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Cell Bio final

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by: Kali Webster

Cell Bio final 327

Kali Webster

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Final exam
Cell Biology
Study Guide
Cell, Biology
50 ?




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This 30 page Study Guide was uploaded by Kali Webster on Saturday April 30, 2016. The Study Guide belongs to 327 at Texas Christian University taught by in Spring 2016. Since its upload, it has received 10 views. For similar materials see Cell Biology in Biology at Texas Christian University.


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Date Created: 04/30/16
CELL BIOLOGY Lecture 1 (1-11-2016) CELL STRUCTURE - Free-living means anything that can survive on its own in a given environment (virus is non-living) - Fundamental functional unit-cell - Differences between the fundamental unit of plants and animals- o Plant cell- cell wall o Animal cell- no cell wall (not as protected from osmotic pressure) - Do all living organisms need oxygen- No (sulfur(live in deep-sea vents)) o Using sulfur as the electron acceptor - Most diverse group of organisms- prokaryotes (bacteria, etc.) - What is the consistency of the cytosol of the cell- Gel-like - Things need to be transported through the cell - Organelles are fixed in space in the cell, limited movement, need to be transported - Loosely-packed part of the DNA (euchromatin), densely-packed part (heterochromatin) - Nucleus holds the DNA^ - Membrane of nucleus- what can leave and enter the nucleus - During replication- the DNA (before the cell divides)- it condenses into the “metaphase chromosome” - ER- transport and modification/processing of proteins- - Golgi- involved in transporting and modification of proteins - Light microscopy- the first- all organisms are made up of cells - Fluorescence Microscopy-tag parts of the cell o Using fluorescent antibodies - Transmission electron microscopy- Look at specific organelles - Atoms are mostly empty space o FIRST CHAPTER- KNOW ORGANELLES AND FUNCTION and STRUCTURE OF DIFFERENT MEMBRANES - Cytoskeleton- gives your cell shape and mobility o Actin, o myosin o microtubules (larger (hollow tubes) than microfilaments (thin threads))  Important during cell division, transporting, etc.  Form the cytoskeleton - Model organisms- organisms used in the lab to represent humans o Ecoli o yeast (saccharomyces Cerevisiae/ Schizosaccaromyces pombe) o Arabidopsis thaliana (mustard plant) o Drosophila melanogaster (fruit fly) o Caenorhabditis elegans (worm- apoptosis) o Mus musculus (mouse) o Danio rerio (zebra mussel) Lecture 2 THE MOLECULES OF LIFE - What kind of interactions are common in the cell and are short-lived o Covalent bonds are permanent- they are not “short-lived” o Hydrogen, ionic, electrostatic bonds aren’t permanent - Fluorescent microscopy- easier to see proteins in the cell, the ability to see objects in the cell is the same as the light microscope because… o The resolving power of a microscope is limited by the wavelength of light o The resolving power is how easy it is to see details inside the cell o The electron microscope, you can see DNA and other minute things in the cell - Which organelles has both an inner and outer membrane? o Mitochondria: Endosymbiosis theory. (The original organism had its own membrane, and then the second organism engulfed it. - Mitochondria divides when it wants to, can’t survive outside the cell. (endosymbionts) o There is a mutual relationship between the cell and mitochondria o KNOW ALL BONDS INVOLVED IN CELL INTERACTIONS, KNOW STRENGTHS, AND WHAT BONDS CAN BREAK - Water is the most abundant in the cell - The presents of life and water are closely linked - Surface tension, due to the H-bonds(weak) and their interactions - Water is polar, so the oxygen take electron, hydrogen gives away electrons - Water can dissolve other polar molecules - If water was ionic, it wouldn’t dissolve - Polarity moves charge about a molecule - Can’t dissolve in water- hydrophobic/ Can- hydrophilic - Proteins will fold to protect the hydrophobic part of the protein - KNOW DIFFERENCE BETWEEN KETOSES (had ketone group) SUGARS AND ALDOSES SUGAR (had aldehyde sugar)-know structures o Glyceraldehyde- 3-C sugar - ribose- five-C sugar -glucose- 6- C sugar - DNA contains ribose (deoxyribose) - Glucose building block for many structures in the body - Ribose, 5 prime C forms a link between a 3 prime C or another ribose (numbering system is important- MEMORIZE) - Glucose and Galactose, and mannose are the isomers, but different substrates (the orientation of the hydroxyl groups are different) - Mannose is used to track proteins/distinguish them - Based on the plane and position of the hydroxyl group (alpha glycosylic bond vs beta) - You can create branches, chained polysaccharides - Chiton- cytoskeletons - Know structures of sugar derivatives (glucosamine, etc.) - Glucose+ fructose= sucrose - Glucose+ glucose= maltose - Galactose + glucose= lactose - Oligosaccharide- less than 100 sugars stuck together. More= polysaccharide LIPIDS - Lipids are hydrophobic - Lipids hydrophilicity gave rise to the first cell - If it’s a long chain of carbons, it’s a long fatty acid - Cell stores fatty acid as a long energy source- generated ATP - Fatty acids are stored in peroxisome where the fatty acids are broken down - Lorenso Oil’s- had a defect that couldn’t transport the fatty acids into peroxisome - Saturated= all single bonds - Unsaturated fatty acid= one or more double bonds forms a kink o Polyunsaturated fatty acids- more than one double bond - Cell membranes are made up of triglycerides o KNOW GLYCEROL STRUCTURE - Fatty acids (3) stuck on a glycerol molecule is a way to store fat triacylglycerol) - Phospholipid bilayer- first enclosed liquid- evolved into the first protocell - Micelle- all hydrophobic ends of the lipids, turn to the inside and form a ball - RNA, in a smaller area to move, and it is an advantage of the formation of the first cell - In a phospholipid, one tail is saturated and one in saturated, gives rise to be fluid cell membrane and not rigid - Non-polar= hydrophobic - Long-chain fatty acids are like steroids because they are looped/folded fatty acid- cholesterol and testosterone(steroids) o Cholesterol is made in the liver which is then modified into different hormones - Amino acids can be acidic, basic, non-charged, etc. o Basic side-chains: lysine, arginine-polar/hydrophilic - Optical isomers (come in a L or