Unformatted text preview: Biochemistry: Mod 1 DNA = phosphate + deoxyribose sugar + A/T/C/G o Contains two strands. The strands are antiparallel (opposite each other). o 5’ → 3’ 3’ ← 5’ RNA = phosphate + ribose sugar + A/U/C/G o Single strand, can fold back onto itself and form pairs between itself (stem‐loop). Each nucleic acid is made up of polymers (many monomers) that are called nucleotides. o Nucleotides contain one or more phosphates, a five‐carbon sugar, and a nitrogen base. o Nucleotides are always made in the 5’ to 3’ direction. o 5 is always the beginning of the strand, 3 is the end where nucleotides are added. DNA organization: DNA is wrapped around proteins called histones → nucleosome → chroma n ﬁber→ chromosomes Steps to the central dogma: o Coding DNA → template DNA → mRNA → tRNA (amino acid) o DNA → transcribed to mRNA → translated to protein o Each step is complementary (opposite) to the previous step, but if you skip a step it will be identical to the previous step. o Example 1. Coding DNA strand 5’ AAA TTT GGG CCC 3’ 2. Template DNA strand 3’ TTT AAA CCC GGG 5’ 3. mRNA 5’ AAA UUU GGG CCC 3’ 4. tRNA Lys Phe Gly Pro Pairing: o DNA: A → T o RNA: A → U DNA replication: o Because DNA is a double helix, one strand can be separated and serve as a template for synthesis of a new strand. o Semi‐conservative: each copy of DNA contains a template strand and a new strand. o Steps of replication: Page 1 of 39 1. The DNA must be separated, creating a replication fork. This is done by helicase. 2. Primase attaches an RNA primer, where the replication is to start. 3. DNA polymerase adds bases to the remaining of the strand until it reaches a stop codon. This is done in fragments, called okazaki fragments. If an error is detected, it removes the nucleotides and replaces them with correct ones, known as exonuclease. o Exonuclease removes all of the RNA primers, and DNA polymerase fills in those gaps. o DNA ligase seals the two strands forming a double helix. DNA → transcribed → mRNA → translated → protein o
o Transcription occurs in the nucleus: o Initiation: RNA polymerase binds to a sequence of DNA called the promoter, found near the beginning of a gene. Each gene has its own promoter. Once bound, RNA polymerase separates the DNA strands, providing the single‐stranded template needed for transcription. Page 2 of 39 o o Elongation: One strand of DNA, the template strand, acts as a template for RNA polymerase. As it "reads" this template one base at a time, the polymerase builds an RNA molecule out of complementary nucleotides, making a chain that grows from 5' to 3'. The RNA transcript carries the same information as the non‐template (coding) strand of DNA, but it contains the base uracil (U) instead of thymine (T). Termination. Sequences called terminators signal that the RNA transcript is complete. Once they are transcribed, they cause the transcript to be released from the RNA polymerase. o Pre‐mRNA must go through extra processing before it can direct translation. They must have their ends modified, by addition of a 5' cap (at the beginning) and 3' poly‐A tail (at the end). Pre‐mRNAs must also undergo splicing. In this process,parts of the pre‐mRNA (called introns) are chopped out, and the remaining pieces (called exons) are stuck back together. Page 3 of 39 Translation occurs in the cytoplasm: o Initiation: The ribosome assembles around the mRNA to be read and tRNA brings in its perspective protein, decoding 3 bases at a time, beginning with the start codon, AUG. o These 3 base pairs of mRNA are called codons. The mRNA base pairs are complementary to the base pairs of the tRNA, called anticodons. o Elongation: The amino acid chain gets longer. The mRNA is read one codon at a time, and the amino acid matching each codon is added to a growing protein chain. When the complementary pairs are formed, they are added to the protein chain by peptide bonds, the result is polypeptides. o Termination: The finished polypeptide chain is released when a stop codon (UAG, UAA, or UGA) enters the ribosome. Gene regulation o Promotor sites: can be turned off or on, enabling or disabling a gene from being replicated. o Alternative splicing: Exons are used to code for protein, introns are clipped out. The order of exons can determine different mature mRNA strands which result in different proteins. o Epigenetics: involves packaging of DNA. DNA is round around histones. These packages are called nucleosomes. How tightly packed they are determines whether or not the gene is on or off. Page 4 of 39 Loosely packed = transcription possible. Tightly packed = transcription impeded. Modifications determine how tightly/loosely packed they are. Many of these modifications are determined by environment/diet. Mutations‐ Mutations originate at the DNA level, but show their effects at the protein level. o Point mutations‐ when one of the DNA bases (nucleotides) are replaced with another, results in a different protein o Example: Coding: 5’ AAA TTT GGG CCC 3’ = Lys Mutation: 5’ TAA TTT GGG CCC 3’ = stop codon o Missense mutations‐ any mutation that causes a change in amino acid o Nonsense mutations‐ leads to a stop codon “stop the nonsense” o Silent mutations‐ doesn’t affect the protein at all, the codon results in the same protein o Frameshift mutations‐ adds one base into the protein sequence, but changes the final protein amino acids by causing a shift. Insertion mutation‐ can change the entire read of the protein Deletion mutation‐ can change the entire read of the protein Conservative mutations‐ where the resulting amino acid is in the same type as the original Non‐conservative mutation‐ amino acid is a different type than the original o
