Year 1 Biochemistry Cellular Composition PDF

#separator:tab #html:true #tags column:12 What does Gibbs free energy tell us? Tells us if a reaction will occur spontaneously  - free energy decreases in spontaneous reactions What does it mean if ∆G > 0? Endergonic (non-spontaneous) reaction reaction very unlikely to occur at that temperature What does it mean if ∆G = 0? Reaction occurs in equilibriumit is used to find out the minimum temperature a reaction can take place in What does it mean if ∆G < 0? Exergonic (spontaneous) reactionThe reaction will always occur at that temperature Gibbs free energy equation:When ∆G = 0 ∆G = ∆H - T∆S∆H = T∆S Explain the trend in disorder in relation to ∆G: As entropy increases - meaning disorder increases therefore lower ∆G (as when ∆G < 0 then the reaction is more likely to occur spontaneously) Describe how water molecules arrange themselves around a protein using hydrophobic interactions: "Water moelcuels close to te surface of hydrophbic proteins have few opportuniteis to form hydrogen bonds between one anotherConsequently, many water molecuels form ordered ice-like ""shells"" around the protein" What is the entropy of the hydrophobic interactions in a protein? How does this effect the reactions regarding to expenses? "The system has a low ∆S thus ∆G is hightherefore Ordered shells have an energetic cost" What can we do to increase entropy without increasing temperature or energy and being more cost effective? If the hydrophobic SA is minimised, the n.o ordered water molecules is reduced.This increases ∆S thus reducing ∆G How deos the hydrophobic effect affect/dominate protein structure? The hydrophobic effect drives hydrophobic molecules together.it is a dominant force in protein folding What is the equation for the dissociation of water? "H2O ⇌ H+ (or H3O+) + OH- " What is the ratio of ions in pure/neutral water? 1:1[H+] = [OH-] What is pH? pH = -log[H+] What is the equation for the equilibrium dissociation constant for water (K)? [H+][OH-] / [H2O] What is/meaning of the Ka for water? the dissociation constant for pure water at 25ºC What is Kw? "the ""ion product"" constant at 25ºCKw for water = 10-14 [H+][OH-] = 10-14 is valid for ANY aq. solution" What is Ka? the equilibrium constant, is a measure of the strangth of an acid or base Equation for Ka: Ka = [A-][H+] / [HA] Equation for pKa involving Ka: pKa = -logKa ORKa = 10-pKa What is the Henderson-Hasslebalch Equation? pH = pKa + log[A-]/[HA] What is the Le Chantelier's Principle? If a dynamic equilibrium is disturbed by changing the conditions, the postion of equilibrium moves to counteract the change What are buffers? they are weak acids/bases that dissociate or associate to realease or absorb pritons, thus resisting/preventing changes in pH Describe how buffers in the blood work: "Bicarbonate ions (HCO3-) is the major buffer system in the plasma and interstitial fluidIn tissues: CO2 + H2O ⇌ H2CO3 (carbonic acid)This immediately dissociates and ionises: H2CO3 ⇌ H+ + HCO3- (bicarbonate)THUS OVERALL:CO2 + H2O ⇌ H+ + HCO3-" Describe/explain how amino acids can act as buffers: "Naturally, amino acids are present as zwitter ions" What are the properties of water due to? (3) Its SizeIts ShapeIts Polarity What does the hydrophobic effect explain to us? why non-polar molelcuels are forced together in aqueous solution What do buffers prevent from happening? Prevent changes in pH at around their pKa Describe the properties of water: (6) High specific heat capacityAdhesion and cohesion (high surface tension)High latent heat of vaporisationExcellent solvent propertiesLower density in solid form than liquid formActs as a transport medium and  a medium for reactions What is the bond angle of water? 104.5º What does it mean when a molecule is polar? when 1 end of the moelecule is different to the other (1 is delta -ive and 1 is delta +ive) - due to separation of electrical charge (due to the different electronegativity levels). Why does oxygen bind to 2 hydrogen moelcules in water? as it is energetically favourable for O to do so How do you get from kCal --- kJ? ÷4.2 In which numbers are covalent bond energies usually in? In the 100s Describe the trend in bond energies going from covalent ---> H-bond ---> hydrophobic interactions --- Van der Waals: Down this series bond energies decrease as the intermolecular forces are weaker What is the van der waals radius? the radium of the whole atom/moelcule How much is 1 Ångström in metres? 1 x 10-10 What is a hydrogen bond? when a hydrogen atom is shared between 2 electronegative atoms (e.g. O or N).Elctrostatic, weak Are hydrogen bonds strong? They are weak individually but very strong all together How do you draw hydrogen bonds? 180º and vertical lines How far are H-bonds? They are 3Å apart Why is water such a good solvent? due to H-bondingmany molecules/substances can dissolve within water What happen to polar molecules in water? they dissolve in water as they can form H-bonds with water What happens to ionic moelcules in water? They dissociate and are surrounded (hydrated) by water forming HYDRATION SHELLS What happen to non-polar molecules in water? They repel water as they don't have similar bond energies  What does it mean when a reaction occurs spontaneously? that the reaction occurs without us putting energy into it what does it mean when a reaction is endergonic (non-spontaneous)? it requires an input of energy for the reaction to occur What happens to ∆G when we increase entropy (∆S)? Why? ∆G decreases this is as there is increased disorder therefore increased sponteneoty therefore a more spontaneous reaction What is the hydrophobic effect? Where non-polar molecules cluster together (decrease SA) in water to minimise the ordered water molecules, increasing entropy (∆S) and lowering ∆Git is the dominant force in protein foldingiot eplains why non-polar molecules are FORCED TOGETHER in aqueous solutions Why is change of entropy the most important driving factor in biological interaction? Due to H-bonding of H2O.proteins have a specific shape as they are dissolved in water What is another unit for M? molL-1 What is the Kw of pure water? 1 x 10-14 What is a STRONG acid? an acid that fully dissociates when in solution What is a WEAK acid? an acid that partially dissociates in solution (forms an ionic salt) Explain the relationship between acid strength and the Ka value: the weajer the acid, the lower the Ka as it takes more energy to stabilise the weak acid to form the salt (to ionise it) When does a buffer work best? at around its pKa (+ or - 1 unit from pKa) Meaning of interstitial fluid: fluid in between cells Describe the overall buffer reaction with HCO3-: describe how you got to this: "In the tissues:      CO2 + H2O ⇌ H2CO3 Carbonic acid immadiates ionises/dissociates:       H2CO3 ⇌ H+ + HCO3- OVERALLCO2 + H2O + H2CO3 ⇌ H2CO3 + H+ + HCO3- Cancel the yellows as they cancel outthereforeCO2 + H2O ⇌ H+ HCO3-" Why can amino acid buffer at 2 different pKa? as they have 2 sides that can buffer (amino group and acid group) at lower pH and higher pH What is mean by the central dogma? "DNA codes for proteins:DNA is the template for RNARNA is the template for proteins" What are ribosomes? the ribosomes is an ancient, complex, molecular machine made from protein and RNAProteins are synthesised o riboso,es using RNA as the template What is protein function determined by? By the 3D shape - which is determined by the amino acid sequence What type of chains are proteins? LINEAR How many standard amino acids are there? 20 (plus post-translationally modified ones) What 2 types of code do all amino acids have? 3 letter code1 letter code List the names and 3 letter codes of ALL 20 amino acids: Alanine - AlaArginine - ArgAsparagine - AsnAspartate - AspCysteine - CysGlutamine - GlnGlutamate - GluGlycine - GlyHistidine - HisIsoleucine - IleLeucine - LeuLysine - LysMethionine - MetPhenylalanine - PheProline - ProSerine - SerThreonine - ThrTryptophan - TrpTyrosine - TyrValine - Val Give the names of ALL 20 amino acids: (and write them down so you know the spelling) AlanineArginineAsparagineAspartateCysteineGlutamineGlutamateGlycineHistidineIsoleucineLeucineLysineMethionine PhenylalanineProlineSerineThreonineTryptophanTyrosine Valine Give the 3 letter codes of ALL 20 AMINO ACIDS: AlaArgAsnAspCysGlnGluGlyHisIleLeuLysMetPheProSerThrTrpTyrVal Give the names and 3 letter codes of the 4 amino acids starting with an A: Alanine - AlaArginine - ArgAspragine - AsnAspartate - Asp Give the names and 3 letter code of the amino acid starting with an C: Cysteine - Cys Give the names and 3 letter codes of the 3 amino acids starting with an G: Glutamine - GlnGlutamate - GluGlycine - Gly Give the names and 3 letter code of the amino acid starting with an H: Histidine - His Give the name and 3 letter code of the amino acids starting with an I: Isoleucine - Ile Give the names and 3 letter codes of the 2 amino acids starting with an L: Leucine - LeuLysine - Lys Give the name and 3 letter code of the amino acids starting with an M: Methionine - Met Give the names and 3 letter codes of the 2 amino acids starting with an P: Phenylalanine - PheProline - Pro Give the name and 3 letter code of the amino acid starting with an S: Serine - Ser Give the names and 3 letter codes of the 3 amino acids starting with an T: Threonine - ThrTryptophan - TrpTyrosine - Tyr Give the name and 3 letter code of the amino acid starting with an V: Valine - Val Describe the chirality of amino acids: "Optical isomersalpha carbons are asymetric so there are 2 stereoisomers: L and D" With which type amino acids can ribosomes form oeptide bonds between? Ribosomes ONLY form peptide bonds between L-amino acids What is the primary sequence of proteins? The sequnce of amuno acids in the polypeptide chain 30625ea35db44ae0aee48edf7d0acacd-ao-1 Lable the parts of this tripeptide: "" "" "" "" 30625ea35db44ae0aee48edf7d0acacd-ao-2 Lable the parts of this tripeptide: "" "" "" "" 30625ea35db44ae0aee48edf7d0acacd-ao-3 Lable the parts of this