D form)- the L form of amino acids is always the one in the body and the only one that will form in the body Lecture 3 Energy, catalysis, and biosynthesis - Amino acids are held together by peptide bonds by condensation reaction - polypeptides are very flexible (single bond and a lot of rotation) - folding involves a process that gives no free energy - KNOW DIFFERENCE BETWEEN THE PYRIMIDINES AND PURINES (WHICH ONE IS WHICH) - Nucleotides consists of nitrogen containing bases - KNOW NUMBERING SYSTEM FOR SUGAR - ATP (nucleotides can be used as energy to make unfavorable reactions to occur) - CoezymeA acts as… - Ribose is a monosaccharide that is the starting material to synthesize nucleotide building blocks - H-bonds are weak, but when thousands come together in a nucleotide strands, the bonds are very hard to break - During the covalent bonds of proteins, they have a high affinity to one another and wont attach to other proteins (low-affinity= proteins won’t bind to each other) - Van Der Waals Attraction: similar to a polar interactions - H-bonds happen because the Hydrogen atom loses an electron(non- covalent)- important in DNA, and two parts of proteins coming together (gives structure in biological systems) - Electrostatic interactions: negative change interacts with the positive charge, this is influenced by water(water breaks up interactions between ions)- these bonds are fairly weak - “rate of dissociation”- low affinity= high rate high affinity= low rate - When oil is put in water, the surface area decreases - Free energy is energy available for useful work that comes from bonds being broken and - It’s easier to go up in energy if it’s one small step at a time - Reactions are sometimes catalyzed by energy carriers - KNOW CATABOLIC AND ANABOLIC PATHWAYS - The world is constantly increasing the entropy (order)G o We are going against the trend of the earth, so we have to put in energy o Light energy is trapped and converted into an energy currency that gives energy to other systems - Biological reactions require enzymes to speed up reactions - IGNORE EQUILIBRIUM RATE CONSTANT - Spontaneous= once the reaction start, it will go to its completion (negative energy value) - The burning of paper is spontaneous (it will occur, but not necessarily fast) - Enzymes lowers the activation energy - The breakdown of ATP is a negative delta G (free energy) - Free energy is a natural property of all molecules in the universe - Coupling reaction, the breakdown of one molecule can be used to make a non-spontaneous reaction to occur - KNOW EQUATION FOR DELTA G - Km is the affinity constant, the bigger Km is, the faster the reaction occurs - If the affinity if higher, Km will be lower - Vmax is the amount of enzyme available o (1/2Vmax)- substrate concentration where the reaction reaches half of its original rate o Vmax- where the maximum amount of enzyme is being used for the substrate - When comparing two graphs showing the affinities of enzymes to their substrate, we look at 1/2Vmax - Steep curve means that the reaction is going fast - You ca make them into linear graphs by using 1/[S] as x and 1/v as the y (don’t need to know that) - KNOW DIFFERENCE BETWEE COMPETETIVE AND NON-COMPETEITVE INHIBITION o Competitive: competes for the active site o Non-competitive: may attach to a different location on the substrate o Inhibitor reduces the affinity of the enzyme to its substrate - Coupled reactions using energy ‘carriers’: o (+) delta G- increasing entropy: happens in our body all the time o Breakdown of ATP is energetically favorable(-7.3 kcal/mole), and it can make an energetically unfavorable reaction occur like the biosynthesis of a molecule (putting two molecules together)- ex. Sugar (-7.3 + 5.3= -2) o ATP breakdown and isn’t used, energy released at heat- fever o Energy carries (NAD,NADP, FADH2, ATP, Acetyl CoA(used to build long- chain fatty acids)): Carries energy for coupling to occur o KNOW FUNCTIONAL GROUPS LINKED TO EACH ENERGY CARRIER  Ingest food, breakdown food, generates energy, links it to energy carries, transfer energy to make unfavorable reactions occur  Energy carriers are hydrolyzed/reduced to create high-energy energy carriers - Biosynthesis: o condensation is used, linking to molecules together, releasing water(unfavorable) o Hydrolysis: takes water and breaks molecules down (favorable) o ATP -> AMP+ + 2Pi -> energy Lecture 4 Shape and Structure of Proteins - Alzheimer’s is the disease involving protein folding - A single base pair mutation in the globin gene results in the change in the protein that leads to Sickle Cell Anemia. This change disrupts the quaternary structure of the protein - The disulfide brides occurs in the Tertiary structure - folding of the beta pleated sheets starts to occur in the secondary structure - Primary: polypeptides sheets - Secondary: alpha helixes and beta pleating sheets - Tertiary: the alpha helixes and beta sheets start to fold (non-covalent bonds) - Quaternary: two polypeptides come together - The First level of folding: - Secondary structure: o A-helix and B sheet come together which is determined by the bond angles - Tertiary: o Different parts of the protein coming together based on the polar amino acids coming together. Come together so that the hydrophobic parts of the amino acids are on the inside (spontaneous) o Polypeptide may conform to form the cell membrane (hydrophobic)  H-bonds, electrostatic, Van Der Waals interactions  Chaperone proteins assist in folding  Looks like a box-> can stuff polypeptides into the box-> helps protein fold by unfolding it(the amino acids in the box pulls at the amino acids of the polypeptides until it forms its ideal structure-tight)-> box opens to release the properly folded protein o Mutations can cause proteins to not fold corrections (prions- also attach to correctly-folded proteins and convert them to incorrectly folded proteins)- Alheimers/parkingsons/mad cow  “Heat-shock proteins”- if you shock a protein with heat, these proteins will start producing these proteins to protect itself - HOMEWORK: - Somatic mutations occurs in a single cell in developing somatic tissue. This cell is in a population of identical mutant cells o Consequence: If the mutation is in the tissue in which the cells are still dividing, then there is the possibility of a mutant clone occurring. - A Germ-line mutation occurs in special tissues that is set aside in the course of development to form sex cells o Consequence: If the mutated sex cell participates in fertilization, then the mutation