o Page 5 of 39 Repairing Mutations o Damage to single nucleotide bases from harmful molecules (chemicals or oxygen). o Repair: Base excision‐ replace with a base that isn’t damaged. 1. DNA repair enzymes recognize the damaged base, removes it. 2. DNA polymerase fills the gap with a new base. 3. Ligase seals the gap. o Damage from UV causes multiple damaged nucleotides, ie causing two thymine’s to fuse together (called thymine dimers). o Repair: Nucleotide excision repair‐ removes 20‐30 nucleotides to fix damage. 1. DNA repair enzymes recognize damage, cuts out damage and surrounding area. 2. DNA polymerase fills in the gap with new bases. 3. Ligase seals the gap. o Base mismatch due to errors in replication. o Repair #1: DNA polymerase proof reading. Removes incorrect base, inserts correct base. o Repair #2: Mismatch repair‐ fixes the mismatch 1. DNA repair enzymes recognize mistake, and remove several bases surrounding the mismatch. 2. DNA polymerase inserts correct bases. 3. Ligase seals the gap. o Double stranded breaks in the DNA from radiation, can lead to cell death o Repair: Page 6 of 39 Homologous recombination‐ a sister chromosome is used as a guide to recombine the strands by copying the chromosome, but this doesn’t always work. Must be done after DNA replication. Nonhomologous end joining‐ if no sister DNA to copy (ie before DNA replication) the non‐damaged sections are joined together and the damaged DNA is lost. Last resort method d/t high risk of mutations. Inheritance o 1‐22 are autosomal chromosomes o If a mutation is on an autosomal chromosome (1‐22) then there is no bias towards males or females. o Sex chromosomes: o Females Xx o Males Xy o X linked mutation is bias because females have Xx and males are Xy. o Allele: a copy of a gene o Genotype: complete set of genes, the genetic makeup of an individual. o Phenotype: all the observable characteristics or traits of an individual, including ones that are not easily seen, such as blood type or color blindness o The genotype (pair of genes) decide the phenotype (observable characteristics) of an individual. o Heterozygous: one allele is dominate while the other is recessive. The dominant allele is observable in the phenotype while the recessive allele is not. (Aa) o Homozygous: two identical alleles (AA or aa) o Dominant: an allele that always expresses its phenotype, even in the presence of a recessive allele, represented by a capital letter. o Recessive: an allele that is only expressed in the phenotype when both alleles of a gene are recessive. Represented by a lower‐case letter. Determining pedigrees o Females are indicated by circles; males are indicated by squares. o Unaffected individuals are indicated by open shapes; affected individuals are indicated by filled shapes. o Recessive vs Dominant o If two unaffected parents have an affected child, they are carrier parents. o Carrier parents = recessive trait o No carrier parents = dominant trait o Autosomal vs Sex linked o Males and females affected equally = autosomal o Only males = sex linked o Autosomal dominant vs sex linked dominant Page 7 of 39 o Affected males with a sex linked dominant trait will pass it on to all of their daughters (females do not inherit father’s Y chromosome, therefore only getting affected X chromosome) Page 8 of 39 Co‐dominance‐ both equally share dominance (red and white) Incomplete dominance‐ neither really stand out (pink) Complete dominance‐ either one or the other is dominant (red OR white) Page 9 of 39 Visual representation with chromosomes Remember that chromosomes come in pairs of two, eliminate the answers that don’t include pairs. Carriers have one of each allele (Rr). If the person actually has the disease, they will have both alleles (rr) or (RR). Autosomal is chromosomes 1‐22, sex linked is chromsomes X and Y. Page 10 of 39 PCR and genetic testing‐ DNA replication in a test tube o What is needed for PCR 1. Template DNA 2. Nucleotides (dNTP’s) 3. DNA polymerase 4. DNA primers o Stages of PCR: 1. Denaturation 95 degrees C Separates the template DNA strands to be able to copy each strand. 