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131 Cards in this Set
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Allosteric enzyme |
an enzyme whose activity is affected by other substances binding to it these substances change the enzyme’s activity by altering the conformation(s) of its quaternary structure |
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Allosteric effector |
a substance that modifies the behavior of an allosteric enzyme allosteric inhibitor allosteric activator |
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Aspartate transcarbamoylase (ATCase) |
made up of two different types of subunits Catalytic subunit: 6 subunits organized into 2 trimers (Aspartate) Regulatory subunit: 6 subunits organized into 3 dimers (CTP, ATP) |
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Feedback Inhibition |
The final product inhibits the first reaction in the series Efficient control mechanism that allows a shutdown of the entire series of reactions when there is excess of the final product Not limited to allosteric enzymes |
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Sigmoidal curve |
indicates cooperative behavior of allosteric enzymes In the presence of CTP (inhibitor), a higher [S] (Aspartate) is needed for the enzyme to achieve the same rate of reaction In the presence of ATP (activator), the rate of the reaction increases even at low [S]. |
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Homotropic effects |
Allosteric interactions that occur when identical molecules are bound (substrate) |
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Heterotropic effect |
Allosteric interactions that occur when different substances are bound (Inhibitor and Substrate) |
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Concerted model |
The substrate binds the R (active) form Shifts equilibrium from T form to R-form (more R-form is produced) Shifting equilibrium is responsible for the observed allosteric effect Binding of Inhibitor: Stabilize T form, shifts equilibrium to T-form, more substrate is needed to shift T-form to-R form (greater degree of cooperativity) Binding of Activator: Removes free R form, Shift toward more R -form to re-establish equilibrium. Less need for S to shift equilibrium in favor of R-form (less cooperativity) Distinguishing feature: The conformation of all the subunits change simultaneously (concerted change) |
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R (relaxed) conformation |
active form; binds substrate tightly |
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T (tight or taut) conformation |
inactive form; binds substrate less tightly |
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Sequential model |
Binding one molecule of substrate to one subunit induces the other subunits to adopt the R state, which has a higher affinity for substrate Binding one molecule of inhibitor to one subunit induces a change in the other subunit to a form that has a lower affinity for substrate Distinguishing feature: binding of substrate induces conformational change from T form to R-form. A conformational change in one subunit makes the same conformational change easier in another subunit |
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Phosphorylation |
a type of covalent modification The side chain –OH group of serine, threonine and tyrosine can form phosphate esters Source of phosphate group is ATP Phosphorylation by ATP can convert an inactive precursor into an active enzyme, or reduce the activity of an enzyme Common example : Na+-K+ pump (Na+/K+ ATPase) found in all animal cells |
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Sodium-Potassium pump |
Transport mechanism that pumps 3 Na+ out of the cell, and 2K+ into the cell, against a concentration gradient Phosphorylation of the sodium–potassium pump is involved in cycling the enzyme between the form that binds to sodium and the form that binds to potassium |
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Glycogen phosphorylase |
Catalyzes breakdown of stored glycogen Exists in two forms – Phosphorylated glycogen phosphorylase a (more active) Dephosphorylated glycogen phosphorylase b (less active). |
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Covalent control |
Phosphorylation converts b-form to a-form. The kinase that puts the phosphates on is controlled by epinephrine (adrenalin) |
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Non-covalent control |
Activated allosterically (T –R) Increase in AMP (signals energy demand). This relaxed form has similar enzymatic properties as the phosphorylated enzyme. Inactivated (R – T) increase in ATP concentration ( sufficient energy stores) Glucose (insulin signals glucose availability) |
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Zymogens |
Inactive form of an enzyme that can be irreversibly transformed into the active enzyme by cleavage of specific peptide bonds Chymotrypsinogen, Trypsinogen (inactive) , Trypsin (active), Procaspases (inactive), Caspases (active) |
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Chymotrypsinogen |
synthesized and stored in the pancreas Activated to chymotrypsin in small intestines |
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Trypsinogen (inactive) , Trypsin (active) |
Activated by enteropeptidase |
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Procaspases (inactive), Caspases (active) |
apoptosis or programmed cell death (cell turnover) |
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Which amino acid residues on the enzyme are in the active sites ? |
Covalently modified versions of specific side chains (labeling) Serine protease: a class of proteolytic enzymes in which the hydroxyl group of serine plays an essential role in catalysis Chymotrypsin is a serine protease (uses S195) S195 is covalently linked to DIPF (enzyme inactivated) Three residues important for catalysis - Serine-195, Histidine-57, and Aspartate 102 These residues are arranged close to each other at the active site |
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How do the critical amino acids catalyze the chymotrypsin reaction? |