will be passed on (may be what occurs when parents are unlike offspring) - If the mutation occurs in the introns, then there will be no seen problem. If, however, the mutation occurs in the exons, then there will be a seen mutation in the offspring(alter gene expression) - Transposons (“jumping genes”): segments of DNA that can move around to different positions in the genome of a single cell. The movement can… o Cause mutations o Increase/decrease the amount of DNA in the genome of the cell, and if the cell is the precursor of a gamete Lecture 5 - Coiled-coils are typically driven by hydrophobic interactions - A change that increased to affinity of Ras or GDP would cause the uncontrolled growth of cancer cells GENOME EVOLUTION - We all evolved from one “ancestor chromosome” o If we compare chromosome 14 from humans and chromosome 12 from a mouse, the genes exons are very similar (some cross over, but most are parallel to each other (synteny- the order of the exons are generally the same) o Transposon(short pairs/DNA elements)- sequences in our genomes that can move around  Some transposons are active, some are inactive  Can cause changes in the genome  Can transcribe to make RNA, which can make DNA, can insert into the DNA of a different of same chromosome (random)  Retrotransposition  First discovered in corn-maize (different kernels have different colors) o A corn gene moved into a color gene - You can trace the beta globin gene to an ancestral globin gene (leghemoglobin) o Which split, and then split again o We have discovered the family tree of the globin gene o Some of the globin genes are turned on or off o Why do we have these different globin genes? How are they functionally different? Why are they expressed at different time? o There must be a strong selective pressure to keep these different globin genes o Overtime, mutations (variation) occurred and the globin genes started to diverge o You can draw a graph, counting the variation between genes and create a timeline o Ribosomal RNA (important to protein synthesis)- old gene, more species that have it  If the gene is new, the presents of it in species is very low  Comparing humans to bacteria, we have similar sequence in the small subunit of rRNA sequence o There are very few differences between humans and monkeys  The change in the sequence doesn’t always change the amino acid (wobble position-the third letter in the codon)  If you change the third level in a codon, it doesn’t always change the code for the amino acid (ex. ATA is the same at ATT)  The protein is more conserved than the nucleotide sequence  KNOW THE MECHANISM OF THE CHANGE OF NUCLEOTIDE BUT NOT THE AMINO ACID SEQUENCE - Exons and start and stop sequences are the most important - What is the logic behind divergence and conservation? o Overtime, mutations have occurred. If the mutation is in the exon, either the cell is killed or fixed. If there is a mutation in the introns, the cell doesn’t have to die, and if it’s not fixed, it usually doesn’t matter o There is no mechanism to select against or for introns o Mutations aren’t directed o The sequences in the introns that are important is…. The splicing sequences.  They are important because of snurfs that are important for splicing introns - In the zoo graph, the green represents the conservation of the gene (it’s maintained in the exon) o You can use the graph to see what genes are conserved, and even if it’s not in the exon, the genes that are conserved are the more important genes - How to genes evolve? o Variation, mutations in genes o When certain genes are turned on or off o Gene duplication (gene is split), when there is mutations in the promotor, the genes will be turned on at different times o Exon shuffling o Transposition (jump into sequences-serve as agents for gene evolution), may make a chromosome shorter, and the sequence that jumped out may jump on another chromosome. o Recombination: during mitosis, the sequences may cross over in the chromosomes are similar which then creates a whole new chromosome (due to transposons) o Horizontal transfer of genes onto another gene/ chromosome (ex. Antibody resistance) o Point mutation leading to changes in gene expression is the lactose gene  Back in the day, humans didn’t drink milk, only babies did from their mother, so the gene to digest milk was only turned on at early stages in development. Everyone was lactose intolerant! Then a variant occurred in the regulatory portion of the gene where adults could digest the lactose, the mutation was inherited because those humans that had it got more nutrients. - The gene in the digest protein and clotting protein are similar because of exon shuffling and a common ancestor - REVIEW WHAT DNA LOOKS LIKE AND THE STRUCTURE - DNA is in the nucleus, the unused parts of the DNA is tucked away in the heterochromatin o KNOW HOW DNA IS PACKAGED o In cell division, the DNA is condensed o Histones are important in gene expression o LOOK UP HISTONES/GENE EXPRESSION Lecture 6 - READ AND LOOK AT THE FIGURES IN THE TEXTBOOK - Promoter doesn’t code for proteins - Stability is inherent to the amino acid sequence - Look at pseudogenes - H2A, H2B, H3, and H4 core histones o Bind to each other to form a bead, DNA wraps around the bead, and the same amount of DNA is wrapped around each one. (electrostatic interactions) o The DNA from one side of the nucleotide to the other is the same length - Linker histone: H1 - The DNA within the chromosomes, the DNA is organized with the nucleus o If it’s a diploid cell, you have two copies of each chromosome in different parts of the cell (localized) o We label DNA with colors  We take DNA that’s complementary to the sequence, and it will bond to the DNA (hybridization)  You take a probe (complimentary to some sequence on the DNA), it will only bind to that specific sequence. We label the probe with color (fluorescence microscopy)  Distinct colors shows that there are clumps of DNA/ chromosomes that are fixed  Fluorescence In situ hybridization - Euchromatin is DNA being used by the cell - Heterochromatin is DNA that is permanent being put away by the cell because it’s not being used - When you need to express a gene (turn it on), it’s dragged into a different location in the cell (middle of the nucleus) o Hypothesis: in the middle of the nucleus is transcription factories for transcription o Nuclear neighborhoods (where it’s either transcribed or just sat there) - We have 3.2 billion base pairs within the human genome - We have about 30,000 nucleotides in