HOSA Carbohydrates (PDF)

**Key points:** - Understand the biochemical reactions that occur in water 10% - Describe the organization of cells 8% - Describe amino acids and peptides 6% - Identify proteins, protein purification, and enzymes 14% - Describe biological membranes 6% - Understand how structures of...

**Key points:** - Understand the biochemical reactions that occur in water 10% - Describe the organization of cells 8% - Describe amino acids and peptides 6% - Identify proteins, protein purification, and enzymes 14% - Describe biological membranes 6% - Understand how structures of nucleic acids convey information 3% - Describe the biosynthesis and replication of nucleic acids 8% - Understand the biosynthesis of RNA 4% - Describe the translation of genetic messages 6% - Understand viruses, cancer, and immunology 6% - Describe the importance of energy and electron transfer in metabolism 10% - Identify storage mechanisms in carbohydrate metabolism 16% - Describe glycolysis 3% **Carbohydrates** - Sugars = Small Carbohydrates (literally hydrates of carbons) **Monosaccharides** - Smaller units that polymerize to make polysaccharides **NOMENCLATURE:** - Based on the number of carbons you can deduce the name: - 3 C is triose - 4 C is tetrose - 5 C is pentose - 6 C is hexose - All carbs have an aldose or ketose functional group - This would be a aldohexose and a ketopentose - Each carbon that has HO and H are chiral centers **FISHER PROJECTION FORMULAS:** - These are dry 3D representations (vertical bond from center away and horizontal bond in front) ---\> draw a cross - These contain stereocenters and exist as a pair of enantiomers (mirror images) - **Vertical Lines**: Represent bonds that go behind the plane of the page (away from the viewer). - **Horizontal Lines**: Represent bonds that come out of the plane of the page (toward the viewer). - If the -OH group is right, it is a D - If the -OH group is left, it is a L - n chiral centers = 2\^n stereoisomers - Swapping the position of two groups on a chiral center (H and OH) results in the inversion of that stereocenter (new molecule) - Only 180 degree rotations are allowed **IMPORTANT MONOSACCHARIDES:** 1. D-Glucose 1. Stored at concentrations of 65-110 mg/dL in our blood 2. Excess stored as glycogen or fat 3. Too much causes hyperglycemic (type 1 or 2 diabetes) 4. Too little causes hypoglycemic 2. D-Fructose 5. Some formula with glucose, but it is a ketohexose 6. Sweetest monosaccharide 3. D-Galactose 7. 6C aldohexose found in lactose 8. Galactosemia ---\> accumulation of galactose (caused by lack of enzymes transferring galactose) 9. Differs from glucose in carbon 4 (hydroxyl group is pointed down in glucose, up in galactose) Glucose + galactose are stereo-isomers (same functional groups, just different hydroxyl groups, same conductivity too) **Haworth Projections** - These are rings known as Haworth structures and are represented in cyclic hemiacetals - It creates a new carbon stereocenter called an anomeric carbon - To do this... Alpha has -OH down, Beta has -OH up **Mutarotation** - The change in specific rotation that accompanies the equilibration of alpha and beta anomers in aqueous solutions - Alpha can turn into beta **Formation of glycosides** - Treatment of a monosaccharide with an alcohol makes an acetal - Water is lost (2 Hs and 1 O) to make OCH3 - A cyclic acetal derived from a monosaccharide is called a GLYCOSIDE (the -OH group is replaced with an -OR group) - Bond from anomeric carbon to OR group is a glycosidic bond - Mutarotation isn't possible because an acetal is no longer in equilibrium with the open-chain carbonyl-containing compound **Reduction to alditols** - Alditols is the product when the CHO group of a monosaccharide is reduced to CH2OH - Sorbitol is found in plants (sugar substitute for diabetics) - Xylitol is used as a sweetening agent in gum and candy **Oxidation to aldonic acids** - Aldehyde group of an aldose can be oxidized by several reagents - Any carb that reacts with a mild oxidizing agent to form an aldonic acid is a reducing sugar (changes the H to O-) **Oxidation of uronic acids** - Enzyme oxidation yields uronic acid (CH2OH turns to COOH) **Phosphoric Esters** - Reaction between phosphoric acid and an alcohol - First step of glycolysis ---\> add phosphate group to carbon-6 **Important Disaccharide** Term: Oligosaccharide (6-10 monosaccharides) **Sucrose** - A sugar composed of glucose and fructose subunits - A table sugar - Isn't a monosaccharide until it reaches equilibrium with its open-chain form, thus sucrose is a non-reducing sugar **Lactose** - Principal sugar present