Stage 1 : Serine oxygen acts as a nucleophile (nucleus-seeking substance), and attacks the carbonyl group of the peptide bond of the substrate The amino group of the peptide hydrogen bonds to the imidazole portion of histidine The carbon-nitrogen bond of the original peptide breaks leaving the acyl-enzyme intermediate Stage 2 (Deacylation stage) : Water becomes hydrogen-bonded to the histidine Water oxygen (acts as a nucleophile) attacks the acyl-enzyme intermediate. The bond between the serine oxygen and the carbonyl carbon breaks releasing the product with a carboxyl group where the original peptide bond used to be Enzyme is regenerated. |
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Cofactor/coenzyme |
a non-protein substance that takes part in an enzymatic reaction. It is regenerated for further reaction Metal ions- electron pair acceptors that can behave as coordination compounds. (Zn2+, Fe2+) Organic compounds- many of which are vitamins or are metabolically related to vitamins |
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Nicotinamide adenine dinucleotide (NAD+) |
Nicotinamide adenine dinucleotide (NAD+) is a coenzyme in many oxidation-reduction reactions in biology. Its structure is composed of Nicotinamide ring Adenine ring two sugar-phosphate groups linked together In oxidation-reduction reactions: Main function: electron transfer reactions NAD+ is an oxidizing agent – it accepts electrons from other molecules and becomes reduced to NADH NADH can be used as a reducing agent to donate electrons. Nicotinamide ring is where reduction-oxidation occurs |
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Chymotrypsin catalyzed reactions |
Fast phase occurs first: the peptide bond is broken and the first peptide is released. The other peptide is covalently linked to the enzyme transiently. Slow stage : second peptide is released, enzyme regenerated. |
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How to identify the critical amino acids in the active site |
Labeling reagents |
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Lipids |
compounds that are part hydrophobic component of membranes, egg yoke, human nervous system
Open-chain compounds with polar head groups, and long non-polar tail groups Fatty acids, Triacylglycerols, Phosphoacylglycerols, Sphingolipids, Glycolipids Fused-ring compounds Steroids |
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Fatty acids |
Carboxyl group at the polar end and a hydrocarbon chain at the non-polar chain (amphipathic) Length of fatty acid plays a role in its chemical character Usually contain an even number of carbons (can contain odd) Fatty acids that contain only C-C bonds, are saturated Fatty acids that contain C=C, are unsaturated: The double bond is nearly always cis (gives a kink) and rarely conjugated Unsaturated fatty acids have lower melting points than their saturated counterparts; the greater the degree of unsaturation, the lower the melting point Rarely found free in nature – they form parts of many commonly occurring lipids |
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Degree of unsaturation |
refers to the number of double bonds. The superscript indicates the position of the double bonds. For example ∆9 refers to a double bond at the ninth carbon atom from the carboxyl end of the molecule |
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Triacylglycerols (Triglyceride) |
An ester of glycerol with three fatty acids Accumulate in adipose tissues (as stored fatty acids for metabolic energy)
Hydrolysis of : Enzyme hydrolysis – lipases Acid or Base (KOH, NaOH) catalyzed hydrolysis Natural soaps are prepared by boiling triglycerides with NaOH, in a reaction called saponification (Latin, sapo, soap) |
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Phosphoacylglycerols (Phospholipids/Phosphoglycerides) |
Phosphatidic acid: One alcohol group of glycerol is esterified by a phosphoric acid rather than by a carboxylic acid Phosphoacylglycerols are the second most abundant group of naturally occurring lipids (found in plant and animal membranes) All have long, non-polar hydrophobic tails, and polar hydrophilic heads (amphipathic) Nature of the fatty acid can vary widely |
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Waxes |
A complex mixture of esters of long-chain carboxylic acids and alcohols Found as protective coatings for plants and animals Plants – coat stems, leaves, fruits Animals- furs, feathers, and skin Myricyl cerotate – component of carnauba wax (Brazilian wax palm). Used in floor and automobile wax Cetyl palmitate: component of spermaceti (wax produced by whales) Used in cosmetics
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Sphingolipids |
Contain a long-chain amino alcohol called sphingosine Found in plants and animals Ceramide: one fatty acid is linked to amino group of sphingosine by an amide bond Sphingomyelin: Primary alcohol group esterified to phosphoric acid which is in turn esterified to choline (amino alcohol) Abundant in cell membranes in nervous system amphipathic Sphingomyelin bares structural similarity to phospholipids |
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Glycolipids |
a compound in which a carbohydrate is bound to an -OH of the lipid by a glycosidic bond Found in cell membranes of nerve and brain cells In most cases, the carbohydrate (sugar) is either glucose or galactose Many glycolipids are derived from ceramides (resulting compound is a cerebroside) Glycolipids with complex carbohydrate moiety that contains more than 3 sugars are known as gangliosides Gangliosides are present in nerve tissues |
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Gangliosides |
glycoprotein with a complex carbohydrate portion Carbohydrate portion contains more than three sugars. One of them is always a sialic acid |
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Steroids |
A group of lipids that have fused-ring structure of 3 six-membered rings (A, B, C) and 1 five-membered ring (D). Examples: cholesterol, testosterone, progesterone Cholesterol is a precursor of other steroids and Vitamin D. Found in biological (animal) membranes (not in prokaryotic membrane) |
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Biological Membranes |