our genome - Percent of DNA sequence in exon (protein-coding sequences)- 1.5% - In prokaryotic and viral genes, there are more coding sequences in their genomes (more efficient) - There are about 13,000 base pairs in human protein coding genomes o The rest are repeat regions (LINEs, SINEs) the amount of unique sequences in 50% o They estimate that there are 100,000- 150 bp microRNAs in the human genome  Important for gene expression - The human genome between people are almost identically - The difference in sequence is small but significant (single nucleotide changes) o Polymorphism (SNPs): small changes in the single nucleotide sequence o If this happens in the regulatory region or the coding sequence in the gene, it can cause a change in the function - Transcriptome: acts to catalog all the transcription that occurs in our genome - Proteome: acts to catalog all the proteins in our genome - Kinome: acts to catalog all the kinases in our genome - Interactome: acts to catalog all the interactions of proteins in our genome- tells us what the protein does DNA REPLICATION AND PACKAGING - REVIEW DNA REPLICATION - KNOW STRUCTURE OF A CHROMOSOME AND THE SIGNIFICANCE OF THEM o Centromere: o Replication origin: where replication begins o Telomere: end of the chromosome - To convince the histones to let go of the DNA, they have to be modifies o Nucleosome remodeling: carried out by the remodeling complex (attach phosphate groups, acetyl groups, or methyl groups) o All the histones have tails- where the remodeling occurs o Conformational change- the protein alters the way it folds  Methylation= creates heterochromatin (gene silencing)  Methylation and acetylation= gene expression  Phosphorylation and acetylation= gene expression  Put on acetyl groups= turns on gene expression (acetylates and deacetylases) Lecture 7 - Okazaki fragments are found on the lagging strand - G/C- pyridine (they have three hydrogen bonds so it’s harder to break apart) - A/T- purines (2 hydrogen bonds, so it’s easier to separate the two strands) - Interphase calls contain chromosomes that are loosely packed and occupy specific regions of the nucleus - Chromatin components are not retained when the classic “beads on a string” structure is the most decondensed chromatic structure (linker histones)- - H1 is the linker histone- octamer (two of each) - An alteration in a change that decreased the rate of hydrolysis of GTP by Ras will cause cancer - Modifications that change on the histones, can be passed down to your children and maybe their children (this can be caused by environmental factors (smoking, BPA)- epigenetic modification- adds functional groups to it - X- inactivation- females have two X chromosomes, so one has to be inactive- randomly o One chromosome is bundled away and cannot be used (heterochromatin)- random o All daughter cells from the inactivated or not are the same, and will be passed down to your offspring DNA REPLICATION/REPAIR - KNOW ENZYMES INVOLVED AND OKAZAKI FRAGMENTS - DNA polymerase can proofread its work - RNA polymerase cannot proofread - Only A with pair with T, and only G with pair with C- self-correcting naturally o Mistakes do happen when the base pairs isomerize  When this happens, the DNA polymerase detects the change and stop, and remove the wrong base  Two active sites on the enzyme: one for polymerizing and one for editing  When you have the wrong base pair, the diameter changes and DNA polymerase can detect it - Because DNA is a double helix, it wraps around itself and generate a coiling tension causing it to super-coil (coiling the coil) o When your pulling the two strands apart, further down the DNA, it gets more “twisty”  When the Helicase gets down to the very twisty part, the DNA helicase doesn’t have enough energy and DNA replication may stop  So the coiling of the DNA is relaxed as its being pulled apart  You relax it by cutting on strand of the DNA, so it’s free to rotate around the phosphodiester bond of the other strand- DNA topoisomerase I  Spontaneous re-formation of the strand occurs  DNA topoisomerase II: resolves intertwined DNA o The cell clips one of the strands, pulls the other strand through and repairs the strand again  There are many inhibitors of topoisomerase’s to treat conditions (antibiotics are specific to bacterial topoisomerase’s) o IDENTIFY THE TARGET OF A SPECIFIC DRUG - Telomere: end of chromosome o There is a problem because you need to remove the primer and replace it with DNA (the gaps are sealed with ligase) o Each round of cell-division, the strand gets shorter and shorter  The running down of the telomere, tells the cell when to die  Replicative cell senescence  Germ cells (constantly dividing, but they never get shorter- this is prevented from happening by an enzyme binding to the uneven end of the chromosome, used the RNA molecule as a template and the 3-prime end is lengthened and then the other strand will come and put down primers (repeated sequences that are lengthened by telomerase) o The gene for telomerase is turned on in germ cells and cancer cells  Cells turn off telomerase to control cell division in somatic cells too - Dyskeratosis congenital: mutation in telomerase genes o How many genes can be effected? - Natural radiation effects your DNA; o BRCA 1 and 2 are involved in DNA repair- mutations are correlated with breast cancer o Mutations are accumulated; the longer you live, the more mutations and the greater chance you’ll have a mutation that is deleterious  Ex. Depurination: where you have a removal of the purine (chemicals/radiation) where you bread the bond between the sugar and the base  Deamination: modification of one base converts it to another base (happens all the time!)  