tripeptide: "" "" "" "" 30625ea35db44ae0aee48edf7d0acacd-ao-4 Lable the parts of this tripeptide: "" "" "" "" 30625ea35db44ae0aee48edf7d0acacd-ao-5 Lable the parts of this tripeptide: "" "" "" "" 30625ea35db44ae0aee48edf7d0acacd-ao-6 Lable the parts of this tripeptide: "" "" "" "" 30625ea35db44ae0aee48edf7d0acacd-ao-7 Lable the parts of this tripeptide: "" "" "" "" Describe the experiment with urea that led us to believe that a protein's shape is determied by its amino acid sequence:Explain the results of this investigaiton: "This means that proteins don't need outside molecules to reform/refold - all information needed to refold is contained within the protein" What is a protein's shape determined by? Its amino acid sequence Its 3D shape is determined by the physiochemical interaction of its amino acids List all the different physiochemical interactions that amino acids can have within a protein: (6) Ionic interactions (salt bridges)Hydrogen bondsVan der Waals (steric) interactionsHydrophobic interactionsCovalent bondsDisulfide bonds What is the side chain/R group of Alanine (Ala)? "" What is the side chain/R-group of Cysteine (Cys)? "" What is the side chain/R-group of Aspartate (Asp)? "" What is the side chain/R-group of Glutamate (Glu)? "" What is the side chain/R-group of Phenylalanine (Phe)? "" What is the structure/R-group of Glycine (Gly)? "" What is the side chain/R-group of Histidine (His)? "" What is the side chain/R-group of Isoleucine (Ile)? "" What is the side chain/R-group of Lysine (Lys)? "" Whats the side chain/R-group of Leucine (Leu)? "" What is the side chain/R-group of Methionine (Met)? "" What is the side chain/R-group of Asparagine (Asn)? "" What is the side chain/R-group of Proline (Pro)? "" What is the side chain/R-group of Glutamine (Gln)? "" What is the side chain/R-group of Arginine (Arg)? "" What is the side chain/R-group of Serine (Ser)? "" What is the side chain/R-group of Threonine (Thr)? "" What is the side chain/R-group of Valine (Val)? "" What is the side chain/R-group of Tryptophan (Trp)? "" What is the side chain/R-group of Tyrosine (Tyr)? "" What are teh 5 amino acids with charged side chains? "" Explain how the charge on a protein can vary:Use lysine as an example: "The charge on a protein varies according to the amino acid sequence and the pH.E.g. lysine's side chain can very between 0 and +1" What happens to the charged amino acids when they get ionised? If H binds to any O- , it becomes an acid (e.g. aspartate ---> aspartic acid) What kind of physiochemical electrostatic interactions do charged amino acid sidechains form? "Ionic bonding - the ""salt bridge""" What are the 4 amino acids with polar sidechains? "" What type of physiochemical electrostatic interactions do polar sidechains form? "Hydrogen bonds " What are the 6 amino acids with aliphatic sidechains? "" What physiochemical electrostatic interactions do aliphatic sidechains form? Van der Waals interactions Describe how Van de Waals interactions come to be: "Atoms behave (almost) as if they are soli spheresAtoms sharing electrons (covalent bonds) are brought unusually close togetherWhen brought together atoms not sharing electrons experience Van der Waals interactions:          - it is not energetically favourable to share electronsincreased energy -= increased repulsion decreased distance between nuclei = increased repulsion - energetically favourable" What are we trying to maximise when we compress a protein? We are trying to maximise the Van der Waals interactions therefore have the maximum hydrophobic effectthis is why hydrophobic proteins fold to the middle/core of a protein (in an aqueous protein) Describe the hydrophobic interactions within a protein: "non-polar side chains tend to be buried in the core of the proteinpolar side chains on teh outside of the molecule can form hydrogen bonds with water " What are the 3 amino acids with aromatic sidechains?What is another thing that is special about these 3 side chains "These 3 sidechains are fluorescent, especially Trp" Would tryptophan's side chain be buried in the core of a protein? Why? Yes and no and maybeIt is difficult to find a place for it (due to the ring) so it is usually faced down with NH on the outside to form interactions (e.g. H-bonds) Describe whats secial about Proline and explain why it can be problematic: "Proline disrubts the backbond hydrogen bonding network in beta sheets and alpha helices and are often found at the extremities of these 2º structuresIt is the only side chain that attaches to an amide nitrogen, thus the backbone is not free to rotate around N thus restricted rotations so can only form specific shapes~10% of prolines are in the cis conformation and introduce kinks in the protein backbone" Describe the covalent bonds that form between cysteine sidechains: "When cystein amino acids are oxidised, they can form disulfide bonds" d085af6cd64348389ed45a5cdd155d25-oa-2 "" "" "" "" d085af6cd64348389ed45a5cdd155d25-oa-3 "" "" "" "" d085af6cd64348389ed45a5cdd155d25-oa-4 "" "" "" "" d085af6cd64348389ed45a5cdd155d25-oa-5 "" "" "" "" d085af6cd64348389ed45a5cdd155d25-oa-6 "" "" "" "" d085af6cd64348389ed45a5cdd155d25-oa-7 "" "" "" "" d085af6cd64348389ed45a5cdd155d25-oa-8 "" "" "" "" d085af6cd64348389ed45a5cdd155d25-oa-9 "" "" "" "" d085af6cd64348389ed45a5cdd155d25-oa-10 "" "" "" "" d085af6cd64348389ed45a5cdd155d25-oa-11 "" "" "" "" d085af6cd64348389ed45a5cdd155d25-oa-12 "" "" "" "" d085af6cd64348389ed45a5cdd155d25-oa-13 "" "" "" "" d085af6cd64348389ed45a5cdd155d25-oa-14 "" "" "" "" d085af6cd64348389ed45a5cdd155d25-oa-15 "" "" "" "" d085af6cd64348389ed45a5cdd155d25-oa-16 "" "" "" "" d085af6cd64348389ed45a5cdd155d25-oa-17 "" "" "" "" d085af6cd64348389ed45a5cdd155d25-oa-18 "" "" "" "" d085af6cd64348389ed45a5cdd155d25-oa-19 "" "" "" "" d085af6cd64348389ed45a5cdd155d25-oa-20 "" "" "" "" What is the primary structure of a protein? The sequence of amino acids in a polypeptide chain What is the secondary structure of a protein? the regular re[eated structures found in many proteins:Alpha-helices and beta sheets are by far the most common What is the tertiary structure of a protein? The conformation (3D shape) of a single polypeptide chain What is the quanternary structure of a protein? The conformation (3D shape) of a protein that consists of >1 polypeptide chain Describe th features/properties of alpha helices: "Around 25% of all soluble proteins are mainly alpha helicesNormally right-handed twist/spirallingTightly packed - no free space insideSide chains pointing out (extend out and up)Alpha helices can assemble to make coiled coilsFavoured residues are metionine, alanine, glutamate and lysine (MALEK)Unfavoured residues are proline glycine and aspartate (PGD)Evey carbonyl oxygen forms a H-bod with the amide hydrogen i+4alpha helices are favoured due to thisthe more bonds we can form, the more stable the structure will be" Draw an alpha helix: "" Describe the hydrogen bonding between backbone atoms in an alpha helix: "Every carbonyl oxygen forms a H-bond witht he amide hydrogen i+4 The oxygens and nitrogens point in opposite directionsThe R-groups extend out and up" "What do the 2 numbers on the drawing mean?" 5.4 B = the rise - the rise every time the helix turns around3.6 residues = the amount of residues we need to go around An alpha helix has the sequence 12345671234567123.. where only 1 and 4 are hydrophobic What is the likely structural consequence/outcome? "The helix will form a dimer due to the hydrophobic effect - hydrophobic molecules like to come togetherHydrophobic sidechains 1 and 4 form a hydrophobic patch/stripe.In aqueous solutions, helices are forced together by the hydrophobic effect" What are the other 2 helices that can be found in proteins other then alpha (VERY RARE): "The 310 helic is occasionally found, i+3         - too tight of a helixAs is the π (pi) helix, i+5        - too loose of a helix" When non-alpha helices are found ehat do they look like? How is this useful to us with proteins? "They are normally short sections and they normally interrupt the alpha helixthis is how we can start to for proteins of different shapes" Describe beta sheets: "Hydrogen bonding between adjecent backbonescan be parallel and anti-parallel (anti-parallel are slightly more favoourable than the parallel)" Draw/sketch beta sheets: "" "Describe the directionality of the side chains in these anti-parallel beta sheets:" "Sidechains are extended out and alternating up and down directionsevery other R-group is upyou can have proteins that 1 side is hydrophobic and the other is hydrophilic due to teh directionality of the sidechains" Which primary sequences can form beta sheets? ANY primary sequence can form beta sheetsEXCEPT proline What is a protein domain? A distinct structural or functional unit within a protein Describe how retinol is transported around the blood: "Inside a retinol-binding protein, a beta-barrel composed of anti-parallel beta strands joined by loops" What are the other 2 common secondary structures? Describe: Loops Turns     - the reverse turn is often found linking beta strands (also known as a beta-hairpin)Loops and turns often form the most variabke and biologically active parts of proteins What is another type of helix other than alpha, 310 and π? Describe: "Collagen triple helix It is composed of 3 polypeptide chains with very conserved sequencesEvery 3rd residue is Gly and there are many Pro and Hyp (hydroxyproline - formed by post-translational modification fo Pro) residuesIt is conformationally restricted due to the high proportion of proline residues" Why in a triple collagen helix does every 3rd residue have to by glycine? "Glycine is the only residue small enough to fit in the middle/core." What can beta barrels be used for? Example: To transport hydrophobic molecules around the bodyE.g. retinol (vitamin A) which is insoluble in water (and therefore also blood) "How is the ""Greek key"" supersecondary motif formed?" "By folding a β-hairpin over 2 β-strands - very common in proteins." What