Cells are encased by membranes composed of bilayers of lipids and proteins. Eukaryotic cells have additional membrane-enclosed organelles (such as nuclei, mitochondria) Membranes separate cell from external environment, role in transport into and out of cell, some enzymes depend on membrane environment for function Composition of lipids in membranes is important for membrane properties polar head groups are in contact with the aqueous environment nonpolar tails are buried within the bilayer the major force driving the formation of lipid bilayers is hydrophobic interaction the arrangement of hydrocarbon tails in the interior can be rigid (if rich in saturated fatty acids) or fluid (if rich in unsaturated fatty acids) |
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Lipid Bilayers |
Principal lipid component is phosphoglycerides Glycolipids, cholesterol (animals), phytosterols (plants) The polar surface of the bilayer contains charged groups The hydrophobic tails lie in the interior of the bilayer
Arrangement of hydrocarbon interior determines bilayer fluidity Saturated hydrocarbons, close packing, ordered, - leading to rigidity Kink(s) in hydrocarbon chain = causes disorder in packing against other chains This disorder causes greater fluidity in membranes with cis-double bonds compared with saturated fatty acid chains |
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Cholesterol |
Presence of cholesterol reduces fluidity (enhances order and rigidity) Fused ring is quite rigid Stabilizes extended chain conformations of the hydrocarbon tails of fatty acids as a result of van der Waals interactions |
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Transition temperature |
With heat, ordered bilayers become less ordered Transition temperature (temperature at which it changes from gel to liquid crystals) is higher for more rigid and ordered membranes, and lower for less rigid and disordered membranes Gel to liquid transition Thickness decreases Surface area increases Mobility of the lipid chains increases dramatically |
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Membrane Proteins |
Several proteins are attached to membranes Transport proteins: help move substances in and out of cell Receptor proteins: transfer of extracellular signals (carried by hormones, neurotransmitters) into cells Types Peripheral proteins: On the surface of the membrane. Integral proteins: cross both sides of lipid bilayer May be completely embedded
Certain proteins are anchored to biological membranes by lipid anchors. Particularly common are the N-myristoyl and S-palmitoyl anchoring motifs N-myristoylation always occurs at an N-terminal glycine residue S-palmitoylation involves thioester linkages at cysteine residues |
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Fluid-Mosaic Model |
Most widely accepted description of biological membranes Lipid bilayer has proteins, glycolipids, and steroids such as cholesterol embedded in it Proteins and a lipid bilayer exist side-by-side (mosaic) without covalent bonds between the proteins and the lipids Proteins tend to have a specific orientation in the membrane, can move along the plane of the membrane |
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Liposomes |
an enclosed phospholipid bilayer structure (artificial membrane). This stable spherical structure can be prepared with a drug inside it and used to deliver that drug to the tissues Gets the drug to a place where it is most effective |
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Functions of Membranes |
Three important functions that take place in/on membranes Transport – allow flow of substances into or out of cells Catalysis – Enzymes (proteins) bound to membranes catalyze reactions Receptors – membrane proteins bind specific biological substances that trigger biochemical responses in cells |
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Passive transport |
substance moves from a region of higher concentration to one of lower concentration (driven by a concentration gradient). Cell does not use energy Simple diffusion: a molecule or ion moves through an opening in membrane Facilitated diffusion: a molecule or ion is carried across a membrane by a carrier/channel protein |
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Active transport |
substance moves from a region of lower concentration to one of higher concentration (against a concentration gradient). Cell uses energy Primary active transport: transport is linked to the hydrolysis of ATP or other high-energy molecule; for example, the Na+/K+ ion pump Secondary active transport: driven by H+ gradient |
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Simple diffusion |
Passive diffusion of small uncharged molecules (CO2, O2, N2) across membrane Driven by a concentration gradient |
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Facilitated Diffusion |
Movement of molecules using a carrier protein Example: glucose passes through glucose permease (carrier protein) into erythrocytes Driven by a concentration gradient, no energy is used |
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Primary Active transport |
Movement of molecules against a concentration gradient Directly linked to hydrolysis of high-energy yielding molecule (e.g. ATP) |
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Secondary Active Transport |
Couple molecule transmembrane transport with an energy source Lactose concentration is higher inside the bacterial cell (requires energy to move lactose into cell Proton pumps: integral membrane proteins that create a hydrogen ion gradient across the membrane Galactose permease allows H+ to flow through it into the cell. One H+ is transported in with a lactose (harness energy for co-transport). |
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Membrane Receptors |
Binding of a biologically active substance to a receptor initiates an action within the cell LDL: low density lipoprotein; principal carrier of cholesterol in the blood stream LDL is a particle that consists of phosphoglycerides, cholesterol, proteins