in milk - Glucose and galactose - D-galatopyranose bonds with a beta 1,4 glycosidic bond to carbon 4 of D-glucopyranose - Beta-D-glucopyranose is equilibrium to its open-chain form, can be oxidized **Maltose** - Juice from sprouted barley and other cereal grains - Two units of glucose join together (alpha 1,4 glycosidic bond) - Can be oxidized **Polysaccharides** **Starch and glycogen** Polymers of ⍺-glucose are used in energy storage -- glycogen is used in animals and starch is used in plants In both glycogen and starch, the ⍺-glucose monomers are connected via 1' -- 4' glycosidic linkages to form helical structures - Starch can exist as linear strands (amylose 20-25%) or be branched (amylopectin 75-80%) due to the presence of additional 1' -- 6' linkages - Glycogen is a more highly branched molecule than amylopectin as it possesses more frequent 1' -- 6' linkages (have both 1' - 4' bonds and 1' - 6' bonds) Branching causes the polysaccharides... - To adopt a more compact structure - Their large molecular size renders them insoluble in water - Can store large amounts of energy **Cellulose** - Polymers of ß-glucose (in alternating arrangement) are used to form cellular structures -- cellulose is a component of plant cell walls - Straight linear chains that can be grouped in bundles and cross-linked with hydrogen bonds - Cross-linked bundles function to increase the structural integrity and mechanical stability (can form microfibrils that are strong) **Acidic Polysaccharides** - Groups of polysaccharides that contain carboxyl groups and sulfuric ester groups - Important in connective tissue - Has amino sugars (glycosaminoglycans) e.g. Hyaluronic acid - Abundant in embryonic tissue/lubricant - Made of D-glucuronic acid joined by beta 1,3 glycosidic bonds Heparin - Sulfonated polysaccharide chains - Anticoagulant activity ---\> stored in mast cells (liver, lungs, and gut) - Binds strongly to antithrombin III (plasma protein involved in clotting) - The larger the molecule, the greater its anticoagulant activity **Lipids** **Classification by function** - 3 major roles: - Store energy - Burning of fats produces twice as much energy as burning carbs - Part of membrane - Water insolubility, compartmentalization - Chemical messengers - Hormones **Classification by structure** 1. Simple lipid 2. Complex lipid 3. Steroids 4. Prostaglandins, thromboxane, leukotrienes **Fatty Acids** - Fatty acids can be linked to the hydroxyl group of alcohols via condensation reactions to produce an ester linkage - Long, unbranched carbon chain with carboxyl group at one end of amphipathic compound (insoluble in water) - Designated as an 18:2 fatty acid; its 18-carbon chain contains two double bonds - 3 most abundant fatty acids in nature are palmitic acid (16:0) stearic acid (18:0) and oleic acid (18:1) **Types of Fatty Acids** - **Saturated** fatty acids possess straight hydrocarbon chains with no double bonds - **Unsaturated** fatty acids have double bonds -- they can be either \*mono-\*unsaturated (1 double bond) or \*poly-\*unsaturated (more than 1 double bond) This causes it to bend Note: Conversion of oil to fat is hydrogenation - Adding hydrogen across double bond of unsaturated fatty acid to produce saturated one **Triglyceride Structure** - Has a glycerol group - Only even-numbered acids are found on triglycerides because the body builds acids entirely from acetate unit (puts the carbon in two at a time) - Triglycerides are composed of three fatty acid chains linked to a single glycerol molecule (3 ester bonds) - 3 fatty acid chains undergo condensation reactions - They are important form of stored energy **Physical State** **Fats versus Oils** Living organism store their lipids as either fats or oils depending on the type of fatty acid involved (trans-saturated or *cis*-unsaturated) - These fatty acids differ in the shape of their hydrocarbon chains (straight or bent) **Fats (Saturated) single bond** - Trans fatty acid has hydrogens on opposite sides, no bends in the chain - However, this tight packaging also increasing the number of intermolecular forces between the fatty acid chains, resulting in a higher melting point e.g. butter, more difficult to metabolize **Oils (Unsaturated) double bond (except coconut oils)** - Unsaturated *(cis)* fatty acids have kinked chains that cause them to be more loosely packed, hydrogens are on the same side - This means there are fewer intermolecular forces and less energy is required to separate the fatty acids, resulting in a lower melting point **Hydrogenation** - The process by which hydrogen atoms are added to unsaturated (double