UV radiation: can use the energy to create bonds (ex. Fusing of two thymine bases)- dimer pulls two bases closer to each other which caused a change of the diameter of the helix  If not fixed, it will make a permanent change  Depurination may cause a frame shift because when it removes a purine, there’s a gap  Repair enzyme goes down the DNA strand and pulls out and looks at each base to make sure it’s right- Base excision repair  Cuts out the DNA of either side of the damage, and remove a stretch of DNA- nucleotide excision repair (NOT ON EXAM): - How does the repair enzyme know which base is the wrong one? o When a C is methylated, then it converts it to T- it assumes that the T got there by mistake, and it will convert the T to a C  There is a higher probability that there will be a mutation is the wrong base is taken out  The enzyme will look for a nick (which only occurs in the new DNA (daughter)) and fix the abnormal base - Why are there mutations? o One DNA is prone to damage (radiation) o The enzymes aren’t 100% accurate o Mutations lead to variation/genetic change which leads to adaptation o Natural section favors an imperfect DNA polymerase Lecture 8 - Xeroderma pigmentosum is caused by defective DNA repair enzymes - Hyperactive telomerase can cause cells to become resistant to replicative cell senescence (doesn’t necessarily cause cancer) - The end result transcription leads to the production of RNA o The end result of translation leads to the production of DNA - What is the differences between exon shuffling and alternative spicing o Alternative splicing only occurs on RNA o Recombination does ‘c (used to produce the exact protein that the cell needs in a given environment by mixing exons) TRANSCRIPTION - The production of RNA from DNA (only messenger RNA is translated into protein) o Only 30,000 RNA is translated into protein (over 100,000 RNA) o RNA polymerase binds to DNA (doesn’t know where to bind-it’s not specific- binds to any DNA that is exposed)  RNA polymerase has a low affinity to DNA o Sigma factor binds to the promoter (promoter= TATA Box(TATATT))- then allows the RNA polymerase to stay on the DNA (increases the affinity)- it unwinds the DNA- runs down the DNA- reads one of the strands of DNA (template strand)- creates an RNA strand that’s complementary to the DNA (identical to the coding strand of DNA, except for the U’s replacing the T’s) – at the end of the gene, there is a termination sequence that makes the RNA polymerase come off (terminator isn’t found on Eukaryotic cells-found in bacteria) o Promotor isn’t found on the RNA- only on DNA (function is to provide an area for the RNA polymerase to bind to the DNA) o There’s no nucleus in prokaryotes  Cotranscriptiontranslation (transcription and translation can occur at the same time)  In eukaryotes, transcription occurs in the nucleus, translation occurs outside the nucleus  RNA polymerase always has the same orientation on the DNA (3 prime to 5 prime direction) - All protein- coding genes (mRNA) are transcribed by RNA polymerase II - RNA polymerase I transcribes more rRNA (ribosomal RNA) - TATA is the minimal sequence in order to transcribe mRNA- if you mutate the TATA box, transcription will stop or slow to a unhealthy level - General transcription factors: bind to any gene with a TATA box on it- non- specific (TFIIB, TFIIF, TFIID) - TF stands for the transcription factor (II strands for o TFIID: has a domain, recognizes and binds to the TATA box, other transcription factors occur o KNOW TFIID, TFIIF, TFIIE, TFIIH (KINASE PHOSPHORALATES THE RNA POLYMERASE- CYD IS PHOSPHORALTED BY TFIIH- GIVES CGANCE TO RNA POLYMERASE II AND MAKES IT STICKY (WRAPS AROUND DNA) TO OTHER PROTEINS-PROTEINS INVOLVED IN RNA PROSESSING) - One gene will be transcribed by multiple RNA polymerases - Rate limiting step is the RNA polymerase attaching to the promotor (starter step) - How to proteins interact/ effect DNA? o DNA binding proteins make contact with the nucleotide sequence o Amino acids attach to specific bases o THINK ABOUT MUTATIONS AND THEIR EFFECT ON THE CELL o We aren’t sure what tells the RNA polymerase on the eukaryotic genes to stop transcribing o RNA polymerase gets phosphorylated multiple times which makes it more sticky o Capping-splicing(introns are removed)-poly A tail (proteins jump of the RNA in this order)  Open reading frame–the ribosome can read the sequence from start to finish  The cap is really important for the initiation of protein synthesis  The tail is important to stabilize the RNA (proteins bind to the tail and makes sure the RNA doesn’t get degraded)  Hyperactivation of a gene can lead to hypertranscription (too much protein)-cancer/death  Structure of cap: another nucleotide that’s modifying (7- methylguanosine)- gets attached to the 5-prime end of the mRNA  Splice the mRNA (there is no capping/splicing or RNA processing in bacteria) only in eukaryotes - Why do eukaryotes have such complex genes with introns? - Introns have to be removed from the RNA (removed from left to right)- splicing (cutting introns and connecting exons together (snRNP’s)) o Spicing reaction involves a reactive adenine which attracts the 5 prime junction (the intron catalyzes its own removal) and slides down to the 3 prime end o snRNP’s are a mix of protein and RNA and direct splicing (U1 and U2 bind to start splicing, and other snRNP’s bring them together so that the reaction can occur)- A attacks the exon-intron junction o From one DNA, you can make 5 mRNA and therefore 5 proteins o Microcephalic osteo… (MOPDI)- inability for the sNRP to do what it’s supposed to do (mutation in a gene encoding U4atac snRNA, a component of the minor U12 dependent spliceosome) - Polyadenylation- addition of a PolyA tail (pre mRNA mRNA) o Customs of the nucleus o Eukaryotes are monosystronic o Prokaryotes are much faster at making proteins, no processing - Genetic code is universal o 1/3 of all genetic diseases… - Anticodon on the tRNA binds to the mRNA and is complimentary to the codon - For each amino acid in the cell, there is one (tryptophanyl) amino acid tRNA synthetase - If it’s the correct tRNA, it’ll bind tightly, if it’s the right amino acid, it’ll bind tightly and they will come together - rRNA (ribozyme) has enzyme activity, and binds the polypeptides together o in the active sight, the rRNA presents adenine - Elogation: o P- site is there the polypeptide with the tRNA sites - eF2 binds to the small ribosomal unit, and uses GTP (pre-initiation complex), binds to the cap (recognizes eIF-4E) on the mRNA (5 prime end), slides down, looking for the start codon (scanning), the large subunit binds to the small subunit, and you now have the whole protein o kozak sequence is around the start codon, and it makes the ribosome stop there and - Termination: when you come to the stop codon, there is a protein, the bond between the polypeptide and the tRNA breaks, and releases the protein - Once the protein comes off, it starts to fold into its final structure - Know the targets of the antibiotics - Misfolded/ old proteins are degraded in the proteasome (ubiquitin attaches and rinds the protein to the proteasome, pushes the protein through and degrades it) Lecture 9 - Epigenetics and mutations are involved in - Start codons are not needed in transcription, only translation - Operons contain a cluster of genes transcribed as a single mRNA, and are only in prokaryotes, only can be regulated by repressor proteins, and only involved RNA polymerase I - The tryptophan operator binds to the tryptophan repressor when the repressor is bound to tryptophan - all genes contain all the genes in the genome o What genes are on and which are off?  