1. LDL binds LDL receptor 2. Receptor + bound LDL = vesicle; pinched off into cell (endocytosis) 3. LDL is released, releases cholesterol to be used in the cell 4. Receptor protein is recycled back to cell surface 5. Oversupply of cholesterol inhibits synthesis of LDL receptor 6. Too few receptors – level of cholesterol in blood increases Atherosclerosis – blocked arteries Heart attacks, strokes |
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Lipid-Soluble Vitamins |
Vitamins are divided into two classes: lipid-soluble and water-soluble Lipid soluble vitamins are hydrophobic A, D, E, K |
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Vitamin A (Retinol) |
Vitamin A (retinol) found in animals Formed from enzymatic cleavage of B-carotene (unsaturated hydrocarbon) B-carotene is abundant in carrots and other vegetables (yellow) Enzymatic oxidation of Retinol (Vitamin A) forms retinal Vitamin A participates in the visual cycle in rod cells the active molecule is retinal (vitamin A aldehyde) retinal forms an imine with an -NH2 group of the protein opsin to form the visual pigment called rhodopsin Rhodopsin absorbs light Isomerization of the 11-cis double bond to the 11-trans double bond All trans-retinal and opsin released (cannot bind opsin) Electrical impulse is generated in the optic nerve and transmitted to brain to be processed as a visual event Enzyme isomerase regenerates 11-cis form leading to formation of rhodopsin Vitamin A deficiency: blindness, night blindness Excess vitamin A: bone fragility |
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Vitamin D |
A group of structurally related compounds that are involved in the regulation of calcium and phosphorus metabolism The most abundant form in the circulatory system is vitamin D3. Formed from action of UV light (from the sun) on cholesterol. Vitamin D3 leads to synthesis of calcium-binding proteins, increase in absorption of dietary calcium in the intestines, and calcium uptake by bones Deficiency – rickets: bones become soft, leading to skeletal deformities |
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Vitamin E |
The most active form of vitamin E is a-tocopherol Required (in rats) for reproduction and prevention of muscular dystrophy Vitamin E is an antioxidant (reacts with oxidizing agents before they can attack other biomolecules) Removes very reactive and highly dangerous free radicals (HOO• and ROO•) |
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Vitamin K |
Important in blood-clotting process Two carbonyl groups (polar groups) & long unsaturated hydrocarbon side made up of repeating isoprene units Number of isoprene units determine type of Vitamin Vitamin K has an important role in the blood-clotting process Required to modify prothrombin (adds a carboxyl group to alter glutamate residues) and other proteins Unmodified prothrombin does not bind calcium Anticoagulants dicumarol and warfarin are Vitamin K antagonists |
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Prostaglandins |
Derived from fatty acids, widely distributed in tissues Have a five-membered ring. Differ from each other by positions of double bonds and oxygen-containing functional groups The metabolic precursor is arachidonic acid (20 carbon atoms: 4 double bonds) Functions: control of blood pressure, induction of inflammation Aspirin, cortisone and other steroids inhibit synthesis of prostaglandins (anti-inflammatory ) |
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Leukotrienes |
Compounds also derived from arachidonic acid Found in white blood cells (leukocytes) Consists of 3 conjugated double bonds An important property is constriction of smooth muscles, especially in the lungs Synthesis of Leukotrienes triggered by allergic reactions (eg; to pollen) Asthma attacks may result from Leukotriene-induced constriction Drugs that inhibit leukotriene C synthesis or block leukotriene receptors are used to treat Asthma |
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Thromboxanes |
Also derived from arachidonic acid Contain cyclic ethers as part of their structure Thromboxane A2 induces platelet aggregation and smooth muscle contraction |
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Why should we eat more Salmon? |
Omega-3 fats found in fish oils inhibit formation of certain prostaglandins and thromboxane A Lower tendency for platelet aggregation, and a lower potential for artery damage |
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Nucleic acids |
are macromolecules formed by the polymerization of nucleotides Types of Nucleic acids Ribonucleic Acid (RNA) Deoxyribonucleic Acid (DNA) |
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Nucleotides |
consist of three parts (covalently linked): a nitrogenous base derived from purine or pyrimidine (nucleobases) a sugar, either D-ribose or 2-deoxy-D-ribose phosphoric acid Nucleotide= Nucleoside + phosphoric acid (phosphoric acid esterified to a hydroxyl group of sugar, example 5’-OH) Name based on parent nucleoside, with a suffix, “monophosphate”, “diphosphate”, triphosphate (example: adenosine 5’-monophosphate) |
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Levels of NA Structure |
Primary structure: the order of bases in the polynucleotide sequence ( the order of bases specifies the genetic code) Secondary structure: the three-dimensional conformation of the polynucleotide backbone Tertiary structure: supercoiling of the molecule Quaternary structure: interaction between nucleic acids and other macromolecules to form complexes (ex: proteins) |
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Pyrimidine and Purine Bases |
Pyrimidine: Cytosine, Thymine, Uracil Purine: Adenine, Guanine |
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Nucleoside |
Base + sugar |
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Primary structure of DNA |
Backbone consist of alternating units of 2-deoxy-D-ribose and phosphate 3’-OH of one sugar is joined to the 5’-OH of the next sugar by a phosphodiester bond (repeating linkage is 3’, 5’-phosphodiester bond) Base sequence is read from the 5’ end (has a phosphate group) to the 3’ end (free hydroxyl group). Single letter A,G,C, and T |
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DNA – Secondary Structure |