bond) fats and oils. - This facilitates its conversion towards a single bond **Complex lipids** - Constitutes the main components of membranes - Classified into PHOSPHOLIPIDS and GLYCOLIPIDS **Phospholipids** - Contains an alcohol, two fatty acids, and a phosphate e.g. Glycerophospholipids (alcohol-glycerol) Sphingolipids (alcohol-sphingosine) **Lipid and membrane structure** - The membrane is made of a lipid bilayer: - Two rows of complex lipid molecules link tail to tail - This is the fluid mosaic model **Glycerophospholipids** - Phosphoglycerides: - Glycerol backbone, two of the hydroxyl group is esterified (on 2nd carbon usually unsaturated acid) - 3rd group esterified by phosphate group, which also esterified to another alcohol - Lecithin: One stearic acid and one linoleic acid, major component of egg York - Contains both polar/non-polar within one molecule (excellent emulsifier) **Phosphatidylinositols: Alcohol inositol bond to molecule by phosphate ester bond** - In higher phosphorylated form it serves as signaling molecules in communication **Sphingolipids** - Myelin sheath contains these - Backbone is sphingosine - It is a long-chain fatty acid connected to ---NH2 group by amide bond ---\> Combination called ceramide - If the fatty acid is stearic acid ---\> may be sphingomyelin - Features are if terminal hydroxyl of sphingosine is esterified to phosphorylcholine or phosphoryl-ethanolamine molecule - It appears in sheath of nerve cells, inside viral membranes, associated with diseases such as multiple sclerosis **Glycolipids** - Complex lipids containing carbs and ceramides - e.g. Cerebrosides: - Consist of ceramide mono- or oligosaccharides - Fatty acid part 18 to 24 carbon, forming beta-1-glycosidic bond with ceramide portion, - Occurs primarily in the brain or nerve synapses - Gangliosides: contains more complex carbohydrate structures **Steroids** - Compounds containing the following fused ring system: - 3 cyclohexane rings and connected to cyclopentane - Not necessarily esters **Cholesterol** - Cholesterol acts as a modulator and adjusts the membrane fluidity - Most abundant steroid in the human body - At high temps it maintains the arrangement of the phospholipids, preventing it from becoming too fluid and porous - At low temperatures is ensures that the phospholipids don't solidity, preventing it from becoming inflexible **Lipoproteins: Carrier of cholesterol** - Transported by lipoprotein: spherically shaped cluster containing both lipid molecules and protein molecules - HDL (high density lipoprotein) - Good cholesterol ---\> 33% protein 30% cholesterol - LDL (low density lipoprotein) - Bad cholesterol ---\> 25% protein 50% cholesterol - VLDL (very low density lipoprotein) - Mostly triglycerides synthesized by liver - Chylomicrons: Carry dietary lipids and synthesized in intestines **Transport of cholesterol:** LDL: - Starts with VLDL (containing triglycerides and cholesterol esters in the core) and is surrounded by phospholipid and protein polar coat - VLDL's diameter shrinks and becomes LDL, satying in plasma for about 2.5 days - Low-density lipoproteins (LDL) transport cholesterol **from the liver to peripheral tissues** HDL: - High-density lipoproteins (HDL) transport cholesterol from peripheral tissues to the liver. - Delivered for synthesis of bile acids and steroid hormones Levels of HDL and LDL: - HDL binds to the liver cell surface, transferring its cholesteryl ester to the cell - May deprive cells of oxygen, causing them to die - When the concentration of cholesterol inside cells is high, LDL synthesis is suppressed Higher levels of HDL are associated with a lower risk of heart disease. LDL, also known as \"bad\" cholesterol, can accumulate in the walls of the arteries, leading to plaque buildup and increasing the risk of heart disease. - Membrane cholesterol function is a growth factor receptor for interleukin-3 (IL-3) - Transmembrane proteins that receive the signal for cell proliferation and differentiation - The amount of cholesterol in membrane near the IL-3 receptor influences the sensitivity of the receptor for IL-3 **Steroid Hormones** **Adrenocrorticoid hormones** These hormones play a crucial role in regulating various bodily functions, including metabolism, immune response, and stress response. - Classified into 2 groups: - Mineralocorticoids (regulate concentration of ions) - Glucocorticoids (control carbohydrate metabolism) Aldosterone (Mineralocroticoid): Enhances readsorption of Na and Cl ions Cortisol (Glucocorticoid): Increases glucose and glycogen in body **Sex hormones** Testosterone: Male hormone Estradoil: Female