Microarray: divided into grids, on each square, you glue mRNA or DNA from one gene, each grid has a sequence of a different gene  You can dye molecules with fluorescence, and you can tell which RNA/DNA is being made, not being made a lot, or at all (off)  Allows us to see what genes are expressed too much (cancer) and which to attack SECTION 2 - DNA binding motifs (shape/folding pattern) o Major groove vs minor groove recognize the sequence due to bonds o If you change a base, you change the binding o Zinc finger motifs bind to DNA o Leucine “zipper” stick on a polypeptide, it will come and stick o Gene- regulating proteins (makes it easier for the general transcription factors on the DNA) o Dimerizing and coming in in different combinations allows the proteins to attach to different sequences o We have about 2,000 gene regulatory proteins, - Helix-loop-helix proteins can interact with a second protein containing a DNA recognition domain or one without a recognition domain (ex. MyoD (apax transcription factor), drives differentiation in muscle cells) o You need two DNA binding domains o Homodimer / heterodimer - Know positive and negative control and - Lac operon (you make all the proteins or none) - Its more efficient for your body to break down glucose (bacteria will reject lacrosse if there is glucose) o If they have glucose, the bacteria won’t turn on lac operon o If given both, they won’t either o If they don’t have lactose, they will shut down lac operon with a repressor (negative control) o If only lactose, cap will bind and lac operon will turn on (cyclic AMP will go up)  When cyclic AMP comes in, there will be a conformational change in CAP, allowing it to bind to DNA  Lactose will bind to the repressor and then RNA polymerase can bind and start transcribing it (activator= positive control) o If none, the cyclic AMP will go up, the operon won’t turn on o KNOW THIS PROCESS - Trip operon regulates the production of tryptophan - Eukaryotes don’t have this - Eukaryotes have gene regulation that involves dozens of transcription factors that come together around the TATA box o They do this by bending the DNA o Complex=enhanceosome (-some meanings “coming together”) o Transcription regulators bind to the mediator (“enhancers”-sequences on the DNA) that are far away from the TATA box and bind because the DNA is bend by proteins o Proteins bind to the DNA to bend it, allowing the enhancers come close to the start-site o Cytokines are produced by this process and activates your immune system o Methylation of DNA (on the cytosine) can shut down gene expression  Methylation patterns can be inherited by daughter cells and be passed on  Example of Epigenetic control (you’re not changing the gene but the  Changing the histones - You can regulate gene expression post-transcriptionally (RNA interference) o Makes microRNA which is then made into SIRNA are complimentary to specific mRNA’s bound by proteins which then splits and may block translation or mRNA rapid degradation o siRNA will knock down the expression of a gene by degrading it (knockdown) NEW SECTION - READ CHAPTER 11 - Eph- protein on the surface of cells o EphB2+ EphB3: if missing a protein, their penis and anus fused together Membrane structure - Bilayer - Transport machinery (Eph)- proteins embedded in the membrane - Lipid bilayer: 5nm thick - Proteins may send signals from one side of the cell to the other, or connect cells together - 3 classes of lipids that make up the membrane o Phospholipids: constructed of fatty- acid tail (hydrophobic), platform which its attached to (glycerol backbone), phosphate, and a head group (polar) (sometimes serine) o Glycolipid: hydrophilic tail, sugar head (outside of the cells-outer leaflet)  Glycolipids cluster due to attractions between adjacent oligosaccharides and adjacent hydrocarbon tails o Cholesterol: all have hydrophobic and hydrophilic groups- steroid ring, and polar head-group, and nonpolar tail  The whole structure interacts with the phospholipids  Doesn’t change the fluidity of the membrane, unless there’s A LOT  Increases degree of lipid packing  Decreases membrane permeability  Decreases membrane flexibility  Enhances the formation of lipid rafts  Lipid rafts: groups of cells that move together  Cholesterol forces phospholipids in specific domains o Microdomains (lipid rafts-proteins in them) - Phosphatidyl-serine: phospholipids (hydrophobic and hydrophilic structures) o When it flips, it signals that the cell is in trouble (phosphatidyl Serine, is mostly inside, when the cell get stressed, initiated programmed cell death, the phosphatidyl serine flips - Water tries to form a “cage” around the hydrophobic molecule (very energetically favorable) o Free- fatty acids take on a micelle (solid ball with hydrophobic in, hydrophilic out) o Phospholipids take on a bilayer (two tails, one saturated, one unsaturated)- liposome  Sphingomyelin and PTC (phosphatidyl-choline) – common in cell membranes - Fluidity: o More saturated, longer= less fluid, o Less saturated, shorter chain length, cis-double bonds= more fluid, heads are closer together  How to you know how fluid it is?  Take phospholipid bilayer, label it with fluorescence (fluorescent tag on a protein)  FRAP: use fluorescent recovery after photobleaching o See how quickly they recovery the bleached area Lecture 2, week 2 - Secondary active transport: o Glucose and Na+ o SGLD1 protein utilizes secondary active transport o There is energy involved - Channels have a weaker bond to the solutes vs pumps - The energy to move ions against their electrochemical gradient can come directly from ATP and the electrochemical gradient - Neutrophils rolling inside the membrane of the capillary o Neutrophils use lectins to bind in the glycolylax membrane o Sugar residues are involved o ATP isn’t involved - Ca2+ getting in and out of the cell o PMCA: in the plasma membrane takes ATP to power it  Takes Ca2+ out of the cell] o SERCA: in the membrane or SR or ER takes ATP to power it  Takes Ca2+ and pumps it back into the SR or ER - Coupled transporters: o Uniport transports one solute, doesn’t couple the transport of one solute with another solute o Symport the two solutes move in one direction (they