2. Annealing 50 degrees C DNA Primers that match the gene were looking for attach to the ends of the piece we want to copy. 3. Elongation 70 degrees C DNA polymerase adds on to the primers, building a copy strand. o Using PCR to detect mutation‐ must copy DNA multiple times (2^n, where n= # of cycles) Make primers that flank the mutation and sequence the product Use primers that stick to the mutation. If the mutation is present, it will stick, if it isn’t present, it won’t stick. Page 11 of 39 Epigenetics is the result of making different proteins and gene expression. It determines what genes function and which do not. o Increased expression means you make more proteins. o Decreased expression means you stop making proteins. o 5 required parts, the first three parts are required to turn a gene on. Promoter‐ Start line for making the protein. Transcription factors‐ foot blocks for the runner (RNA polymerase) to start with. RNA polymerase‐ the runner, makes the mRNA Need all three of the above to turn gene on (increase expression) Nucleosomes‐ the packaging of DNA. Spacing determines whether or not the promoter is visible for a gene to be turned on or off. If it is loosely packed, the promoter is visible and can be accessed by RNA polymerase =Increased expression. If it is tightly packed, the promoter cannot be accessed and therefore the gene will remain turned off= Decreased expression. Methylation‐ CH3 (Methyl) is added to DNA or nucleosomes. Turns gene off by causing nucleosome to become tightly packed. Without it, gene remains on. Acetylation‐ Gene expression is turned on because nucleosomes are widely spaced apart and transcription factors can get in to start transcription. Page 12 of 39 Page 13 of 39 Biochem Mod 2 Amino acids o Amino acid back bone‐ same in all amino acids Central carbon (alpha carbon)‐ carbon in the center that holds the amino acid together as other groups bind to it. C‐H, CH Amino group‐ contains nitrogen and hydrogen. NH2, NH3+ Carboxyl group‐ has two oxygens and one carbon, gives the amino acid its acid properties. COO‐, COOH o Side groups (R group) are different which effect how the amino acid acts, will classify them as charged, polar, or hydrophobic. To determine what their classification is, first look for: Charged amino acids‐ R group will have ‐/+ which means negatively or positively charged. If charged, will take precedence. If not charged, look to see if its polar. Polar amino acids‐ will have SH, OH, NH at the end of the R group. If not polar, then the amino acid is non‐polar. Non‐polar amino acids‐ will have CH at the end of the R group. Hydrophobic amino acids‐ will have several hydrogen molecules in the R group, each will end with an H atom. o Bonds Amino acids will have three types of bonds Charged amino acids make ionic bonds (only + with ‐) Polar amino acids will make hydrogen or disulfide bonds o OH and NH make hydrogen bonds o SH makes disulfide bond (the strongest type of bond, can only bond with itself so very few of them) o “OH look it’s a Northern Hemisphere/Southern Hemisphere Polar Bear!” Non polar amino acids will make hydrophobic interactions o CH “Can’t have water” (weakest bond, but many of them) Strongest to weakest: disulfide, ionic, hydrogen, hydrophobic What breaks the bonds (denaturing) Charged: pH and salt changes Polar: pH and salt changes, disulfide bonds have to be broken by reducing agents Non polar: broken by heat Page 14 of 39 Alanine: be able to recognize structure. Is hydrophobic and ionized (will have + or ‐) Page 15 of 39 Protein structure‐ chains of amino acids o Linking amino acids together‐ Forming peptide bonds (the backbone of an amino acid) Primary structure: chain of amino acids by peptide bonds, does not denature The carboxyl group and amino group of two amino acids bond together by using 2 hydrogens and one H2O. A water molecule is lost in this process, known as dehydration. o Amino group + carboxyl group Secondary structure: shaped that is formed when hydrogen bonds are added between carboxyl and amino groups. Forming of alpha helix and beta sheets within the backbone, held together by hydrogen bonds. Tertiary structure: three‐dimensional folding, the result of different secondary structures interacting with one another via their R groups/side chains. These interactions include hydrophobic interactions, hydrogen bonds, ionic bonds, and disulfide bonds. Proteins can now function at this stage. Disruption of its hydrophobic state is the simplest way to denature. Quaternary structure: more than one amino acid/polypeptide/protein, held together by R groups/side chains. Not all proteins need this structure. Page 16 of 39 Protein folding o Chaperones help fold proteins o Can misfold, or take another shape (conformation), results in it being non‐functional o Denature: environmental change that causes the protein to misfold or unfold by breaking side chain bonds/secondary structure, but does NOT break the primary structure o Degradation: breaking apart of the primary structure/peptide bonds by hydrolysis o Aggregation: proteins clump together abnormally due to hydrophobic interactions either by unfolding or mutation. o Hydrophobic interactions: when