The three dimensional conformation of the backbone Double helix: two polynucleotide chains wrapped around each other coiled in a right-handed manner antiparallel Proposed by James Watson & Francis Crick, in 1953 |
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Right and Left handed helices |
Right- handed helices: helix winds upwards in the direction in which the fingers of the right hand curl when the thumb is pointing upward Left-handed helices: helix winds upward in the direction in which the fingers of the left hand curl when the thumb is pointing upward |
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Base Pairing |
A major factor stabilizing the double helix is complimentary base pairing by hydrogen bonding between T-A and between C-G T-A base pair has 2 hydrogen bonds G-C base pair has 3 hydrogen bonds |
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Other conformations (forms) of DNA |
B-DNA considered the physiological form, a right-handed helix, diameter 11Å, 10 base pairs per turn (34Å) of the helix A-DNA a right-handed helix, but thicker than B-DNA, 11 base pairs per turn of the helix, base pairs are not perpendicular to helix axis, lie at an angle of ~ 20o , has not been found in vivo Z-DNA a left-handed double helix, usually found in purine-pyrimidine alternating sequences (CGCGCG), may play a role in gene expression |
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Tertiary structure (Prokaryotic DNA) |
Prokaryotic DNA is Circular: a type of double-stranded DNA in which the 5’ and 3’ ends of each stand are joined by phosphodiester bonds Supercoiling |
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Supercoiling (Prokaryotic) |
Further coiling and twisting of DNA helix. Positive supercoil: overwound circular DNA, have more than the normal number of turns of a helix Negative supercoil: underwound circular DNA, have fewer than the normal number of turns of a helix |
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Supercoiling in Eukaryotic DNA |
More complicated than prokaryotes Eukaryotic DNA is bound to a number of proteins Chromatin: a complex of DNA and protein found in eukaryotic nuclei. Resembles “beads on a string” Histones: the principal proteins in chromatin. Basic proteins (H1, H2A, H2B, H3, H4) Each bead is a nucleosome |
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Nucleosome |
a globular structure in chromatin in which DNA is wrapped around an aggregate of histone molecules |
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Denaturation of DNA |
Disruption of secondary structure commonly by heat (melting) Strands unwind, then separate to form single strands Midpoint of transition (melting) curve = Tm the higher the % G-C, the higher the Tm (why?) Renaturation (annealing) is possible on slow cooling |
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RNA |
consist of long, unbranched chains of nucleotides joined by phosphodiester bonds between the 3’-OH of one sugar and the 5’-OH of the next the sugar unit is D-ribose (it is 2-deoxy-D-ribose in DNA) the pyrimidine bases are uracil and cytosine (they are thymine and cytosine in DNA) in general, RNA is single stranded (DNA is double stranded) RNA molecules are classified according to their structure and function |
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Messenger RNA (mRNA) |
carries coded genetic information from DNA to ribosomes for the synthesis of proteins single stranded a complementary strand of mRNA is synthesized along one strand of an unwound DNA, starting from the 3’ end |
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Transfer RNA (tRNA) |
Transports amino acids to site of protein synthesis a single-stranded polynucleotide chain between 73-94 nucleotides intramolecular hydrogen bonding occurs in tRNA (A—U and G—C ) Different types found in living cells, frequently several tRNAs for each amino acid (carried at its 3’ end) |
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Ribosomal RNA (rRNA) |
found in ribosomes, the site of protein synthesis only a few types of rRNA exist in cells. Ribosomes consist of 60 to 65% rRNA and 35 to 40% protein |
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Small nuclear RNA (snRNA) is a recently discovered RNA |
Found in nucleus of eukaryotes Small (100-200 nucleotides long) Associates with proteins to form small nuclear ribonucleoprotein particles (snRNPs) snRNPs help with processing of initial mRNA transcribed from DNA |
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Micro RNA (miRNA) |
small (~ 22 bp) non-coding RNA molecule found in plants, animals and some viruses functions in RNA silencing and post-transcriptional regulation of gene expression. miRNAs function by base-pairing with complementary sequences within mRNA molecules silencing these molecules |
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Information Transfer in Cells |
Replication = process of duplicating DNA Transcription = process of formatting RNA on a DNA template Translation = process of protein synthesis in which the amino acid sequence of the protein reflects the sequence of the bases in the gene that codes for that protein Information encoded in the nucleotide sequence of DNA is transcribed through synthesis of an RNA molecule RNA sequence is determined by the DNA sequence Sequence of RNA is read by protein synthesis machinery, and translated into amino acids in proteins |
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Central dogma of biology |
DNA—RNA —Protein |
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Semiconservative Replication |
Replication involves separation of the two original strands and synthesis of two new daughter strands using the original strands as templates each daughter strand contains one strand from the original DNA, and one newly synthesized strand |
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Challenges in replication |
1. Separating the two DNA strands Strands must be unwound Protection of single strands from nucleases 2. Synthesizing DNA from 5’ to 3’ end two antiparallel strands must be synthesized in the same direction on antiparallel templates 3. Guarding against errors in replication Correct base is added to the growing strand |
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In which direction does replication go?