hormone, form from testosterone by aromatization of A ring, regulates cyclic changes in uterus and ovaries **Bile salt** They are **produced in the liver, directly from oxidation of cholesterol**. Bile salts are important in solubilizing dietary fats in the watery environment of the small intestine. - Strong detergent (one end strongly hydrophilic, other hydrophobic) - Can break down products of cholesterol **Prostaglandins, Thromboxane, Leukotrienes** These three substances are types of eicosanoids, which are lipid molecules derived from the oxidation of essential fatty acids. - Prostaglandins: involved in inflammation, pain, and fever; also regulate blood pressure, blood clotting, and the smooth muscle tone of the uterus. - Thromboxane: helps form blood clots and regulates platelet aggregation. - Leukotrienes: contribute to inflammation, allergic responses, and anaphylaxis; also regulate smooth muscle contraction, mainly in white blood cells **Proteins** **Functions** 1. Structural material 2. Catalysis 3. Movement 4. Transport 5. Hormones 6. Protection, storage, regulation **Amino Acids** - 20 common nature amino acids make up the protein - Classified into nonpolar, polar neutral, acidic, and basic - EXCEPT glycine, all amino acids exist as 2 enatiomers Polar: Acidic, Basic, Neutral Nonpolar: Most hydrocarbons Acidic: COOH Basic: Amino Neutral: Alcohol, Cysteine **Amino acids as Zwitterions** A zwitterion is a molecule that contains both a positively and negatively charged group within its structure. In the case of amino acids, this occurs when the amino group (-NH2) is protonated, creating a positive charge, while the carboxyl group (-COOH) is deprotonated, creating a negative charge. - Amino acids are zwitterions also in solid state and in the presence as ionic compounds - 20 amino acid fairly soluble in water, if no charges then smaller ones considered to be soluble When placed in a acidic/basic environment: - Positive ion at low pH, negative ion at high pH - In both cases, the amino acid is still an ion (still soluble) - **Isoelectric point: the pH at which a molecule carries no net electrical charge or is electrically neutral in the statistical mean** Amino acids can react with acid by taking a proton e.g. ---COO- becomes ---COOH, ---NH3+ becomes -NH2 **Amino acids combine to form proteins** - It would be amino acid + amino acid = dipeptide + water (H2O) - The new covalent bond between the C---N is called a peptide bond - Is reversible through a hydrolysis reaction - In a polypeptide an amino acid is called residues C-Terminus: Amino acid at end of peptide that has free alpha-carboxyl group N-Terminus: Amino acid at end of peptide that has a free alpha-amino group **Amino Acid Characterisitcs** - The side chains of lysine, histidine and arginine (basic) are positively charged at or near neutral pH - Charged amino acids are often found in the active site of proteins - phe, tyr, trp contain aromatic ring, allowing it to locate and measure protein, also involved in neurotransmission TYR: - COnverted to neurotransmitters called catecholamines, including epinephrine **Uncommon Amino Acids** - These are derived from common amino acids Hydroxpryoline and hydroxylysine: - Found in connective protein such as collagen Thyroxine: - Found in thyroid gland **Protein properties** Structure: Due to interactions of atoms in the backbone, peptide groups are planer due to partial resonance The isoelectric point of hemoglobin is pH 6.8 Others, such as serum albumin, have more acidic groups than basic group Solubility: Repulsion---**When these molecules get too close to each other, repulsion between the molecules occur**. As the molecules move farther apart from each other, the potential energy due to repulsion decreases. **Primary Structure** - The first level of structural organization in a protein is the order / sequence of amino acids which comprise the polypeptide chain - The primary structure is formed by covalent peptide bonds between the amine and carboxyl groups of adjacent amino acids - Primary structure controls all subsequent levels of organization because it determines the nature of the interactions between R groups of different amino acids - Peptide bond/link between amino acids **Secondary Structure** - The secondary structure is the way a polypeptide folds in a repeating arrangement to form α(alpha)-helices and β(beta)-pleated sheets - This folding is a result of hydrogen bonding between the hydrogen and oxygen atoms in the same chain as they are attracted to each other - Secondary structure provides the polypeptide chain with a level of mechanical stability (due