enzyme substrate is added and the transcript-probe-antibody complex is detected by the substrate
- This process is used to detect where transcripts are expressed
Immunohistochemistry
uses an antibody to detect a specific protein and measure its expression. The antibody used in this process is recognized by a second antibody. The second antibody is linked to either an enzyme or a fluorescent molecule.
Immunohistochemistry
uses an antibody to detect a specific protein and measure its expression. The antibody used in this process is recognized by a second antibody. The second antibody is linked to either an enzyme or a fluorescent molecule.
Hematopoietic stem cells
differentiate into types of blood cells
Intestinal stem cells
basis for the constant renewal of cells lining the intestines
Mesenchymal stem cells
capable of differentiating into a wide range of cell types — adipocytes, osteoblasts, & hepatocytes
Totipotent
able to differentiate into any cell type; zygote → morula
Pluripotent:
differentiates into any of the germ layers (ectoderm, mesoderm, endoderm); obtained from the internal cell mass of the blastocyst
Multipotent
adult stem cells; differentiates into several types of cells w/in a relatively limited functional scope
Oligopotent
can only derive into a few types of cells
Phenotype
physical manifestation of a genetic trait; characteristics visible to the naked eye; usually appearance, but could be something like the efficiency w/ which cells carry out a certain metabolic pathway is an example of a phenotype.; ie. The flower is red
Genotype
combination of genes responsible for the phenotype; I.e. RR and Rr are genotypes for the red flower color
◦ A certain phenotype can correspond to multiple genotypes, but the converse doesn’t hold
Wild-type (w⁺)
the default phenotype or genotype that’s present in most members of a species; doesn’t have the mutation
Complete dominance
one copy of a dominant gene is enough to induce the dominant phenotype (I.e. RR and Rr both produce red flowers)
Co-dominance
two dominant alleles can be expressed at the same time
Incomplete dominance
a heterozygote displays a blended phenotype
Locus
a specific place on a chromosome
Hemizygous
when only one copy of a given allele is present; can be the result of nondisjunction in an organism w/ aneuploidy or in regard to genes on the X & Y chromosomes
Leakage
genes traveling b/t species
Penetrance
the likelihood that the carrier of a given genotype will manifest the corresponding phenotype
Expressivity
the intensity or extent of the variation in the phenotype
Hybridization
Process of two complementary, single-stranded DNA or RNA combining together, producing a double-stranded molecule through base pairing
Technique is used for interbreeding between individuals of genetically distinct populations
Gene pool
the combined set of all genes/alleles in a population; describes the genetic status of a population
Mendel’s 2ⁿᵈ law of independent assortment
the inheritance of one gene does not affect the inheritance of another gene; also applies to genes on the same chromosome b/c of crossing over.
Independent assortment-
during metaphase I, homologous chromosome pair up along the metaphase line in random orientation, some on the left and some on the right. During anaphase I, the homologous chromosomes are pulled apart and those on the right go to one daughter cell; those on the left go to the other
Recombination frequency (θ)
how often a single crossover will occur b/t 2 genes during meiosis
Crossing over
= occurs during prophase I; @ the chiasma (points of crossing over, essentially random); made possible b/c of pairing of homologous chromosomes = tetrads; which is formed by a process called synapsis
2 strand double cross over
what happens; results; makes
chromatids exchange alleles @ first, but then they exchange them back, resulting in no net recombination; results in no genetic recombination; 0/4 recombinants
3 strand double cross over
what happens; results; makes
chromatids exchange alleles during a crossover, then one of the crossover chromatid exchanges w/ a different chromatid; results in genetic recombination; 2/4 recombinants
4 strand double cross over
what happens; results; makes
the chromatids exchange, then 2 totally different chromatids on the same chromosome exchange; genetic recombination; 4/4 recombinants
when males are more disproportionately affected
X-linked inheritance
when males and females are equally affected
autosomal inhertiance
when the phenotype doesn’t skip a generation
autosomal dominant
when the phenotype skips a generation(s)
autosomal recessive
Synaptonemal complex
protein complex that glues the tetrad together
◦ The synaptonemal complex is the highly organized filament structure that is built during prophase I and is designed to carry out the process of crossing over.
◦ A group of proteins that holds together homologous chromosomes
Tetrad
a pair of four chromatids (synaptic pair)
Sex-linked characteristics
inheritance that takes place for genes on the X chromosome
Y chromosome genes
Therefore, for all intents and purposes, Y chromosome genes only contribute to male sex determination
Sex determination
determined by the 23rd chromosome set; Mom → X chromosome; Dad → Y chromosome; XX = daughter; XY = son
SRY
(sex-determining region Y) = codes for the transcription factor that initiates testis differentiation (male gonad formation); absence of the Y chromosome, all zygotes = female; Y chromosome present = male
Cytoplasmic/extranuclear inheritance
gene transmission outside of the nucleus; (inheritance of things other than genomic DNA)
◦ All cellular organelles like mitochondria is inheritsd from the mother
◦ found in most eukaryotes; commonly occurs in the cytoplasmic organelles like mitochondria and chloroplasts or from cellular parasites like viruses or bacteria
Mutation
error in DNA sequence; ∆ in DNA by means other than recombination
Random mutation
Random changes in the DNA sequence (could be due to radiation, chemicals, replication error, etc.)