Origin or Replication
Replication Fork |
Replication is bidirectional Origin of replication: the point at which the DNA double helix begins to unwind at the start of replication Replication fork: the point(s) at which new DNA strands are formed Prokaryotes: 1 origin of replication, 2 replication forks Eukaryotes: Several origins of replication; 2 replication forks at each origin |
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Addition of a nucleotide to a growing DNA chain |
DNA synthesis occurs in the 5’ to 3’ direction from the perspective of the growing chain The 3'-hydroxyl group at the end of the growing DNA chain is a nucleophile. It attacks the phosphorus adjacent to the sugar in the nucleotide which is added to the growing chain. Pyrophosphate is eliminated, and a new phosphodiester bond is formed. |
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Semi-discontinuous model for DNA replication |
DNA synthesis occurs in the 5’ to 3’ direction (from the perspective of the growing chain) The leading strand is synthesized continuously in the 5’ -> 3’ direction toward the replication fork The lagging strand is synthesized semi-discontinuously (Okazaki fragments) also in the 5’ -> 3’ direction, but away from the replication fork The lagging strand fragments are joined by the enzyme DNA ligase |
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Requirements for DNA synthesis |
All four deoxyribonucleotide triphosphates: deoxythymidine triphosphate (dTTP), deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), magnesium ions (Mg2+) DNA (template) Primer: a short RNA strand to which the growing polynucleotide chain is covalently attached in the early stages of replication Multiprotein complex called replisome (13 proteins in bacteria) DNA polymerase: the enzyme that catalyzes the successive addition of each nucleotide to the growing DNA chain There are at least five types of DNA polymerases (Pol) in E coli Pol II, Pol IV, Pol V: repair enzymes |
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Proteins required for DNA replication |
Helicase: unwinds DNA duplex DNA gyrase: puts negative supercoils ahead of replication fork Single-strand binding protein (SSB): protects single strand DNA from nucleases Primase: copies a short stretch of DNA template strand to produce RNA primer sequence DNA pol III: Each half of the replicative polymerase dimer is bound to its template strand by a β-subunit sliding clamp. DNA pol I and DNA ligase act downstream on the lagging strand to remove RNA primers, replace them with DNA, and ligate the Okazaki fragments |
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3 essential activities of E. coli DNA polymerase I |
1. 5' to 3' polymerization of DNA - makes phosphodiester bonds to create DNA strands 2. 3' to 5' exonuclease - proofreading function to remove nucleotide errors arising during replication 3. 5' to 3' exonuclease - to remove RNA primers |
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Proofreading |
the removal of incorrect nucleotides immediately after they are added to the growing DNA during replication DNA replication takes place only once each generation in each cell Errors in replication (mutations) can be lethal to organisms Errors in replication occur spontaneously only once in every 10^9 to 10^10 base pairs Errors in hydrogen bonding occur once in every 10^4 to 10^5 base pairs Pol I has proofreading, and repair functions. It can remove an incorrect nucleotide, and add the correct nucleotide |
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Mutations in DNA |
Common mutagens: UV light, radioactivity, some chemical agents UV irradiation causes dimerization of adjacent thymine bases - a cyclobutyl ring is formed between carbons 5 and 6 of the pyrimidine rings. Normal base pairing is disrupted by the presence of such dimers. The normal shape of the DNA is distorted |
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Oxidation Damage |
Oxygen radicals, in the presence of metal ions such as Fe2+, can destroy the sugar rings in DNA, break the strand |
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Nick translation |
DNA Polymerase I can fill in the gap after removing the nucleotides DNA polymerase I can remove up to 10 nucleotides from a 3’-OH single strand nick |
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Mismatch repair |
1. The newly synthesized DNA has a mismatch (G-T) 2. MutH, MutS, and MutI, link the mismatch with the nearest methylation site, which identifies the strand as the parental (correct) strand 3. Exonuclease I removes DNA from the strand between proteins 4. DNA polymerase III fills in the missing bases, DNA ligase seals the gap
Prokaryotes alter their DNA at certain locations by modifying bases with added methyl groups |
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Base-excision repair |
A damaged base is removed from the sugar–phosphate backbone by DNA glycosylase, creating an AP (apurinic/apyrimidinic) site. AP endonuclease removes the sugar and phosphate from the nucleotide An excision exonuclease removes the AP site and several nucleotides. DNA polymerase I fills the gap DNA ligase seals the phosphodiester backbone |
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Nucleotide-excision repair |
Commonly used to repair damage that deforms the DNA structure (pyrimidine dimers caused by UV light, chemicals ) ABC excinuclease binds to the region and cuts out a large piece of DNA, including the lesion. DNA polymerase I and DNA ligase then resynthesize and seal the DNA. |
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Eukaryotic DNA Replication |
A higher level of complexity There are multiple origins of replication More proteins and enzymes are involved The timing must be controlled to that of cell division In human cells, a few billions base pairs of DNA must be replicated once, and only once per cycle Cell cycle times vary from less than 24 hours to hundreds of days |