to the presence of hydrogen bonds) - Hydrogen bonds (weak) between oxygen of COOH on 1 amino acid and hydrogen of amine group **Tertiary** - The tertiary structure is the way the polypeptide chain coils and turns to form a complex molecular shape (i.e. the 3D shape) - Made up of various alpha-helices and beta-pleated sheets - It is caused by interactions between R groups - H-bonds (between R-groups) - Disulfide bridges ---\> Very strong bond occurring between cysteine - Ionic bonds ---\> Between - and + charged R groups - Hydrophobic interactions ---\> Weak bond between non-polar R groups - Relative amino acid positions are important (e.g. non-polar amino acids usually avoid exposure to aqueous solutions) - Tertiary structure may be important for the function of the protein (e.g. specificity of active site in enzymes Amino acid polarity impacts protein tertiary structure - Looks like a globular protein due to polar R groups being oriented out of water and non-polar R group being oriented in the water - Seen in integral proteins, contains polar and non-polar proteins **Quaternary** - Quaternary structures are found in proteins that consist of more than protein chain inked together - Insulin (two chains) and collagen (three chains) are examples of proteins with a quaternary structure - Conjugated protein - Contains a non-polypeptide subunit - **Haemoglobin** is a conjugated protein as each subunit associates with an iron-containing haeme group - Non-Conjugated - Not all proteins will have a quaternary structure -- many proteins will only consist of polypeptides - **e.g. Insulin and collagen (strong fibrous material found in bones/ligaments)** **Hemoglobin** - Tetramers, consiting of 4 chains (2 alpha, 2 beta) - Able to bind 4 oxygen, having positive cooperatively (when one O is bound, the next one is easier) - AT any oxygen pressure, myoglobin will have a higher % of saturation than hemoglobin - Myoglobin: Oxygen storage in muslce - Hemoglobin: Transports oxygen, requires both strong bonds and easy release **Protein denaturation** Heat and pH can disrupt and break bonds, altering the 3D shape and denaturation occurs Heat produces kinetic energy pH ---\> H+ interrupting ionic bonds **Enzymes** **Enzymes are biological catalysts** - They are large molecules that increase the rates of chemical reactions without undergoing change - They make reactions faster by lowering the activation energy - Ribozyme: Enzymes made of nucleic acid Trypsin is a digestive enzyme produced in the pancreas and released into the small intestine to facilitate protein digestion. **Nomenclature** **Oxidoreductases:** catalyze oxidation and reduction **Transferases:** catalyze transfer of a group of atom hydrolyses: catalyze hydrolysis reactions L**yases:** catalyze addition of two groups to a double bond/ removal of two groups to create a double bond I**somerases:** catalyze isomerization (rearrangement) reactions ligase: catalyze the joining of two molecules **Enzyme activity** - Measures how fast an enzyme is able to catalyze a reaction - The molecule being acted on is called a substrate **Enzyme substrate concentration** - Constant concentration of a substrate leads to increased enzyme concentration (rate is linear) - Constant concentration of ENZYME, increases concentration of substrate (rate reaches a point and slows) **Temp** - When optimal temp is reached, a decrease occurs **pH** - Enzymes will denature in extreme pH **Lock and key model** - The lock and key model is **a theory of enzyme action that explains how enzymes fit their substrate**. - The active site of an enzyme is structured to fit a specifically shaped substrate. **Induced-fit model** - The induced-fit model **states a substrate binds to an active site and both change shape slightly, creating an ideal fit for catalysis**. **Enzyme inhibition** **Irreversible inhibitor:** bind to active site and never let go **Reversible inhibitor:** can also unbind **Competitive inhibitor:** bind to active site of enzyme **Noncompetitive inhibitor**: bind to different site despite from active site, change the shape of enzyme, slowing down the rate (substrate can still bind, but no catalysis) - Maximum rate is achieved at low substrate con. with no inhibitor - But at a high substrate con. when an inhibitor is present, it will be displaced and equilibrium will be achieved **Active Sites** 1. Pyruvate Kinase - Pyruvate kinase is **an enzyme that catalyzes the conversion of phosphoenolpyruvate and ADP to pyruvate and ATP in glycolysis** and plays a role in regulating cell metabolism. - Nonpolar = CH2 group lies in a hydrophobic pocket, K+ on