are the 3 types of supersecondary motifs? "" Why are supersecondary structures important? Superseconday structures are the building blocks of most proteins How are supersecondary structures stabilised? The helices' and sheet's primary sequences must be complementary to each other "Describe the properties/features of this supersecondary structure 9and supersecondary structures in general):" "The same domains are found in seemingly unrelated proteins4 helix bundles like the 1 on the right are found in many different proteinsHere 2 alpha-alpha motifs combine into a 4-helix bundle domain in cytochrome B562 Note the heme prosthetic group bound betwee the helices" What is triose-phosphate isomerase structure formed from (structurally)? "from 8 parallel β-strands and 8 parallel α helices to form an αβ barrel.Around 10% of all known enzyme structures take this form" Descirbe the immunoglobin (Ig) fold:(one of the most common domains found in nature) "In the immunoglobin fold 7 anti-parallel β strands fold to form a β-sandwichIt's similar to an extended Greek key" Why do proteins fold? To satisfy the sum of their molecular interactions What happens if a protein adopts an incorrect structure? Misfolding proteins can lead to a lot of diseases (especially neurodegenerative diseases). What does Anfinsen's dogma state? That protein structure is determined by its primary structure What deos molecular recognition depend on? Ligand binding What is molecular recognition? How a molecule recognises/knows what it needs to bind to What substances can be ligands? Atoms, ions, molecules or macromolecules Give examples of ligand binding: (6) Substrate bind to enzymesSingle-stranded DNA binds complementary DNATranscription factors bind to DNADrugs bind to enzymes (or other types of protein)Hormones bind to specific receptorsAntibodies bind to antigens Describe allosteric interactions?: "Allosteric proteins bind to multiple ligands" Describe non-allosteric interaction: "Non-allosteric proteins only bind to 1 ligand" What is affinity? A measure of how tightly a protein binds to a ligand Describe the functions of Hb in comparison to Mb: Hb transports O2 from lungs ---> releases in tissues. It also transports CO2 and H+ from tissues ---> releases in lungs.Mb stores (binds and releases) O2 in musclesBoth Mb and Hb can bind, transport and detoxify NO, which is a vasolidator and inhibitor of platelet aggregationBoth Mb and Hb are globular proteins with tightly-packed cores Describe the structure of Hb in comparison to Mb: Both are globular proteins with tightly-packet cores.Haemoglobin (Hb):4 polypeptide chains.       - In ADULTS: 2 alpha-chains and 2 beta-chains (HbA1).       - In FOETUSES: 2 alpha-chains and 2 gamma-chains (HbF)alpha-chains: 141 amino acidsbeta-chains: 146 amino acidsEach chain binds a haeme prosthetic groupMyoglobin (Mb):1 polypeptide chain and 1 haeme group.154 amino acids~25% identical to Hb chainsThe conformation (the 2º and 3º structure) is very similar to Hb chain What transition metals are epecially essential to biology? IronCobaltCopperMolybdenum Why are transition metals essential to biology? "As metal ions bind a discrete n.o ligands to form ""coordinate"" (""dative covalent"") bonds which share a pair of electrons donated by the same atoms" What are the common donor atoms in metal ion-ligand complexes? N, O , S What is a monodentate ligand? "they donate a single pair of electrons to the metal ion" What is a polydentate ligand? a ligand that donates ≥2 pairs of electrons to metal ions Describe the Haeme prosthetic group: "It is a porphryin ring that sits in a hydrophobic cavity on the surface of the subunitO2 binds to the Fe2+ ion in the centre of the ringThis ion is always in the Fe2+ ferrous (2+) state.It is polydentate, binding up to 6 ligands: 4 haeme N, 1 His side-chain (the ""proximal histidine"") and O2." When does the Ferrous (Fe2+) irin oxidise to ferric (Fe3+) iron in Hb? Only if haeme is stripped from the protein What is a special of property of haeme (HINT: in the absence vs presence of O2)? "The Fe2+ has 5 ligands and a larger atomic radius than in the O2-bound stateThe larger radius means that the Fe2+ doesn't fit in the plane of the porphyrin ring in deoxy-Hb       - the Fe2+ is pushed down out of the plane of the porphyrin ringThe O2 forms a H-bond ith another His side chain - the ""distal histidine"" that stabilises the O2-Fe2+ interaction" What stabilises the O2-Fe2+ interaction in the Haeme group in Hb? "The O2 that forms a H-bond with another His side chain - the ""distal histidine""" How well conserved protein is Mb? Mb is a very well conserved proteinMb is well conserved between species In general what does it mean if 2 proteins are >30% identical? They will have a similar backbone shape 086ccbb1686e472c9c898670868bb084-ao-1 "" "" "" "" 086ccbb1686e472c9c898670868bb084-ao-2 "" "" "" "" 086ccbb1686e472c9c898670868bb084-ao-3 "" "" "" "" af8d645ba13444588bcbed50bfe177dc-oa-1 "" "" "" "" af8d645ba13444588bcbed50bfe177dc-oa-2 "" "" "" "" af8d645ba13444588bcbed50bfe177dc-oa-3 "" "" "" "" af8d645ba13444588bcbed50bfe177dc-oa-4 "" "" "" "" Describe the law of mass action:The equilibriumWhat is vf = [P].[L].ka?What is vr= [P:L].kd? "The velocity of the forward reaction is dependent on ka and the concentration of the protein and ligand The velocity of the reverse reaction is dependent on kd and the concentration of the protein:ligand complex" Define the dissociation constant Kd:Units: kd/ka therefore = [P][L]/[P:L]It describes the concentrations of P, L and P:K at equilibriumUnits: moles.L-1 What is the fraction Y? The fractional saturation - how many ligand binding sites are occupiedY vaires between 0.0 and 1.0 What does it mean if Y=1.0? All ligands are bounded to proteins What is the most important equation in biology to find Y? Y = [L]/(Kd + [L])Described how well a lignad binds to a protein What is another equation for Y (not the most important in biology)? Y = [P:L]/([P:L] + [P]) What is the factional saturation when [ligand] = Kd? 0.5 What type of curve is the curve described by this equation?Y = [L]/(Kd + [L]) "A Hyperbolic curve" What does the Y mean when [L] is low? "At low [L], very few binding sites are occupied" What does the Y mean when [L] = Kd? "When [L] = Kd, half the binding sites are occupiedThe Kd is the [L] rerquired to occupy half the binding sites" What does the Y mean when [L] is much greater than Kd? "When [L]>>Kd most of the bidning sites are occupied" Describe what the Y vs [L] curve would look like for when 2 different proteins can bind to the same ligand with different affinities: "Both curves will eventually reach Y=1, the bthe Kd=8 (red) protein is at a lower fractional saturation than the Kd=4 (blue) protein at any given [L]" Describe the effect on Y whena ligand (eg. drug) can bind to 2 different proteins with edifferent affinities: "At any given [drug], the Y of the desired target will be > than that of the non-specific target" Why do some drugs cause side effects? "They may bind to the wrong proteinperhaps drug might bind to non-specific target as well, causing side-effectsas at any given [drug], Y of desired target > non-specific target " What can O2 binding to Mb and Hb be measured with? "Using a spectrophotometer" What is P50 in a Y vs pO2 graph? Dissociation constantP50 = [O2] required to occupy 1/2 the binding sites Give the equation for Y in Mb binding sites: Y = pO2 / (P50 + pO2) What do we measure [O2]/pO2 in? torr (mm Hg) What type of curve is the oxygen binding to Hb? Sigmoidal What type of curve is the oxygen binding to Mb? Hyperbolic What is P50 in an Oxygen bidning to Hb sigmoidal curve? 26 torr What is P50 in an Oxygen bidning to Mb hyperbolic curve? 2 torr What is the pO2 in:Lungs:Resting tissues:Exercising tissues: ~100 torr~40 torr~20 torr Why is Hb a better delivery protein than Mb? As the sigmoidal curve allows Hb to deliver ~x10 as much O2 to exercising tissues as MbMb is useless as a delivery protein as it wouldn't release any O2, whilst Hb is good at realeasing O2 where needed (due to sigmoidal curve and Kd)P50 of Hb is x13 larger than Mb What are the 2 structural forms of Hb? Describe: "Deoxyhaemoglobin      - Tense form (T-form)      - Low affinity for O2 Oxyhaemoglobin      - Relaxed form (R-form)      - High affinity for O2 " What does it mean when Hb has positive cooperativity? Why does Hb have positive cooperativity? Binding of O2 to 1 subunit increases the binding affinity of the other sitesthe sigmoidal binding curve indicates thisReleasing O2 from 1 subunit makes it easier to release the other 3 O2 from the other 3 subunits Describe how cooperativity can be diagnosed: "Using a Hill Plot" Describe how the R-state and T-sate bindign curves look like and how that relates to how the observed Hb binding curve looks like: "" Describe the concerted model for cooperativity: "All subunits exist in either the T or the R stateThe P50 for the R state < P50 for T stateAs O2 binds the eq. shifts from favouring the T-state to favouring the R-stateAs pO2 increases, R state is favoured As pO2 decreases, T state is favoured" What does allosteric mean when referring to Hb? Binding to Hb at a site other than the O2 binding site Describe the function of the 2,3-bisphosphoglycerate (2,3-BPG) allosteric effector: "It is present in low concentrations in all cells but approx. eqimolar eith Hb in RBCsIt is over-prodiced in response to high-altitude, airway obstruction or congestive heart failureBinds more tightly to deoxy-Hb (T-state) than oxy-Hb (R-state)Without 2,3-BPG Hb wouldn't release O2, thus we would suffocateBPG stabilises the T-state and so increases the P50 BPG + oxy-Hb ↔ deoxy-Hb-BPG + 4O2 " Descfribe how 2,3-BPG explains how O2 is tranferred to foetal blood: "BPG has a lower affinity for HbF than Hb so HbF has a smaller P50 for O2 than Hb HbF binds O2 more tightly than Hb" What is the bohr effect? "When protons (H+) bind preferentially to deoxy-Hbdeoxy-Hb-H+ + 4O2 ↔ Oxy-Hb + nH+ n is a number between 1.7 - 2.7" Describe the Bohr