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Initiation of DNA replication cycle in Eukaryotes |
Origin recognition complex: multi-protein complex that binds DNA and serves as point of attachment for other proteins Replication activator protein: an activation factor Replication licensing factors: replication can not proceed until they are bound Phosphorylation by cyclin and cyclin-dependent kinases Initiates replication Phosphorylated RAP and RLF are released (from ORC) and subsequently degraded. G2 phase: DNA has been replicated During mitosis DNA is separated into the daughter cells |
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Structure of the PCNA homotrimer |
Proliferating cell nuclear antigen (PCNA) is part of Pol δ It is the eukaryotic equivalent of the part of Pol III that functions as a sliding clamp (B-subunit). |
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Eukaryotic Replication Fork |
DNA polymerase α: Primase activity and addition of first few nucleotides After a few nucleotides are incorporated, DNA polymerase δ, with its associated proteins bind and do the majority of the synthesis. Separate proteins (FEN-1 and RNase H1) degrade the RNA primers after replication. |
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Telomeres |
Replication of linear DNA poses particular problems at the ends of the molecules The ends of eukaryotic chromosomes have special structures called telomeres (series of repeated DNA sequences) which protect the end of the chromosome from degradation |
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Telomerase |
an enzyme containing a section of RNA that is complementary to the telomere sequence. It uses this RNA as a primer to synthesize DNA at the ends of the chromosome Removal of the primer shortens the DNA but it is now longer by one repeat unit The telomerase extension cycle is repeated until there is an adequate number of DNA repeats for the end of the chromosome to survive |
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Comparison of DNA Replication in Prokaryotes and Eukaryotes |
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General Features of RNA synthesis |
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Transcription |
Template strand (antisense, noncoding); DNA strand used as a template for the synthesis of RNA RNA polymerase binds and transcribes the template strand RNA polymerase moves from 3’ end to the 5’ on the DNA template strand The mRNA is formed from the 5’ end to 3’ end Nontemplate strand (coding/sense): The DNA strand that has the same sequence as the RNA that is synthesized |
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Transcription in Prokaryotes |
Simplest of organisms contain a lot of DNA that is not transcribed RNA polymerase needs to know which strand is the template strand which part to transcribe where first nucleotide of gene to be transcribed is located Promoters: DNA sequence that provide direction for RNA polymerase (close to 3’ end of template strand) Controls frequency with which a gene is transcribed |
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Elements of a bacterial promoter |
Promoters have at least three components Transcription start site (TSS): site used to initiate RNA synthesis -10 region (Pribnow box): an essential part for transcription to occur -35 region: Important in the control of RNA synthesis UP element: a promoter element that is 40- 60 bases upstream of the transcription start site Element: general term for a DNA sequence that is somehow important in controlling transcription |
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consensus sequences |
Promoter regions in a number of prokaryotic genes contain base sequences that are common |
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Initiation and Elongation in Transcription |
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Factor independent termination (Intrinsic termination) |
controlled by specific sequences Two G-C rich regions (inverted repeats) A-T rich region Inverted repeats form a hairpin loop which destabilizes the association between RNA pol II and the DNA template A-T rich region forms A-U base pairs which also destabilizes the association between RNA pol II and the DNA template, terminating transcription |
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Factor-dependent (Rho protein) |
rho () protein binds a recognition site on mRNA, and moves along it toward the transcription bubble. When the Rho protein reaches the transcription bubble, at the termination site it causes dissociation (removal) of RNA pol from DNA template. |
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Transcription control in Prokaryotes |
Enhancers and silencers: DNA sequences that transcription factors bind, to increase or reduce the level of transcription Alternative σ factors: Expression of different σ-subunits that direct RNA pol to different promoters Transcription attenuation: controls transcription after it has begun by adjusting the level of transcription based on the quantity of a related product Operons: a group of genes that are controlled by the same promoter Genes are only transcribed in the presence of an inducer (a small molecule) Example induction of β-galactosidase (hydrolyzes lactose to galactose and glucose)
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Catabolite repression in the Lac Operon |
Catabolite repression: repression of the synthesis of lac proteins by glucose Promoter has two regions Binding site for RNA polymerase RNA polymerase binding site overlaps with repressor binding site Binding site for a regulatory protein, catabolite activator protein (CAP) Binding of CAP depends on cAMP (“hunger signal”) Cap-cAMP complex bound to CAP site , RNA pol I binds Positive regulation It takes the presence of lactose and the absence of glucose for the lac operon to be active