the other side coordinates with pohsphate group **Catalytic power of enyzmes** His \> Cys \> Asp \> Arg \> Glu presence in active site, most are acid or base The presence of these residues in the active site is significant because they can participate in catalysis by forming covalent bonds or stabilizing transition states **Regulation** **Feedback control** - Formation of a product inhibits an earlier reaction **Zymogens** - Some enzymes in the body are produced in an inactive form, with a small part of their polupeptide chains needed to be removed to activate - These include proenzymes/zymogens - e.g. Trypsin and Chymotrpsin **Allosterism** - Allosteric regulation occurs when molecules bind to sites other than the active site, causing conformational changes that affect enzyme activity. - Regulated by allosteric enzymes - These allosteric modulators can be either activators (increasing enzyme activity) or inhibitors (decreasing enzyme activity). - Allosteric enzymes usually contain 2 chains, one for regulatory site and active site **Protein Modification** - Refers to a change in the primary structure, an addition of a functional group - Phosphorylation (addition of phosphate groups) can regulate enzyme activity by turning them on or off - These modifications are often reversible and play key roles in cellular signaling pathways **Isozymes** - Isozymes are different forms of the same enzyme that catalyze the same reaction but have different protein sequences. - They are often found in different tissues or cellular compartments and can be regulated differently. - A key example is lactate dehydrogenase (LDH), which has different isozymes in heart and skeletal muscle tissue. **Medicine** **Enzyme** **Medical Use** **Clinical Application** --------------- ------------------------ --------------------------------------------------------------- Trypsin Digestive Aid Treatment of pancreatic insufficiency and digestion disorders Thrombin Blood Clotting Surgical procedures to control bleeding Streptokinase Blood Clot Dissolution Treatment of heart attacks and deep vein thrombosis Asparaginase Cancer Treatment Treatment of acute lymphoblastic leukemia Lactase Lactose Digestion Management of lactose intolerance **Nucleic Acid** - Two types are RNA and DNA **Bases** Purines: Adenine and guanine Pyrimidines: Cytosine, thymine, uracil **Sugars** - In DNA, deoxyribose has one less OH group than ribose. - RNA contains ribose sugar, while DNA contains 2\'-deoxyribose. - These sugars form part of the backbone structure of nucleic acids. Nucleoside: Combination of sugar and base (made of adenine and ribose called adenosine) **Phosphate** - Phosphate bond with nuceloside is called a nucleotide (structure of RNA and DNA) **Primary, Secondary, Higher-order structure** Primary structure is the sequence of nucleotides (A, C, G, and T) that make up the DNA molecule. Secondary structure is the local arrangement of these nucleotides, including base pairing and hydrogen bonding, which forms the double helix in its most stable form ---\> B-DNA Higher-order structure, also known as tertiary structure, is the overall 3D arrangement of the DNA molecule, including supercoiling, wrapping around histone proteins, and looping. - Condensed into chromatin in a solenoid form - In nucleosome ---\> 8 histones form a core, with 147 base-pair dna strand aorund it **RNA Types** 1. Messenger RNA (mRNA) carries the genetic code that determines the order of amino acids in a polypeptide sequence. 750 nucleotides on average 2. Transfer RNA (tRNA) is responsible for transporting amino acids to the ribosome according to the mRNA sequence 3. Ribosomal RNA (rRNA) is RNA complexed with protein in ribosomes. 1. 35% protein, 65% ribosomal RNA 2. Smaller unit consists of one large RNA molecule and 20 different proteins 3. Larger unit contains 2/3 RNA and 35-50 proteins 4. Small Nuclear RNA (snRNA) is RNA in the nucleus, complexed with proteins to form smaller nuclear ribonucleoprotein particles 4. Helps with processing intial mRNA, splicing 5. Catalyzes by RNA protein ---\> led to discovery of ribozymes 5. Micro RNA (miRNA) inhibits tranlsation and promotes degradation 6. Small interfering RNA (siRNA) is used to eliminate expressio nof an undesirable gene **Genes** **Exons** are the coding sequences in DNA that are responsible for encoding proteins. They are the functional parts of a gene that are preserved in the final mRNA transcript. **Introns**, on the other hand, are non-coding sequences that are spliced out of the pre-mRNA transcript by ribozymes. They do not encode proteins - In humans, 3% of DNA codes for proteins, but