effect: "deoxy-Hb-H+ + 4O2 ↔ Oxy-Hb + nH+Le Chantelier's Law: if the [H+] increases, the eq. shifts to the left. As the pH drops, O2 is releasedHb acts as a buffer" Describe how in the Bohr effect the H+ binds: "Protons (H+) bind preferentially to doexy-HbOnly in the T-state (deoxy-Hb) are the 3 residues oriented to enable the formation of 2 salt bridges that help to stabilize the T-state" Describe the CO2 binding in the Bohr effect:What does this mean for the alloestic effect? "CO2 can bind directly to the N-terminal groups of Hb by forming carbamateThe carbamate partipates in salt bridges that help to further stabilise the T-stateCO2 reduces the affinity for O2 beyond that caused by a drop in pH aloneThis illustrates a general rule: allosteric affects are cumulative" Describe the mutations that can occur in Hb (include at least 1 example): "In general, mutations that occur on the surface of Hb have no phenotype (they are ""silent"" mutations).Mutations in the interior are rarely silent and often result in hemolytic anaemia>1000 variants are knownMost are single residue substitutionsExamples:E6V mutationHammersmith beta F42S (increased P50)Bristol beta V67D (increased P50)Boston alpha H58Y (increased methemoglobin formation)Kansas beta N102T (disrupts H-bond that stabilises R-state)" Describe the E6V mutation in Hb: "The E6V mutation in Hb produces HbSThe most famous mutation in Hb is glutamic acid ---> Valine at position 6 in the beta chainThe E6V mutation a hydrophobic patch on the surface of Hb.This can interact with an existing hydrophobic patch on the surface of T-state Hb and results in sickle cell anaemia HbS forms fibres Sickle cell reduces the 1/2 life of RBCs These cells block  capillaries, causing inflammationa dn painPeople who are homozygous for the mutaitonn are often seriously debiliated and don't survive into adulthood" 20 amino acids: Glycine3 letter code1 letter codeStructurePropertiesInteresting facts (2)Mnemonics "GlyG- Very small (smallest)- Hydrophobic- Not liked by alpha helix1) In collagen triple helix2) Makes backbone flexibleGlyyyycine is tiiiiiny" 20 amino acids: Alanine3 letter code1 letter codeStructurePropertiesInteresting facts (1)Mnemonics "AlaA- Small- Hydrophobic - Nicely fits into alpha helixOften sought in site directed mutagenesisSimilar to serine but doesn't have OH" 20 amino acids: Valine3 letter code1 letter codeStructurePropertiesMnemonics "ValV- A bit bulky due to the 2 methyls- Very hydrophobic - binding pocket for elastaseThe 2 methyls of side chain look like ""V"" for Valine" 20 amino acids: Proline3 letter code1 letter codeStructurePropertiesInteresting facts (2)Mnemonics "ProPThe strangest amino acid- Makes a loop- Binds to backbone- hydrophobic1) Can form cis peptide bonds (cis-prolyl isomerase)2) Not liked by alpha helixProline is PRO because only she/he can do cis bonds " 20 amino acids: Leucine3 letter code1 letter codeStructurePropertiesInteresting facts (1)Mnemonics "LeuL- Very hydrophobic- Very bulky (but not aromatic!)Isomer of isoleucineLeucine is Lucy with long hair = long side chains" 20 amino acids: Isoleucine3 letter code1 letter codeStructurePropertiesInteresting facts (1)Mnemonics "IleI- Very hydrophobic- Very bulky (but not aromatic!)Isomer of LeucineStructure is only slightly different from leucine" 20 amino acids: Methionine3 letter code1 letter codeStructurePropertiesInteresting facts (1) "MetM- Very hydrophobic Has sulphur but can't do disulphide bonds...(Sulphur locked)" 20 amino acids: Phenylalanine3 letter code1 letter codeStructurePropertiesInteresting facts (1)Mnemonics "PheF- Hydrophobic and aromatic- BulkyForms π-π stacking interactionsPhenylalanine for phenyl ring + Phe = F" 20 amino acids: Tyrosine3 letter code1 letter codeStructurePropertiesInteresting facts (1)Mnemonics "TyrY- Hydrophobic with a bit of polarity on OH- Aromatic1) Receptor Tyrosine KinasesTyrosine = Y, similar to phenylalanine" 20 amino acids: Tryptophan3 letter code1 letter codeStructurePropertiesMnemonics "TrpW- Hyrdophobic with a bit of polarity on NH- AromaticNOT charged" 20 amino acids: Serine3 letter code1 letter codeStructurePropertiesInteresting facts (1)Mnemonics "SerS- Polar- OH group- A bit smallCatalytic triat in serine proteasesSimilar to alanine but with OH" 20 amino acids: Cysteine3 letter code1 letter codeStructurePropertiesInteresting facts (3)Mnemonics "CysC- Polar- Sulphur- A  bit small1) forms disulphide bonds2) Important for 3º/4º structure3) Catalytic residue in proteasesCySSS for Ssssulfur" 20 amino acids: Threonine3 letter code1 letter codeStructurePropertiesMnemonics "ThrT- Polar- OH group- A bit biggerA bit similar to Valine" 20 amino acids: Asparagine3 letter code1 letter codeStructurePropertiesInteresting facts (1) "AsnN- -CONH2 - LongerHas an amide in its sidechain" 20 amino acids: Glutamine3 letter code1 letter codeStructurePropertiesInteresting facts (1) "GlnQ- -CONH2 - LongerHas amide in its sidechain" 20 amino acids: Lysine3 letter code1 letter codeStructurePropertiesInteresting facts (1)Mnemonics "LysK- +ive- LooongIMPORTANT - lots of ionic interactionsStrange: Lys = K" 20 amino acids: Arginine3 letter code1 letter codeStructurePropertiesInteresting facts (1)Mnemonics "ArgR- +ive- Long- ""like aromatic""IMPORTANT - think ""ion channels""R for Arginine" 20 amino acids: Histidine3 letter code1 letter codeStructurePropertiesInteresting facts (1) "HisH- +ive or neutral - depending on local environmentTHE MOST IMPORTANT - in catalytic triad of proteases" 20 amino acids: Aspartic acid3 letter code1 letter codeStructurePropertiesInteresting facts (1)Mnemonics "AspD- Always -ively charged (be careful)IMPORTANT - catalytic triad""Like"" asparagine" 20 amino acids: Glutamic acid3 letter code1 letter codeStructurePropertiesInteresting facts (1)Mnemonics "GluE- Always -ively chargedIMPORTANT""Like"" glutamine" List all the hydrophobic amino acids in order of increasing bulkness:Name3 letter code1 letter code Glycine, Gly, GAlanine, Ala, AValine, Val, VProline, Pro, PLeucine, Leu, LIsoleucine, Ile, IMethionine, Met, MPhenylalanine, Phe, FTyrosine, Tyr, YTryptophan, Trp, W List all the hydrophilic amino acids in order of increasing chai length:Name3 letter code1 letter code Serine, Ser, SCysteine, Cys, CThreonine, Thr, TAsparagine, Asn, NGlutamine, Gln, Q List all the +ively charged amino acids:Name3 letter code1 letter code Lysine, Lys, KArginine, Arg, RHistidine, His, H List all the -ively charged amino acids:Name3 letter code1 letter code Aspartic acid, Asp, DGlutamic acid, Glu, E Why do proteins need to be purified? To remove anything thst interferes with your experiments       - Interfering compounds can include inhibitors, activators, aggregated or denatured proteins       - Often we need to remove proteins, such as isoforms, with related activitiesImpure proteins are very resistant to forming the crystals that are required for X-ray crystallography Describe the sources of proteins used for purification: Before molecular biology was possible, proteins were purified from tissues or cells that naturally contained a high proportion of protien of interestMolecular biology has increased the potential sources of proteins:ProkaryotesUnicellular eukaryotesMammalian cells Explain the ADVANTAGES of using prokaryotes in protein production:Example: Escherichia coli (E.coli)ADVANTAGES:V easy to genetically manipulateV east to grow in large quantities in liquid culturepotentially V high yields of protein Explain the DISADVANTAGES of using prokaryotes in protein production:Example: Escherichia coli (E.coli)DISADVANTAGES post-translational modificatiols will probably be different to those used in eukaryotic cells            - eg. mammalian-like protein glycosylation not possiblePoor at folding proteins           - e.g. multi-subunit proteins or ones with a high-disulfide bond content Explain the ADVANTAGES of using unicellular eukaryotes in protein production:Example: YeastADVANTAGES:Easy to genetically manipulateEasy to grow in large quantitiesPotentially high yieldsPost-translational modifications more similar to mammalian ones - including glycosylation Explain the DISADVANTAGES of using unicellular eukaryotes in protein production:Example: YeastDISADVANTAGES:Only moderate ability to fold complicated proteins Explain the ADVANTAGES of using mammalian cells in protein production:Example: HEK cells (Human Embryonic Kidney)ORHeLa cells (Henrietta Lacks)ADVANTAGES:Full range of mammalian post-translational modificationsAbility to form and assemble complicated proteins Explain the DISADVANTAGES of using mammalian cells in protein production:Example: HEK cells (Human Embryonic Kidney)ORHeLa cells (Henrietta Lacks)DISADVANTAGES:Less easy to genetically manipulateHard to grow in large quantitiesPoor yields of proteinV expensive growth media How do we know if the protein you want is present in a sample? Test for activity by doing an assayAn assay is a test for the presence of something What is the most common technique for purifying large quantities of protein? Describe: "Column chromatographyThe solution containing the protein mixture, the ""liquid phase"", is added to a porous solid matrix, the ""stationary phase""The matrix - based on polymers such as agarose or cellulose - interacts with proteins according to their physiochemical properties" What are the 4 types of column chromatography? Gel filtration chromatography - separates on the basis of protein size Ion exchange chromatography - separates on the basis of protein charge  Hydrophobic interaction chromatography - separates on the basis of protein hydrophobicityAffinity chromatography - separates on the basis of specific protein interactions, such as enzyme binding to a substrate, or an antibody to an antigen Describe gel filtration chromatography: "Also known as Size Exclusion or Molecular Exclusion ChromatographySeparates proteins according to size       - Big proteins come out first and small proteins last - as small molecules enter to aqueos spaces within beads wheareas large