can use ATP)  SGLT1 glucose and Na+ o Antiport the two solutes move in different directions (they can use ATP)  Na+, K+ pump o THERE IS NEVER NOT AN ELECTROCHEMICAL GRADIENT o LOOK AT THE EXAMPLES OF TRANSMEMBRANE PUMPS - Channels: o Solute moves down its electrochemical gradient o K+ pump  Vestibule K+ ions are surrounded by water  Too big to go through the pore  Water needs to be ripped off  Selectivity filter filter that allows one some ions to go through  Lined by carbonyl Oxygens (C=O: :O=C)  Distance of oxygens are the selectivity filter - In the previous example of a K+ channel, Na+ can’t go through because… o Na+ is smaller than K+ and thus doesn’t interact with the selectivity filter tightly enough - Most channels are closed, and they all need some sort of signal to open - At the end of hair cells, there is a K+ channel at the end of the filaments o The hair cells vibrate on the tectorial membrane o The stereocilia on the hair cell goes one way, they open, goes the other way, they close o The liquid on the outside of the hair cells has a higher concentration than on the inside of the hair cells K+ rushes in - If there is no K+ outside of the hair cells… o Sensory cells release neurotransmitters (acetylcholine) o Ca2+ rushes in, and allows the vesicles to fuse and for the neurotransmitter to be released  Sag Gets ramped up with K+ comes in Lecture 3 week 2 (2/19/2016) - PMCA is a uniporter- Plasma Membrane Calcium ATPase (only transfers Ca2+) - Sometimes channels can randomly open without a stimulus to cause them to open o If they are a “gated” channel, they spend most of their time in the closed position - The Tip-links at the end of cochlear hair cell stereocilia are mechanically- gated channels o All skeletal muscle cells use mechanically gated channels - Only certain cells have the ability to generate action potentials (neurons, muscle cells, ) o You need many voltage gated channels o KNOW FIGURE IN BOOK ON VOLTAGE GATED Na+ CHANNEL  Electrical potential (say -70 to -40mV)causes channels to open Na+ rushes in voltage changes(depolarized) channel opens very very quickly (the channel is open but inactive because the loop of the protein that’s part of the channel is plugging the hole) K+ rushes out (repolarization)when the cell is back to – voltage, the channel then closes  At .25 ms, not many Na+ come in (it takes A LOT to make more Na+ inside the cell than outside)  Resting potential-70mV (now the Na+ channel can close)  When Na+ comes in, the Na+ goes up and down-steam on the edge of the membrane  soon the voltage gets more positive and K+ rushes out  Most of the Na+/K+ voltage gated channels are down- stream of the initial segment of the neuron  The propagation of the Na+ channels is one way  Voltage-gated K+ channels are much slower than Na+ channels  K+ channels open when Na+ channels are inactivated  Na+ K+ ATPase pumps are pumping 3 Na+ out and 2K+ out (resetting the membrane)  Na+ doesn’t stick around  “What happens if this is a resting neuron and you come right in the middle and zap it with a voltage…” the voltage goes both ways because none of the Na+ channels are closed o Ca2+ channel (voltage-gated)  Mostly closed until electro-potential is a little more positive on the inside  At the end of the membrane and comes in in order to fused the vesicles to fuse (there are neurotransmitters in these vesicles) these then are released into the synaptic cleft  bind to ligand- gated channels on another neuron Na+ that’s high on the outside comes rushing out (DEPOLARIZATION!)  But how does the electrical-potential go from the cell body to the initial segment? Week 3 - Chapter 15 - KNOW ORGANELLS AND FUNCTION - 54% cytosol, 22% mitochondria, 12% ER, 6% nucleus, 3% Golgi apparatus - All organelles are surrounded by a lipid bilayer like the plasma membrane - The address in the protein is always an amino acid which tells the protein where to go o Proteins enter the nucleus through nuclear pores o Nucleus is very rigid/stiff cytoskeleton o Go through a meshwork of filaments to get into the nucleus o NLS sequence- “take me to the nucleus” o Nuclear importer will bind to the protein with the NLS, and it will dumb its cargo into the nucleus o Ran-GTP (when importer and nuclear protein comes into the nucleus) causes the importer to dump its cargo  When importer makes it to the cytosol, the Ran-GTP is hydrolyzed, Ran-GDP is then fallen off o NF-AT (T-cells) normally in the cytosol, phosphorylated, the nuclear sequence is hidden, sits in cytosol, when phosphate groups are taken off (calcineurin-phosphatase (when calcium is there)),  when calcium levels are low (nucleus) calcineurin falls off and NF-AT is taken out of the nucleus - proteins unfold to enter mitochondria and chloroplasts o membrane is more fluid o transport proteins in the membrane o two lipid bilayers  some proteins need to be in lumen, matrix, inner membrane, or outer membrane o cytosol to matrix  TOM (transporter of outer mitochondrial membrane),  TIM (inner) Multi-pass proteins  When the protein arrives to the transport protein, the protein has to be stretched out in a long peptide strand (chaperone protein help the protein to not fold back (Hsp70)) ATP (NEEDED) causes the removal of Hsp70  Once protein is in the matrix, peptidase will cleave the signal sequence off the protein - Peroxisomes o Proteins enter peroxisomes from both the ER or cytosol o Peroxisomes are detoxifiers o Form myelin when myelin is wrapped around the axon, the proteins are pushed to the side  Na+ cannot get out until it passes the myelin chief (Na+ jumps and causes the action potential to go faster)  Phospholipid is made in the peroxisome which makes myelin  Zellweger Syndrome (mutates in the Pex2 gene)’ Week 2, Lecture 2 - Cathrin has adapter protein (adaptin) recognizes cargo proteins o Phosphatidylinositol’s reacts with dynamin and sequences, you end up with vesicle dynamin falls offadaptin falls off vesicle  Dynamin requires energy in the form of  Mutation in dynamin loses functioning in peripheral nerve neurotransmitter release (Charcot-Marie-Tooth) destroys neurotransmitters to make muscles contract - Vesicles has to dock in order to embed proteins in the plasma membrane o RAB vesicle docking  Interact with vesicle by phosphatidylinositol’s  Tethering protein recognizes RAB and brings it close to the target membrane  Requires Ca2+ to come in the  SNARE proteins (in vesicle and membrane) SNAREs interact  In the presence of Ca2+, they twist together and pull membrane pieces of vesicles and target