over 70% of DNA doesn't have a purpose **Satellites** are repeating DNA sequences that can range from hundreds to thousands of base pairs in length. - Smaller ones are called mini-satellites ---\> associated with cancer when mutated **Medical Application** **Antisense RNA** Antisense RNA molecules are complementary to specific mRNAs. By binding to their target mRNA, they can prevent translation through mechanisms such as steric hindrance or by recruiting RNase enzymes that degrade the mRNA **Micro RNA** miRNA prevents mRNA from trnaslating ---\> treatment of hepatitis C **Small interfering RNA** Binds to mRNA, often leading to translational repression or degradation of the mRNA. Prevents Ebola virus from making protein **Crispr** - (Clustered Regularly Interspaced Short Palindromic Repeats) is a groundbreaking technology that allows for precise editing of genes. - Guided by RNA, the CRISPR-Cas system can specifically target and cut DNA at a desired location, allowing for the modification or deletion of genetic sequences. **DNA replication** Each strand of DNA has a 5\' end and a 3\' end; they are antiparallel. DNA polymerases replicate DNA by moving along a template strand and synthesising a new complementary strand - DNA polymerases add new nucleotides by joining the 5'-end of a new nucleotide to the 3'-end of the existing chain - Hence DNA is *synthesised* in a 5' → 3' direction, as the 3'-terminal is the end that is being extended Because double stranded DNA is antiparallel, this means that the extension of the two new strands will occur in opposite directions **DNA replication is a semi-conservative process whereby pre-existing strands act as templates for newly synthesised strands** - The process of DNA replication is coordinated by two key enzymes -- helicase and DNA polymerase **Helicase** - Helicase unwinds the double helix and separates the two polynucleotide strands - It does this by breaking the hydrogen bonds that exist between complementary base pairs - The two separated polynucleotide strands will act as templates for the synthesis of new complementary strands **DNA Polymerase I (repair) III (replication)** - DNA polymerase synthesises new strands from the two parental template strands - Builds polymers; adds nucleotides to build the new strand of DNA. - Free deoxynucleoside triphosphates (nucleotides with 3 phosphate groups) align opposite their complementary base partner - DNA polymerase cleaves the two excess phosphates and uses the energy released to link the nucleotide to the new strand - Only works from 5' - 3' **Ligase** - **Function**: Joins Okazaki fragments on the lagging strand. - **Details**: Seals nicks between the fragments, ensuring a continuous DNA strand. **Topoisomerases (Gyrases)** - **Function**: Alleviate supercoiling and tension ahead of the replication fork. - **Details**: They temporarily break single or double strands of DNA to relax the supercoiled structure, facilitating smooth progression of the helicase. **Isomerase** - Facilitates relaxation of supercoiling in DNA **Primase** - **Function**: Synthesizes short RNA primers. - Placed about every 50 nucleotides in the lagging-strand synthesis. - Primases catalyze the synthesis of short oligonucleotides, which consist of 4 to 5 ribonucleotides. - Needed to intiate the synthesis of both daughter strands **DNA Amplification** **PCR** The reaction occurs in a thermal cycler and uses variations in temperature to control the replication process via three steps: 1. *Denaturation* -- DNA sample is heated (\~90ºC) to separate the two strands 2. *Annealing* -- Sample is cooled (\~55ºC) to allow primers to anneal (primers designate sequence to be copied) 3. *Elongation* -- Sample is heated to the optimal temperature for a heat-tolerant polymerase (Taq) to function (\~75ºC) **Taq polymerase** is an enzyme isolated from the thermophilic bacterium *Thermus aquaticus* - As this enzyme's optimal temperature is \~75ºC, it is able to function at the high temperatures used in PCR without denaturing - Taq polymerase extends the nucleotide chain from the primers -- therefore primers are used to select the sequence to be copied **Strands** - **Coding Strand**: - Also known as the non-template strand. It has the same sequence as the RNA transcript (except for the substitution of uracil (U) for thymine (T)). - Orientation: 5\' to 3\' direction. - **Template Strand**: - The strand that is read by RNA polymerase or DNA polymerase to synthesize RNA or DNA, respectively. The RNA or new DNA strand is complementary to this strand. - Orientation: 3\' to 5\' direction, which allows for the synthesis