molecules can't" Describe ion exchange chromatography: "Separates proteins according to their charge" Explain what titration curves describe (according to proteins): "They describe how a protein's charge varies with -pH Proteins with many acidic residues will be more -ively chargedProteins with many basic residues will be more +ively charged" Describe the shape of a titration curve of a protein (overall charge vs pH): "" Define the isoelectronic point (pI): "The pH at which a protein has no overall charge (zwitterionic)When pH>pI, the protein is -ively chargedWhen pH<pI, the protein is +ively chargedThe pI of manny proteins is ~5 or ~9" How are titration curves useful to us when we are identifying proteins and purifying proteins in column chromatography? "By knowing the isoelectronic point we know the pH at which the protein is zwitterionic.Therefore by altering the pH of the buffer, we can alter which proteins bind to the column" Describe are the 2 types of Ion Exchange Chromatography (IEX) and media: "We cna choose IEX media with a permanent +ive or -ive chargeBy changing the pH of the buffer proteins can be made to bind to an IEX column - or not bind2 common IEX media:        1) DEAE cellulose (+ive)        2) CM cellulose (-ive)" What are the 2 way to elute (unbind) a protein from an IEX column? "Change the pH of the buffer flowing through the column to change the charge on the protein (seldom used, it's more difficult to control)Increase the buffer's [salt] to elute the protein via ""charge shielding""        - Elution can be a step-wise e.g. sequentially apply buggers containing 0.1, 0.25, 0.5, 1.0M NaCl        - Or can be done via a gradient of steadily increasing salt e.g. 0 ---> 1.0M NaClGradient elution has a higher resultion than step-elution." Describe the A280 method of protein detection: "MOST  IMPORTANT!An A280 signal - or any other non-specific method of detecting proteins - tells you how much protein is presentBut it CAN'T tell you how many types of protein are present" How do you tell if a chromatography fraction contains only 1 protein? "Use sodium dodecyl sulfate polyacrylamide gel electrophoresis ""SDS-PAGE""SDS-PAGE separates proteins on the basis of their subunit size  It's the standard check of purity for chromatography fractions" What is SDS-PAGE? Sodium Dodecyl Sulfate Polyacrylamide Gel ElectrophoresisIt separates proteins on the basis of their subunit size  It's the standard check of purity for chromatography fractions Describe how to carry out SDS-PAGE electrophoresis: "An acrylamide gel has pores about the same size as most proteins and acts as a molecular sieve Mixtures of SDS- and heat-treated proteins are applied to the top of the gelSDS- gives proteins a -ive charge, heat makes them the same shapeA voltage is applied to the gel and the proteins migrate towards the +ive electrode (anode) accroding to their sizeProteins run from the top ---> bottomAfter electrophooresis the gel is stained (often with coomassie blue ) to visualise the proteins" When is SDS-PAGE most effective? Why? Most effective when combined with activity assaysAs SDS-PAGE only tells you the proteins that have the same molecular weightThats why SDS-PAGE combined with an activity assay is a good way to assess purity What is the problem with total protein detection methods (e.g. A280)? They will only tell you the concentrstion of proteins, not how pure they are What are enzymes? Biological catalysts, usually proteins, that speed up chemical reactions in living organisms without being consumed in the reactionThet are typically proteins ranging in sizeThey often contain bound co-factors (sometimes called coenzymes) which contribute to the catalytic mechanisms (e.g. biotin, metal ions, thiamine pyrophosphate, NAD+/NADH, niacin, haem,etc.) Describe the similarities and differences between chemical catalysts and enzymes as bioogical catalysts: "LIKE catalysts used in chemical reactions, enzymes:catalyse (by more than 106-fold) the rate at which chemical reactions attain equilibrium, but without affecting the equilibrium position of the reaction  are unchanged in the overall reaction that they catalyseUNLIKE chemical catalysts, enzymes have evolved to have:high substrate specificityVery high catalytic powerCatalytic power under ""gentle"" reaction conditions (e.g. pH6-8, 37ºC) " Define kcat: it is equivalent to the n.o substrate molecules in a single molecule of enzyme can bind and convert to product every second (when substrate is unlimited) What are the 3 things we can measure to determine enzyme activity? the rate of production of productthe rate of loss of the substratethe rate of production or loss of a co-factor involved (i.e. NAD+/NADH)All of this can be done in an enzyme assay What is the formula for the enzyme activity? "" What results do we usually expect as well as expectations for enzyme activity during an enzyme assay? reaction rate to be proportional to the enzyme concentration               - double enzyme concentration = rate doublesat higher enzyme concentrations the substrate may be used up during the measurement (the absorbance tails off).                - It is important to measure the initial rate by drawing a tangent to the curve at a time shortly after adding the enzyme. the key experiment for characterizing the kinetic parameters of an enzyme, its interaction with substrate, and the effects of inhibitors, is the measurement of reaction rate as a function of substrate concentration (keeping the enzyme concentration constant) Define an enzyme activity unit: the amount of enzyme which transforms 1 µmol of substrate per minute at 25ºC Define the specific activity of an enzyme: the n.o enzyme units per milligram (mg) of proteina measure of purity What are the 2 things we want to maximise in each enzyme purification? the recovery of the enzymethe purity of the enzyme        - the specific activity should increase! Formula for % recovery: "" Formula for degree of purification: (specific activity purification) / (specific activity crude) What are the 3 ways that substrances can inhibit enzymes/uses of enzyme inhibition? Biologically - one of themechanisms for controlling enzyme-catalysed reactions in vivo Therapeutically - many drugs (and toxins) are enzyme inhibitorsMechanistically - our understanding of enzyme mechanisms often involves studying the effect of inhibitors What are the 2 types of inhibitors? Irreversible inhibitors - the enzyme is irreversibly inactivated by covalent or tight binding of inhibitor, usually to enzyme active siteReversible inhibitors - competitive or non-competitive  Describe the differences between competitive and non-competitive inhibitors: "COMPETITIVEcompetes directly with the substrate for binding to the active siteit is usually a substrate analoguethe inhibitor's effect can be overcoe at high enough [S]e.g. HIV protease and crixivan (anti-A.I.D.S. drug)e.g. Prostaglafin synthase with IBUPROFEN blocking active site accessNON-COMPETITIVE :doesn't compete directly with the substrate for binding to active site (substrate and inhibitor can both be bound at once)." Describe competitive inhibition: "competes directly with the substrate for binding to the active siteit is usually a substrate analoguethe inhibitor's effect can be overcoe at high enough [S]" Describe non-competitive inhibition: "doesn't compete directly with the substrate for binding to active site (substrate and inhibitor can both be bound at once).Km is therefore little affected" How can competitive and non-competitive inhibitors be distinguished kinetically? "Compare double reciprocal (Lineweaver-Burk) plot for th enzyme reaction in the presence of an inhibitor, and its absence, respectively" How do you determine Km and Vmax using a Lineweaver-Burk Plot: "" Describe the  difference between irreversible and reversible enzyme inhibitors (using examples): "IRREVERSIBLE:the enzyme is irreversibly inactivated by covalent or tight binding of inhibitor, usually to enzyme active sitee.g. ACh with DIPF - toxin/poisone.g. prostagladin synthase (cyclooxygenase; COX) with aspirin - therapeutice.g. trypsin with pancreatic trypsin inhibitor - regulation (of protein digestion in the gut)REVERSIBLE:competitive or non-competitivecompetitive - competes directly with the substrate for binding to the active site        - e.g. HIV protease with Crixivan (anti-A.I.DS. drug)            - e.g. prostagladin synthase with IBUPROFEN blcoking active site accessnon-competitive - doesn't compete directly with the substrat for binding to active site (substrate and inhibitor can both be bound at once). Km is therefore little affected." Describe the effect of competitive inhibitors on the values of Km and Vmax: "" Describe the effect of non-competitive inhibitors on the values of Km and Vmax: "" How do we measure the effects of competitive and non-competitive inhibitors on the values of Km adn Vmax using enzyme assays? "Can be distinguished kinetically!We compare double reciprocal (Lineweaver-Burk) plot for the enzyme reaction in the presence of an inhibitor, and its acsence, respectively" Why can't you use A280 chromatogram to identify types of enzymes? As ALL PROTEINS  absorb light at 280nm 4fe61be1bbce43f1af57063485254bd0-ao-1 Lable the bacteria cell: "" "" "" "" 4fe61be1bbce43f1af57063485254bd0-ao-2 Lable the bacteria cell: "" "" "" "" 4fe61be1bbce43f1af57063485254bd0-ao-3 Lable the bacteria cell: "" "" "" "" 4fe61be1bbce43f1af57063485254bd0-ao-4 Lable the bacteria cell: "" "" "" "" 4fe61be1bbce43f1af57063485254bd0-ao-5 Lable the bacteria cell: "" "" "" "" 4fe61be1bbce43f1af57063485254bd0-ao-6 Lable the bacteria cell: "" "" "" "" e562fa74418f4874a76515738ce7498b-ao-1 Lable this eukaryotic cell: "" "" "" "" e562fa74418f4874a76515738ce7498b-ao-2 Lable this eukaryotic cell: "" "" "" "" e562fa74418f4874a76515738ce7498b-ao-3 Lable this eukaryotic cell: "" "" "" "" e562fa74418f4874a76515738ce7498b-ao-4 Lable this eukaryotic cell: "" "" "" "" e562fa74418f4874a76515738ce7498b-ao-5 Lable this eukaryotic cell: "" "" "" "" e562fa74418f4874a76515738ce7498b-ao-6 Lable this eukaryotic cell: "" "" "" "" e562fa74418f4874a76515738ce7498b-ao-7 Lable this eukaryotic cell: "" "" "" "" e562fa74418f4874a76515738ce7498b-ao-8 Lable this eukaryotic cell: "" "" "" "" e562fa74418f4874a76515738ce7498b-ao-9 Lable this eukaryotic cell: "" "" "" "" e562fa74418f4874a76515738ce7498b-ao-10 Lable this eukaryotic cell: "" "" "" "" e562fa74418f4874a76515738ce7498b-ao-11 Lable this eukaryotic cell: "" "" "" "" e562fa74418f4874a76515738ce7498b-ao-12 Lable this eukaryotic cell: "" "" "" "" e562fa74418f4874a76515738ce7498b-ao-13 Lable this eukaryotic cell: "" "" "" "" e562fa74418f4874a76515738ce7498b-ao-14 Lable this eukaryotic cell: "" "" "" "" e562fa74418f4874a76515738ce7498b-ao-15 Lable this eukaryotic cell: "" "" "" "" e562fa74418f4874a76515738ce7498b-ao-16 Lable this eukaryotic cell: "" "" "" "" e562fa74418f4874a76515738ce7498b-ao-17 Lable this eukaryotic cell: "" "" "" "" e562fa74418f4874a76515738ce7498b-ao-18 Lable this eukaryotic cell: "" "" "" "" In higher organisms which cell organelles have lipid membranes that define them? NucleusMitochondriaERGolgi apparatusLysosomesPeroxisomesChloroplasts What are peroxisomes? A small organelle present in the cytoplasm of many cells, which contains the reducing enzyme catalase and usually some oxidasesThey carry out oxidative reactions ca0f09d2826c44a2bdddd81b78f2e9f7-ao-1 Lable this phospholipid bilyaer proposed by the fluid mosaic model: "" "" "" "" ca0f09d2826c44a2bdddd81b78f2e9f7-ao-2 Lable this phospholipid bilyaer proposed by the fluid mosaic model: "" "" "" "" "What is suggested by this fluid mosaic model?" "Protein floating in a sea of lipidproteins are at low concentrationproteins fit perfectly into bilayerbilayer has constant thicknessbilayer is symmetricalLipids are all the sameMembrane is the same everywhere you look" UPDATE THE FLUID MOSAIC MODELDescribe the reality properties of surface membranes (lipid bilayer): (6) Many proteins form complexes      - long-lived or short-lived      - protein-protein      - protein-lipidMembranes are 'patchy'      - different types of protein      - different types of lipidThe bilayer deformsMany membranes are densely packedCellular membranes can be very different from one anotherMany membranes are asymetric UPDATE THE FLUID-MOSAIC MODEL: (6) "1) Many membrane proteins form complexesThese complexes are often important for function2) Membrane proteins and lipids form complexesThese complexes are often important for funciton3) Simple lipid mixtures spontaneously form domainsa natural feature of lipids that form membranesNO ENERY INPUT required for these to form4) Biological membranes have visible 'patches'proteins cluster together (proteins with lipids)Membrane thickness is then variableMembrane distorts to  match protein5) Many biological membranes are densely packed with protein6) Cellular membranes can be different from each other7) Many membranes are assymetric" c76c1726ba5144bbb8ff1fc205c9128f-oa-1 Lable this more realistic fluid-mosaic model: "" "" "" "" c76c1726ba5144bbb8ff1fc205c9128f-oa-2 Lable this more realistic fluid-mosaic model: "" "" "" "" c76c1726ba5144bbb8ff1fc205c9128f-oa-3 Lable this more realistic fluid-mosaic model: "" "" "" "" c76c1726ba5144bbb8ff1fc205c9128f-oa-4 Lable this more realistic fluid-mosaic model: "" "" "" "" What is the overall conclusion/evaluation of th fluid-mosaic model? It is useful but too simple Describe the properties & features of lipids in the bilayer (phospholipids): "Lipids are amphipathic - hydrophilic and hydrophobic parts in the same structureLipids spontaneously self-associate because of hydrophobic effect and vdw interactions between the fatty acid tailsLipids are chemically diverse Different biological membranes use different lipidsLipids are dynamic therefore membrane is in dynamic motion" Meaning of amphipathic: (in lipids) hydrophilic and hydrophobic parts in the same structure Describe/explain why cholesterol is impoortant in eukaryotic membranes: "Polar -OH group gives it a weak amphipathic character Important in controlling membrane fluidity and lipid packing" Describe membrane symmetry: "Many membranes are asymmetric The 2 monolayers (inner and outer) have different lipid compositionsThis is functionally very importantMany cytosolic proteins bind to specific lipid headgroups" Describe lipid dynamics in membranes: "Lipids rarely 'flip-flop' from 1 side of the membrane to the other (prevented by enzymes called flipases/flopases)However, lipids diffuse laterally at 10-8cm2s-1 The hydrocarbon chains are flexible and dynamic Lipids rotate freelly around their long axis - on their headsLipids are constantly in motion" Describe membrane fluidity as a property of a lipid bilayer: "Biological membranes are fluid; alllows proteins to move and interactBelow a certain temperature ilayers chanfe from 'fluid' → 'rigid gel' phase↓Lateral diffusion of lipids and proteins in gel phase" What happens to bilayer when above transition temperature? "It is in the 'fluid' phaseallows proteins to move and interactallows the lateral diffusion of lipids and proteins" What happens to bilayer when below transition temperature? "In the 'rigid gel' phaselateral diffusion of lipids and proteins is greatly reduced in the gel phase" Describe the relationship between transition temperature and chain length: "The transition temperature is lower if chains are shorter or have double bonds" Describe/Explain the evidence of lateral diffusion in the membrane - Sedai virus: "Sedai virus used to fure mouse cells with human cellsCell-surface proteins labelled with green (mouse) and red (human) fluorescent markersInitially, fluorescent labels stayed separatedAfter 40 mins, fluorescent labels we inter-mingledCONCLUSION: free diffusion of the cell-surface proteins within the hybrid membrane" Describe/Explain the evidence of lateral diffusion in the membrane - FRAP: "Evidence from Fluorescence Recovery After Photobleaching (FRAP)Label lipids or proteins with fluorescent markersBleach small area with laserMeasure rate of recovery = rate of lateral diffusion of protein or lipidCONCLUSION: free diffusion of lipids and proteins within the membrane" What are the 2 types of proteins in membranes? IntegralPeripheral/extrinsic Describe the properties of integral membrane proteins: "Embedded in the bilayerTightly bound to the membrane by hydrophobic forcesCan only be separated from membranes using agents that disrupt membranes        - therefore hard to study their biochemistry as it's difficult to separate them from the membrane  Pass through the membrane once (""single-pass"") or several times (""multi-pass"")Generally insoluble in aqueous buffers" Describe the properties of peripheral/extrinsic membrane proteins: "Bind to membrane surface only, via binding to integral membrane protein and/or directly with the lipid headgroup regionsInteract via H-bonds or salt bridgesCan be easily dissociated from the membrane under mild conditionsIncludes lipid anchored proteinsOften soluble in aqueous buffers" Describe the structure of integral membrane proteins: "α-helical or β-barrel, with structured or unstructured loops outside the membraneα-helical proteins the most commonβ-barrel ONLY found in bacterial outer membranes, mitochondria and chloroplastsStructures are rarely (if ever) mixed within the bilayer region" Describe the physical chemistry of integral membrane proteins: "integral membrane proteins contain lots of hydrophobic amino acidsPresent a hydrophobic surface to the lipid acyl chains" Explain how the hydrophobicity of integral membrane proteins affect the ability to study them: The surface hydrophobicity of integral membrane proteins makes them difficult to study They are often very unstable outside of membrane environment Describe the transmembrane 2º structure α-helix structure in membrane: "Most common formTypically 20-25 amino acids long Thermodynamically stable 2º structure       - All main-chain H-bonds are satisfied (C=O, N-H)       - Unsatisfied H-bond groups are unfavourable in membrane       - Hydrophobic residues face the lipid acyl chain Helices can be predicted from amino acid sequence " How can helices be predicted? "From amino acid sequencesCan predict TM α-helices 'by eye' or with bioinformatics programmesScan for segments >18 amino acids dominated by hydrophobic residues " Describe the transmembrane 2º structure β-sheet in the membrane: "Antiparallel β strandsAlternating polar and hydrophobic amino acids       - harder to predict from sequenceHydrophobic residue acids face the bilayer core, polar residues face the protein interior Intra-chain H-bonds between strandsFold into β-barrels" Compare a transporter to a channel: " TransporterChannel Transport down OR against concentration gradientONLY allow diffusion down concentration gradient" Describe the shape of the graph for active and parssive transpport of a raate vs concentration graph: "" What are the 3 type sof ways to transport small solutes? "UniportSymportAntiport" Describe the function of solute transporters: "Move small molecules across lipid membranes        - Sugars, amino acids, peptidesOften require co-transport of ions (or other small molecules)Uniport, symport, antiport" Describe/explain how cells maintain ion/electrochemical gradients across the plasma membrane of eukaryotes: "Na+/K+ ATPase found in plasma membrane of ALL animal cellsIt is an active transporterHydrolysis of 1 ATP drives export of 3Na+ and import of 2K+          - 'P-type' transporter - ATP hydrolysisCells expend >30% of all ATP on driving this pumpThis generates a chemical and electrical difference at the membraneTherefore an inward-directed electrochemical Na+ gradient" How is the sodium membrane potential at membranes harnessed? "The sodium gradient is electrochemical  The movement of Na+ down this gradient is thermodynamically favourable with a -ive ∆G This free energy can be harnessed to drive other transporters" What do membranes use to maintain an inward-directed electrochemical sodium gradient in eukaryotic cells? Na+/K+ ATPase Give an example of sodium transporters in health and disease: "Glucose uptake depends upon Na+:Glucose symporters: SGLTs They promote glucose import into cell from bloodstreamThis prevents the excretion of glucoseLvels of intestinal SGLTs are regulated by dietary carbsInhibitors of SGLTs can now be used to treat diabetes:↑ glucose excretion in urine↓ blood glucoseAnd can also...