membrane together  Inner leaflet of target membrane and outer leaflet of vesicle bind together after SNAREs start twisting then you end up with inter leaflet of vesicles forming into the outer leaflet of target membrane  Synaptotagmin is the Ca2+ sensor  interacts with vesicle SNARE and allows the SNAREs to interact (ESSENTIAL FOR VESICLE TO FUSE TO MEMBRANE)  SNARE proteins are targets for toxins (tetanus, Botox, etc.) o RAN in the nucleus Week 2, lecture 3 - Calcium, action potential, voltage-gated channel, and sodium potassium pumps is required for neurotransmitter release - Which is not needed for vesicles fusion: RAB - Proteins embedded in the membrane usually have - Carbohydrates are added in the lumen of the ER and the lumen of the Golgi - Lipids link to proteins in the ER and the Golgi where sugars are added (glycolipids) o GPI-linked proteins (glycolphosphatidylinosital) o We almost never find GPI-linked proteins in the cytosol side of the cell membrane or the outside of the Golgi/ER membrane - Chaperone proteins (BIP/HSP70) bind to proteins to help them fold properly o Proteins that fold incorrectly mutations in the chaperone proteins o Proteins aggregate together and they clog up cells o ER and cytosol - If they fold incorrectly the cell ramps up production of new proteins - TOW protein aggregate together, microtubules fall apart, then the proteins aggregate together, and neuron doesn’t work  Alzheimer’s o Cell then turns up transcription and translation of the TOW proteins ER gets bigger - Sensory mechanisms when the signals detect a change in the folding, there is a signal that goes into the cytosol (heat shock proteins are an example of this) - What happens in the Golgi modification starts at the cis part is closest to the ER and goes to the trans side - Targets: o Plasma membrane secrete o Secretory pathways  Constitutive pathway just dumping things out  Regulated secretion pathway is signaled - Endocytic pathways: o Phagocytosis--. Macrophage destroys bacterium (surrounds its membrane around) brings to (driven by receptor by signals in the macrophage reorganizes the cytoskeleton and pushes its own membrane around) o Pinocytosis bring in fluid and small molecules outside of the cell  Primary mechanism to recycle the membrane o Receptor-driven endocytosis  Cholesterol in the cell cholesterol is packaged in LDL binds to LDL receptorsrecruits clathrin in the cell membrane forms vesicle with receptor and LDL in them  Recycling pathway vesicles fuse and form large endosome PH drops and LDL with receptor breaks free and then be able to signal again at the plasma membrane o What do you do with everything inside the vesicle?  Recycle back out (LDL receptor/aquaporin’s)  Bring it to lysosome and degrade it  Trans-cytosis: happens in the gut  IgA has an important function in the lumen but made on the underside of the epithelial tissue of the gut  IgA binds to receptor membrane buds off vesicles have receptor (IgA molecule in them) fuse on the top membrane in the gut goes out and antibody is not in the lumen o Lysosome: all acidic enzymes in the lysosome  You put an ATP-driven pump on the lysosome and brings H+ inside the cell  Week 4- Lecture 1 - Innate immunity o What your body immediately does when a pathogen is in your body o The macrophage/monocyte  Primary function phagocytosis  Secondary function activation of T-lymphocytes (part of the adaptive response)  Makes cytokines (hormones) o Macrophage recognizes any pathogen and gram negative bacteria (all have LPS that’s recognizes by macrophage) o Flipase stops functioning phosphatidylserine is put on the surface of the cell o Major sites of entry of pathogens into your body  Respiratory, gastrointestinal, and urogenital systems (90% of macrophages are focused on these areas)  Bone marrow “stem cells” make neutrophils, macrophages and monocytes  If you are lacking adaptive immunity response,  #1 Macrophage recognizes pathogen initiates immune response engulfs pathogen in phagosome degrades pathogen  #2 receptor recognizes bacterial component triggers production of cytokines, goes through Golgi, goes into lysosome and goes out of the cell as inflammatory cytokines  Have to have Ca2+ binds to cynaphogamin, SNARES twist, need RAB to make lysosome, dynamin squeezes  Extracellular pathogens: bacteria, viruses, fungi, parasites are phagocytosed into cell  Pathogen comes in within 4 hours, you have initial response innate response tries to finish the pathogen adaptive response is there incase innate response can’t finish it (activated of B and T cells)  Complement receptors coat the pathogen and internalize the pathogen and there is a signaling event that leads to transcription of cytokines, and/or phagocytosis occurs and degrades the pathogen  Sometimes the pathogen can get out of the phagosome and doesn’t get into lysosome and stays into cytosol  PAMP- recognize pattern that can be on multiple pathogens o LPS is a patter, glolipids, sugars (manose)  Pseudopodia is when the actin fibers are able to remodel the cytoskeleton to wrap around pathogen  Signal recognition  Pathogen gets phagocytized destroyed in the phagosome proteins are degraded into amino acid chains fit into protein that is made in the ER shipped into Golgi RAB allows vesicles to fuse to the phagolysosome  MHC is two proteins that present the peptide on the surface of the cell T-cells recognize the peptide and initiates the killing of the pathogen o All leads to inflammatory response (make cytokines) o Blood-born macrophage: monocyte  Virus is utilizing what’s in the cell to make its own proteins proteasome take the long chain of amino acid and chews them up the chewed up pieces(short peptides) are then shuttled into the ER and recognized by MHC class 1’s goes to the plasma membrane and bring the peptides and can be recognized by T- cells  MHC molecules are always presenting self-peptides, but sometimes is presenting viral peptidesThis is called self- peptide  TAP transporter for antigen(peptides) into the ER lumen  Calnexin and BIP are chaperone proteins and help to make the protein/MHC fold  2 kinds of T-cells that recognize peptides that the MHC presents  Power of 8: o CD8 recognize pathogens presented by MHC class 1 o CD4 recognizes peptides on MHC class 2  IkB inhibits IKK by blocking NFkB  RANGtp binds to transporter and NFkB is released back out of the nucleus and is then GDP, and falls off Week 4, lecture 2 - Neutrophil sometimes called polymorphonueclear cell - Mature neut


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