a protein is exposed by unfolding, causing its hydrophobic parts to be exposed to water o Misfolding of proteins leads to Alzheimer’s: Intracellular tangles and extracellular plaques (senile plaques) are caused by aggregated amyloid‐beta fibers which accumulate in the brain. Connections between tau is lost leading to progressive neurodegeneration. Page 17 of 39 Enzymes o One of the most important types of proteins in our cells. o o Known as catalysts, they speed up reactions and use less energy ↑rate of reac on, ↓ac va on energy Can do the same reaction over and over They only act on specific substrates‐ a molecule that is specific for that enzyme Binds via its active site, a binding platform for its specific substrate Have a high degree of specificity‐ they catalyze only one type of reaction, and most act on only one substrate Molecules that are different in shape or function bind to the enzyme. Therefore, the enzyme and its substrate are complementary, like a “lock and key”. While an enzyme and its substrate are complementary, many enzymes will adjust their active site slightly to improve the fit of the substrate. Known as induced fit, “hug”. Reusable‐ Enzyme cycle Enzyme + specific molecule/induced fit = reaction/product, then release of product Enzyme pathways o Enzyme reaction: substrate + enzyme = product o Enzyme pathway: Substrate + enzyme 1 = product → Enzyme 1 product (now substrate) + enzyme 2 = enzyme 3 The product of one enzyme can be the substrate of another. o Control of enzyme activity An organism must be able to control its enzyme availability and activity. 1. Control of enzyme availability‐ depends on its rate of synthesis and degeneration. Changes over time. 2. Control of enzyme activity‐ can be inhibited due to too much product or presence of inhibitors, and can be modulated (modified) through structural alterations. o Control by modification: phosphorylation and dephosphorylation (attachment/removal of a phosphate group) Page 18 of 39 o Control by enzyme inhibition: Feedback inhibition – similar to the drug induced form of noncompetitive inhibition, when excess product is detected, the pathway is stopped by inhibition. The product at the end of the pathway binds to allosteric site on the first enzyme at the beginning of the pathway to stop the process. o Medical inhibition with drugs Competitive inhibition‐ appears similar to the substrate, binds to the active site on the enzyme to prevent substrate from binding Can be overcome by adding additional substrate, if there’s too much substrate and not enough inhibition the substrate will win. Noncompetitive inhibition‐ binds to the allosteric site (away from the active site) Changes shape of protein and therefore changes the active site, causing the substrate to not be able to fit into the enzyme. Page 19 of 39 Biochem Mod 3 Myoglobin and Hemoglobin o Myoglobin‐ storage of oxygen, holds onto it 1 subunit, one polypeptide chain with tertiary structure, has only one heme therefore can only carry one oxygen molecule Can grab oxygen quickly, stores oxygen within muscles tightly (high affinity) Is not affected by changes in pH Since it is stored in muscles, can be used to detect MI, rhabdomyolysis, etc. because it will be released when muscle is damaged. o Hemoglobin‐ transport of oxygen 4 subunits, quaternary structure Responsible for transport of oxygen from the lungs to the tissues, but not very good at grabbing oxygen (lower affinity). Heme‐ iron, where O2 binds, has 4 so it can carry 4 oxygen molecules at one time Tense or "T" state Deoxygenated, heme is bent out of shape (dome shape), its 4 subunits are spread apart, low affinity. Low affinity = decreased ability to grab oxygen, will require more and more oxygen to bind Once one subunit binds to oxygen, the shape of all 4 subunits change to the relaxed state. This is called cooperativity, and it will bind to O2 faster now. Relaxed or "R" state Oxygenated, heme is flat and relaxed, subunits are closer together Has an increase in affinity so its ability to grab oxygen is better. Once one of the subunits grabs oxygen, the others tend to grab oxygen more quickly. Deoxygenated (tense state) Oxygenated (relaxed state) Page 20 of 39 Oxygen binding curve o ‐Dotted line is myoglobin, is hyperbolic shaped, can grab oxygen even at low levels (high affinity). Once it grabs one oxygen, it is full to capacity because it can only carry one at a time. ‐Solid line is hemoglobin, is S shaped (sigmoidal). Has 4 subunits that must cooperate together (cooperativity). Has low affinity therefore unable to grab oxygen unless there is plenty available. Page 21 of 39 Bohr Effect o How pH allows hemoglobin to function o Use CHART. Low pH (acidic) causes the following: C = ↑CO2 H = ↑H+ (acid) A = acidic R= release of O2 from hemoglobin, right shift in curve T= tense state in the tissues High pH (basic) causes the following (opposite of low pH): C = ↓CO2 H = ↓H+ (acid) A = basic R...
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