↓ BPSupport weight loss" How do prokaryotes generate an electrochemical gradient? "In the bacterial inner membrane, e- transport generates an electrochemical proton gradient The protonmotive force (PMF)Drives ATP synthase and many other bacterial transportersMain cellular source of ATP" Describe an example of a proton-coupled transporter in prokaryotes: "Lactose permease, LacYSugar transporter from E.coli Cotransports H+/sugarWell-studied model system" Describe the central dogma:Does all RNA get used in translation only? "No, it could be used for:Translation (protein synthesis)Structural roles, catalysisGene control" State the main difference between prokaryotes and eukaryotes that contributes to protein synthesis: " ProkaryoteEukaryote no membrane enclosed nucleuscontains membrane encolded nucleus" Meaning of genome: all the genetic information of an organism What can the genome be divided into? chromosomes What is a gene? the basic unit of inheritancea section of DNA that codes for protien What does a nucleotide consist of? "A nitrogen-containing baseA 5C sugar≥1 phosphate group" What are nucleotides? the subunits of the nucleic acids What does a nucleoside consist of? "A nitrgoen-containing baseA 5C sugar" What is used to determine the 5' and the 3' end of the DNA structure? "The 5' C and the 3' C on the pentose sugar ring" What is the difference in the pentose ring structure between DNA and RNA? " DNARNA β-D-2-deoxyriboseβ-D-ribose" Describe the difference in bases between RNA and DNA: " DNARNA A, T, C, GA, U, C, G" What i the structure of a purine? "Nitrogen rich heterocyclic double ring structure" What is the structure of a pyrimidine? "Nitrogen rich heterocyclic single ring structure" Which bases are purines?What are the differences between those bases? "Adenine and GuanineDifference sidechains" Which bases are pyrimidines?What are the differences between those bases? "Cytosine, Uracil, ThymineDifferent side chainsBetween uracil and thymine is the methyl group" Describe the structure of Adenine: "" Describe the structure of Guanine: "" Describe the structure of Cytosine: "" Describe the structure of Uracil: "" Describe the structure of Thymine: "" Describe 1 way that DNA bases can be modified:Why is it important? "Methylation - it switches off geneimportant as an epigenetic markerDNA bases are also a site of damageChanges how the genetic material is used by the cell but DOESN'T change the genetic material itself" How are nucleotides joined together? "By phosphodiester linakge between the 5' and 3' carbon atoms" Is RNA stable? Why? No it is relatiely unstablethe Carbon 2' hydroxyl acts as a nucleophole to break the phosphodiester linkage between the nucleotide How are the 2 DNA strands paired/connected? "Via base pairing - H-bonds between basesPurine with pyrimidineA and TG and C" What is Chargaff's rules for base pairing? "The ratio of pyrimidine bases to purine bases is 1:1%G = %C%A = %T" Describe the structure of DNA rules: "2 DNA strands pair via H-bonds between basesChargaff's rule: ""The ratio of pyrimidine bases to purine bases is 1:1""      - %G=%C and %A=%TStrands have polarity and run antiparallel Bases stack due to hydrophobic interactions" What is the denaturation of DNA? The conversion of duble helical DNA into single strands What causes the denaturation of DNA? High THigh pHChemicals (e.g. Urea) What is the annealing of DNA? The renaturation of DNAThe conversion of single strands into double helical DNADirected by AT and GC H-bonding Describe quantitavely the main featrues of the DNA helix: "" Describe what are the DNA's major and minor grooves: "The glycosidic bonds are no precisely opposite one another therefore slightly assymetric which creates longer distance on 1 sideMajor groove: Wide and shallow      - lots of protein-DNA interactions happen within the major groove as its easier to access the genetic infoMinor groove: Narrow and Deeppink, purple and white = backboneorange = bases" Why is long DNA stiff or flexible? Why? "FlexibleAs a polymer, DNA has elastic propertiesPersistence length - the lenght of DNA along which a thermally excited bend of 1 radian (~57º) typically occurs; a basic mechanical property quantifying the stiffness of a polymer" What is the persistence length? The length of DNA along which a thermally excited bend of 1 radian (~57º) typically occursa basic mechanical property quantifying the stiffness of a polymer Is short DNA stiff or flexible? Explain why (2 reasons): "StiffElectrostatic repulsion of phosphates      - bending brings those phosphates closer together and the electrostatic repulsion will push them apart againCompressive base stacking      - bending is hard becase it is energetically favourable htat bases are stacked up nicely" Describe the 3 main protein-DNA interactions: Use examples: Proteins can:Recognice and bind to specific DNA sequences       - e.g. TATA-binding protein (TBP)Recognise structural features, such as DNA damage      - e.g. DNA glycosylase bound to 3-methyladenineBind DNA non-specifically       - e.g. histone protiens in a nucleosome What does it mean when DNA replication is semi-conservative? "1 strand is old and 2 strand is new" What is DNA synthesised from? From dNTP building blocks using a DNA template What are the 5 different E.coli polymerases? DNA polymerase I - DNA replication and repairDNA polymerase II - DNA repairDNA polymerase III - Main DNA replicating enzyme DNA polymerase IV & V - DNA repair Describe how DNA polymerases core enzyme fold (e.g. in hands): "DNA polymerase iteractions with:ssDNA on the side of the fingersdsDNA in the palm of the hand" Describe the features and properties of DNA polymerase III: DNA polymerase III is the main replicating enzyme in E.coli 9 protein subunitsDNA synthesis activity:     - Independent of sequence     - 250-1000 nucleotides (nt) per second     - Capable of synthesising 1000s of nt before dissociating3'-5' exonuclease activity     - proofreading to minimise copying mistakes What are the 4 DNA polymerase requirements? DNA polymerase uses all 4 dNTPs (i.e. dATP, dGCP, dCTP, dTTP) as the immediate precursors of DNA synthesisDNA polymerase synthesises a complementary copy of hte DNA template DNA polymerase can only add a dNTP to the 3' end of a pre-existing DNA or RNA molecule, so it needs a primer with a free 3'-hydroxyl group DNA polymerase requires Mg2+ to function Describe how transcription begins:Use an example: "It starts at the origin (oriC)Section of DNA easily melted due to lots of A&T therefore less H-bondingThen DNA undergoes conformatiional changes and a transcription bubble is formedDNA helicase unwinding action requires energy as it is a lot more energetically favourable for the 2 DNA strands to remain together" Describe the function of DNA helicase in transcription: "DNA helicase unwinds dsDNA (double-stranded) to produce ssDNA (single-stranded) for replicationthis is done in 2 opposite directions at the same timethis requires energy as it is more energetically favourable for the 2 DNA strands to stay together" What do single stranded binding (SSB) proteins do? "They bind ssDNA non-specifically and cooperatively, removing 2º strucureprevents annealingprevents hairpin structures from happening" What does primase do in transcription?Use an example: "Primase synthsizes  RNA primers de novo E.g. DnaG E.coli Primase is a specialised RNA polymerase Makes short RNA primers (5-10 nt)No proofreadingNo specific sequences for initiationFrequency of priming different on leading/lagging strands" Describe the funciton of primase DnaG in E.coli: "Primase is a specialised RNA polymerase Makes short RNA primers (5-10 nt)No proofreadingNo specific sequences for initiationFrequency of priming different on leading/lagging strands" What happens in a bi-directional replication fork (briefly): "Starts at origin (oriC)InitiationBidirectional replication established" Describe what happens during DNA polymerisation: "DNA polymerase adds deoxynucleotide triphosphates (dNTPs) to the 3' end of the growing chain.DNA polymerase makes DNA in the 5' to 3' directionEach addition creates a new 3' OH for the next addition" What is the role of Mg2+ in the catalytic site of DNA polymerase III? "Stabilising the phosphates Activating the OH to make it a better nucleophile" What are the 3 things that the catalytic site of DNA polymerase III requires? "Aspartates (acidic residues)Mg2+ Triphosphate (high energy bond)" What is a nuclease? An enzyme that cleaves phosphodiester bonds What is an Exonuclease? An enzyme that cleaves nucleotides one-at-a-time from ends of polynucleotides What is an Endonuclease? An enzyme that cleaves nucleotides within polynucleotides What is the difference between an exonuclease and an endonuclease? " ExonucleaseEndonuclease Cleaves nucleotides one-at-a-time from ends of polynucleotidesCleaves within polynucleotides" Describe the process of DNA proofreading: "3'-5' exonuclease (in DNA polymerase III) removes incorrect nucleotide" How does the structure of RNA differ from that of DNA? "RNA differs from DNA in several ways:Single-stranded vs. double-strandedContains ribose sugar (DNA has deoxyribose)Uses uracil (U) instead of thymine (T)Can form complex structures (e.g., hairpins) for various functions" A molecular biologist is studying the role of RNA in protein synthesis. During her experiments, she observes that certain RNA molecules can catalyze reactions similar to enzymes. She notes that these RNA structures often contain hairpins and bulges, which contribute to their stability and function. Additionally, she focuses on how base pairing