Base substitution errors
one base is mutated into a different base
Inversion mutation error
a stretch of DNA (segment or chromosome) breaks off, the reattaches in the opposite orientation
◦ can occur when a mistake takes place in the directionality of a chromosome; a segment is reversed from end to end
Insertion mutation error
a segment of DNA is moved from one chromosome to another
addition mutation error
1 or more nucleotides added to the genome
deletion mutation error
1 or more nucleotides removed from the genome; also deletion of large chromosomal regions
◦ Leads to either a loss of heterozygosity or a reduction in gene dosage
Amplification mutation error
generally ↑ the gene dosage by leading to more transcription of the genes in question
Duplication mutation error
adding extra copies of a region
Translocation mutation error
chromosomal regions are moved around; a sequence of genes switches places from one chromosome to another; involves a reciprocal switch
◦ Balanced translocation = the exchange of genetic material is even
◦ Unbalanced translocation = exchange is unequal
transposons
non-coding genetic elements that can move from chromosome to chromosome; comprises > 40 of human genome; generally non-problematic unless inserted somewhere that breaks up a coding sequence
Mispairing
incorrect nucleotide is inserted on one strand of the dsDNA
Genetic Drift
- Random changes in a population (random changes in allele frequencies)
- Effect of genetic drift increases as the population size decreases
- The role of chance (strong selective pressures are absent) in determining the reproductive fitness of various alleles
Synapsis
the pairing of homologous chromosomes during prophase I
Crossing over
the exchange of genetic material during synapsis
Hardy-Weinberg Equilibrium Assumptions: (6)
◦ Organisms must be diploid and sexually reproduce
◦ Random mating
◦ Very large population size
◦ Alleles are randomly distributed by sex
◦ No mutations occur
◦ No migration into or out of the population
Hardy Weinberg equation
1 is 100%; p & q = the only two alleles of a gene present in the population
◦ p+q=1
test cross
In a test cross, an individual with a dominant phenotype is crossed w/ an individual w/ the recessive phenotype.
- IF the dominant phenotype organism is homozygous → F1 generation won’t have any individuals w/ the recessive phenotype
- IF the dominant phenotype is heterozygous → ∼ 50% will manifest the recessive phenotype.
Backcross
a hybrid is crossed with a parent or something close to the parent genetically
◦ Goal = obtain offspring more similar to the parent
◦ Mating b/t the offspring and the parent; preserves parental genotype
Parental (P) generation
on the pedigree this is the row that represents the parents
filial generation; explain F1 and F2
Filial 1 (F1) = children: the row below the parents Filial 2 (F2) = grandchildren = row below F1; children of F1 and grandchildren of P
Genetic recombination occurs between _____ and is used to figure out ____
occurs b/t maternal & paternal sister chromatids
- You can use genetic recombination to figure out the distance b/t genes on a chromosome
- More likely for recombination to occurs over a larger distance (further apart the two genes are, the more likely it is that they recombine)
Centimorgan (cM)
measures the distance on a chromosome; aka map unit
Biometry
using stats to understand biological data
T-test
a type of inferential statistic used to determine if there is a significant difference between the means of two groups
Correlation test
a statistical technique that can show whether and how strongly pairs of variables are related
Analysis of variance (ANOVA)
a collection of statistical models used to analyze the differences among group means in a sample
Natural selection
survival of the fittest; a population’s gene pool is changed over time; favoring traits that are more advantageous to the survival of the population (selective pressures); selection doesn’t take place on the individual level for specific traits
Fitness concept
reproductive success
◦ Conceptual measure of an individual’s potential for reproducing, passing on their traits to progeny ensuring their genes go on
Selection by differential reproduction
individuals who produce more viable offspring = selected for
Stabilizing selection
both extremes are selected against
Directional selection
only one extreme phenotype is selected against and the other extreme is favored
Disruptive selection
the median phenotype is selected against
Speciation
the evolutionary process of forming new and distinct species out of a shared ancestral population.
Species
a group of organisms that can successfully breed to form fertile offspring
Polymorphism
naturally occurring differences in form b/t members of the same population
Adaptation & speciation
a process where certain individuals or subpopulations develop evolutionary strategies specific to certain microenvironments, or niches; sets the stage for speciation
Adaptive radiation
the rapid rise of a # of different species from a common ancestor
Inbreeding
breeding b/t genetically closely related individuals; can → ↑ recessive mutation manifestations
Outbreeding
breeding among genetically distant members of a population
Bottlenecks
an external event dramatically ↓ the size of a population in a way that is random in regard to alleles
founder effect
results from bottlenecks that suddenly isolate a small population → inbreeding & ↑ prevalence of certain homozygous genotypes
◦ Causes: migration
◦ Population w/ a non-random sample of genes
• So founder effect = isolation while bottleneck = severe ↓ in population size .
Divergent evolution
◦ Same lineage, evolving apart to be more different
◦ Produces homologous structures (bat’s wing & horse’s hoof)
◦ I.e. bats and horses. Both share the same lineage (mammals) but the the limb of the bat became wings while the horse developed hooves.
Parallel evolution
◦ Same lineage, evolving closer together to be more similar, using similar mechanisms.
◦ I.e. feeding structure in different crustacean species. The feeding structure came from mutation of pair of legs, turning them into mouth parts
Convergent evolution
◦ Different lineage, evolving closer together to be more similar, using different mechanisms.
◦ Produces analogous structures (bat’s wing & butterfly’s wing)
◦ I.e. bats and butterflies: Both have wings, but they came from totally different lineages and evolved through different mechanisms/mutations.
Coevolution
◦ Two species evolve in response to each other.
◦ I.e. predator/prey or host/parasite species