of RNA or DNA in the 5\' to 3\' direction. **Telomeres** Telomeres are repetitive nucleotide sequences located at the ends of chromosomes, consisting of the sequence TTAGGG repeated many times. - **Function**: - They protect chromosome ends from degradation and prevent fusion with neighboring chromosomes. Telomeres play a crucial role in maintaining the stability of the genome **Gene Expression and Protein synthesis** **Central Dogma** - Info containedi n DNA molecules is transferred to RNA molecules - Then from RNA molecules the info is expressed in a protein - In some viruses with a RNA genome, replication proceeds from RNA to RNA - In retrovirus ---\> RNA is transcribed to DNA **Transcription** - DNA transcribes into RNA (mRNA) The template strand, also known as the (-) strand or antisense strand, serves as a template for RNA synthesis. Coding strand is (+), sense strand is complementary to the template strand and is used to synthesize the final RNA product. **3 kinds of polyerase catlyst the transcription:** 1. RNA polymerase 1 ---\> catlyst most formation of rRNA 2. RNA polymerase 2 ---\> catlyze mRNA formation 3. Polymerase 3 ---\> Catalyze tRNA formation as well as one ribsomal subunit an other regulatory RNA Structural genes include exons and introns **Regulatory portion isn't trabnscibed but has a control elements:** 1. Promoter: They play a crucial role in initiating transcription by binding to RNA polymerase and other transcription factors. 1. Contains a consensus sequence, such as the TATA box, which is typically 26 base pairs upstream. 2. Enhancer: DNA sequences that can be located far away from the promoter, but still influence gene expression by binding to transcription factors. **Elongation**: After intiation, RNA polymerase zips up bases At the end, a termination sequence occurs When the inititation starts, polyermase 2 in an unphosphorylated form, performs elongation upon phsphorylation. Post-transcription process: - The addition of a 7-methylguanosine (m7G) cap at the 5\' end - The attachment of a poly-A tail containing 100-200 adenine residues at the 3\' end - The removal of introns through splicing - Methylation to prepare the mRNA for translation and function **Translation** - mRNA serves as a template - Correspondence between 3 bases and one amino acid is called a genetic code - In prokaryotes, transcription and translation occur simultaneously in the cytoplasm ranslation (polypeptide synthesis) involves four key steps: initiation, elongation, translocation and termination - Prior to translation, tRNA molecules are fused to specific amino acids via a reaction involving ATP hydrolysis - The function of the ATP is to create a high energy bond that is transferred to the tRNA molecule - This 'charging' of the tRNA creates stored energy that is used by the ribosome to form a peptide bond **Initiation** The ribosome binds to the 5\' end of the mRNA, and starts moving towards the 3\' end of the strand until it reaches the start codon (AUG). **Just the small subunit of the ribosome** - The small ribosomal subunit binds to an initiator tRNA molecule that recognises the start codon (AUG) - The initiator tRNA is structurally distinct from other tRNA molecules and is only used to begin translation **Elongation** - The large ribosomal subunit has three tRNA binding sites -- an **A** site *(aminoacyl)*, a **P** site *(peptidyl)* and an **E** site *(exit), only two at a time though as one exists, one enters* - The initiator tRNA is bound to the central P site and a second tRNA molecule pairs with the next codon in the A site - The amino acid in the P site is covalently attached via a peptide bond (condensation reaction) to the amino acid in the A site - The tRNA in the P site is now deacylated (no amino acid), while the tRNA in the A site carries the peptide chain **Translocation** - The ribosome moves along the mRNA strand by one codon position (in a 5' → 3' direction) - The deacylated tRNA moves into the E site and is released, while the tRNA carrying the peptide chain moves to the P site - Another tRNA molecules attaches to the next codon in the now unoccupied A site and the process of elongation is repeated **Termination** - Elongation and translocation continue in a repeating cycle until the ribosome reaches a stop codon - These codons do not recruit a tRNA molecule, but instead recruit a release factor that signals for translation to stop - The polypeptide is released and the ribosome disassembles back into its two independent subunits **Termination** When the codon reads stop, releasing factor then cleaves the polypeptide chain from the last tRNA