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The trp operon in E.coli |
Five proteins A-E make up four enzymes that catalyze synthesis of tryptophan Trp R encodes a repressor protein that binds tryptophan High levels of tryptophan, repression occurs Low levels of tryptophan, five proteins are synthesized |
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Transcription Attenuation |
Hairpin loops: Secondary structures formed in mRNA that are responsible for termination of transcription 1•2 pause structure: hairpin loop which forms and causes RNA polymerase to pause 3•4 terminator: hairpin loop which forms and causes premature release of the RNA transcript 2•3 antiterminator: hairpin loop which forms allowing transcription to continue |
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Attenuation mechanism in trp operon |
When trp levels are high, ribosome passes over the trp codons quickly, Pause structure forms causing premature abortion of the transcript as the terminator loop is allowed to form. When tryptophan levels are low, the ribosome stalls at the trp codons, allowing the antiterminator loop to form, and transcription continues |
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Transcription in Eukaryotes |
More complex Three primary RNA polymerases (each recognizes a different set of promoters) RNA polymerase I: transcribes most ribosomal RNA (rRNA) RNA polymerase II: transcribes messenger RNA (mRNA) RNA polymerase III: transcribes transfer RNA (tRNA) Numerous protein factors control transcription initiation No operons - Genes to be transcribed depend on transcription factors Elongation involves the addition of the 5'–phosphate of ribonucleotides to the 3'–OH of the elongating RNA (pyrophosphate is release) Specific termination signals terminate transcription. |
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Pol II Promoters |
Upstream elements: specific proteins bind this region to activate (enhancers) or suppress (silencers) transcription TATA box: located ~25 – 100 bases before the transcription start site. Has consensus sequence TATAAT or TATAAA Necessary for transcription in some genes, Orients the RNA polymerase correctly in others Initiator element (lnr): includes TSS (+1) and surrounding sequences Downstream element: a region for possible regulation (not as common as upstream elements) |
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Transcription factor |
any protein that is not a subunit of RNA polymerase, but can regulate transcription |
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Sequence of events in Pol II transcription |
A transcription factor (TFIID) binds the TATA box – first step in assembly of the pre-initiation complex Poll II is phosphorylated before transcription starts. |
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Regulation of Transcription |
DNA looping brings enhancers (or silencers) into contact with transcription factors and polymerase to activate or suppress transcription |
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Response Elements |
short sequences of DNA within a promoter region that are able to bind specific transcription factors (produced under certain cell conditions) and regulate transcription. Usually located in the promoter region of different genes, but can all be activated by the same stimuli, to produce a coordinated response Heat shock response element (HSE) is present in heat shock protein genes. In response to high temperature, the heat shock transcription factor (HSTF) will interact with HSE, to activate the transcription of heat shock proteins (Hsp70, Hsp90) |
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Comparing Eu and Pro RNA |
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Zinc Finger motif |
arises from zinc interacting with two closely spaced cysteines and two closely spaced histidines Zinc finger proteins follow the major groove of DNA |
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Basic-Region Leucine Zipper Motif |
A 30-amino acid segment with a periodic repetition of leucine residues at every seventh position Other regions of the protein are rich in lysine and arginine residues |
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Posttranscriptional Modification of Eukaryotic mRNA |
1. 5' end capping: Addition of a N-methylated guanine group to the 5’ end. - Capping protects RNA from exonucleases. 2. Polyadenylation: A long sequence of adenosine residues (polyadenylate “tail”) that is usually100-200 nucleotides long, is added to the 3’ end of the mRNA - polyadenylation protects the mRNA from nucleases and phosphatases 3. RNA splicing: the process of removing introns (non-coding sequences). - RNA is transcribed from genes that contain introns which must be removed before RNA becomes biologically active
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Capping of 5’-end of mRNA |
Capping of the 5’ end with a methylated guanylate residue, bonded to the next residue by a 5’ -> 5’ triphosphate. The 2’hydroxyl group of the neighboring ribose sugar is also frequently methylated (sometimes next neighbor as well) |
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The splicing reaction |
Specific sequences make up the splice sites: GU at 5’ end of intron, AG at 3’ end of intron, Adenine (A) in the branch site Advantage: A single gene can code for multiple proteins - Tau ( found in brain of Alzheimer patients) has six isoforms Abnormalities in the splicing process can lead to various disease states. - Some forms of β-thalassemia are caused by mutations in the sequences required for intron recognition (leads abnormal processing of the β-globulin primary transcript) |
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Ribozymes |
Proteins are not the only biological molecules with catalytic properties. Some RNAs, called ribozymes (ribonucleic enzymes), also catalyze certain reactions. Some Ribozymes catalyze their own splicing (self-splicing) |