between adenine and uracil plays a critical role in maintaining the integrity of these structures. In the context of RNA secondary structures, what is the significance of hairpins and bulges? "Hairpins and bulges create helical regions that stabilize the RNA molecule and influence its interactions during processes like translation." A laboratory technician analyzes the biochemical composition of E. coli cultures. The results show that 50% of the dry weight is protein, with significant amounts of RNA and smaller proportions of carbohydrates and DNA. The technician notes the roles of different RNA types in protein synthesis. What role does ribosomal RNA (rRNA) play in E. coli? Options:A. It acts as a template for DNA replication.B. It functions as a signal for regulating gene expression.C. It carries amino acids to the ribosome.D. It plays a role in protein synthesis within ribosomes. "It plays a role in protein synthesis within ribosomes." During a molecular biology experiment, researchers are studying the transcription cycle of a gene. They focus on how core RNA polymerase initiates transcription, elongates the RNA strand, and terminates the process. They also analyze promoter recognition and the mechanisms involved in Rho-dependent and Rho-independent termination. What is the primary difference between Rho-dependent and Rho-independent termination? Options:A. Rho-dependent termination involves ATP hydrolysisB. Rho-independent termination includes DNA unwindingC. Rho-independent termination is based only on RNA sequenceD. Rho-dependent termination needs a specific protein factor "Rho-dependent termination needs a specific protein factor" What structural feature allows RNA to form complex tertiary structures? Options:A. The presence of intronsB. Base pairing and interactionsC. The presence of a double helix structureD. The presence of ribose sugar "Base pairing and interactions" What are hairpins in RNA, and why are they important for transcription termination? "Hairpins in RNA are secondary structures formed by GC-rich sequences that create a loop. They are crucial for transcription termination as they signal RNA polymerase to pause and detach, effectively ending transcription. This process is part of Rho-independent termination." What are the different classes of RNA, and what roles do they play in the cell? "Different classes of RNA and their roles:mRNA (messenger RNA): carries genetic information from DNA to ribosomes for protein synthesistRNA (transfer RNA): brings amino acids to ribosomes during translationrRNA (ribosomal RNA): forms the core of ribosome structure and catalyzes protein synthesissnRNA (small nuclear RNA): involved in RNA splicing and processing" During a lab experiment, students are tasked with analyzing the transcription process in prokaryotic cells. They observe that RNA polymerase binds to a specific promoter region on the DNA strand and begins synthesizing RNA in the 5' to 3' direction. The students note the presence of various types of RNA being produced, including mRNA, tRNA, and rRNA. However, they also introduce a transcription inhibitor into the reaction mixture, which halts RNA synthesis midway through the process. How would the introduction of a transcription inhibitor affect the types of RNA synthesized during the experiment? "The transcription inhibitor would prevent RNA polymerase from proceeding with RNA synthesis, resulting in incomplete transcripts and potentially affecting the production of all RNA types (mRNA, tRNA, rRNA)." During a molecular biology lab, students are studying the transcription mechanisms of prokaryotic cells. They observe that core RNA polymerase binds to DNA at specific promoter regions, initiating the synthesis of RNA. The team also discusses how termination can occur through both Rho-dependent and Rho-independent mechanisms. They note the importance of understanding these processes for genetic engineering applications. What is the primary difference between Rho-dependent and Rho-independent termination during transcription? "Rho-dependent termination requires the Rho protein to bind to the RNA transcript and facilitate detachment from the DNARho-independent termination relies on the formation of a hairpin structure in the RNA transcript followed by a series of uracils, leading to dissociation." What are the four nucleoside triphosphates (NTPs) required for RNA synthesis? Options:A. AMP, GMP, CMP, UMPB. ATP, GTP, CTP, dTTPC. dATP, dGTP, dCTP, dTTPD. ATP, GTP, CTP, UTP "ATP, GTP, CTP, UTP" What mechanisms do cells use to regulate transcription in response to environmental changes? "Cells regulate transcription in response to environmental changes through: Promoter strength: Variations in -10 and -35 sequences affect RNA polymerase bindingSigma factors: Specific factors help RNA polymerase recognize different promoters Feedback mechanisms: Products of transcription can inhibit or enhance further transcription" What happens during the elongation phase of transcription? Options:A. RNA polymerase requires a primer to initiate synthesisB. RNA polymerase synthesizes DNA from RNA templatesC. RNA polymerase unwinds the DNA double helixD. RNA polymerase synthesizes RNA using the DNA template "RNA polymerase synthesizes RNA using the DNA template" What is the role of sigma factors in transcription initiation? Options:A. Sigma factors facilitate RNA polymerase binding to promotersB. Sigma factors help in the processing of mRNA after transcriptionC. Sigma factors are involved in the termination of transcriptionD. Sigma factors enhance the stability of RNA polymerase during elongation "Sigma factors facilitate RNA polymerase binding to promoters" What percentage of E. coli dry weight is made up of RNA? Options:A. 10%B. 30%C. 50%D. 20% "20%" How does the secondary structure of RNA contribute to its function as a ribozyme? "The secondary structure of RNA, including hairpins and bulges, allows it to fold into specific shapes. These shapes enable RNA to act as a ribozyme by providing the necessary active sites for catalysis, enhancing its structural and catalytic functions." What is the central dogma of molecular biology? Options:A. DNA → Protein → RNAB. DNA → RNA → ProteinC. RNA → DNA → ProteinD. DNA → RNA "DNA → RNA → Protein" Describe the role of mRNA in the process of translation. "mRNA (messenger RNA) carries genetic information from DNA to ribosomes, where translation occurs. It serves as a template for assembling amino acids into proteins, ensuring the correct sequence based on the genetic code. This process is essential for protein synthesis in cells." Where does transcription begin on the DNA template strand? Options:A. At the enhancer regionB. At the 3' end of the DNA strandC. At the promoter regionD. At the terminator sequence "At the promoter region" During a molecular biology experiment, researchers are studying the transcription cycle of a gene. They focus on how core RNA polymerase initiates transcription, elongates the RNA strand, and terminates the process. They also analyze promoter recognition and the mechanisms involved in Rho-dependent and Rho-independent termination. How does core RNA polymerase recognize the promoter region? Options:A. By binding to specific DNA sequencesB. By interacting with ribonucleoside triphosphatesC. Using a secondary protein for guidanceD. Through random association with DNA strands "By binding to specific DNA sequences" A bacterial cell is undergoing transcription to synthesize RNA. The RNA polymerase binds to the promoter region of the DNA, unwinding the double helix and synthesizing a complementary RNA strand in the 5' to 3' direction. Various inhibitors are present that could affect this process. What type of RNA is primarily synthesized during transcription? Options:A. Ribosomal RNA (rRNA)B. Messenger RNA (mRNA)C. Small nuclear RNA (snRNA)D. Transfer RNA (tRNA) "Messenger RNA (mRNA)" " What is the significance of the ""bubble"" formed during transcription? " "The ""bubble"" formed during transcription is significant because it: - Allows RNA polymerase to access the DNA template - Facilitates the synthesis of the RNA transcript - Separates the DNA strands temporarily for copying This process is essential for accurate gene expression." Explain the concept of promoter strength and its impact on transcription levels. "Promoter strength refers to how effectively a promoter initiates transcription. - **Strong promoters**: closely match consensus sequences, leading to high transcription levels. - **Weak promoters**: less similar, resulting in lower transcription levels. This affects gene expression and protein production in cells." Discuss the importance of Mg2+ in RNA polymerase activity. "Mg²⁺ is crucial for RNA polymerase activity as it stabilizes the negative charges on the phosphate groups of NTPs, facilitating the formation of phosphodiester bonds during RNA synthesis. It also helps in the proper positioning of the substrate for efficient transcription." What is the typical direction of RNA synthesis by RNA polymerase? Options:A. 3' to 3' directionB. 5' to 5' directionC. 3' to 5' directionD. 5' to 3' direction "5' to 3' direction" A researcher is studying the biochemical composition of E. coli for a project on prokaryotic cellular structures. They discover that in terms of dry weight, E. coli consists of approximately 50% protein, 20% RNA, 10% carbohydrates, and 3% DNA. The researcher notes that ribosomal RNA (rRNA) plays a significant role in protein synthesis and accounts for a large portion of the total RNA content. Based on the provided biochemical composition, what percentage of E. coli's dry weight is attributed to nucleic acids? "Approximately 23% (20% RNA + 3% DNA)." Which type of RNA is involved in protein synthesis and makes up a significant portion of ribosomes? Options:A. Ribosomal RNA (rRNA)B. Transfer RNA (tRNA)C. Small nuclear RNA (snRNA)D. Messenger RNA (mRNA) "Ribosomal RNA (rRNA)" Discuss the evolutionary significance of the central dogma in molecular biology. "The central dogma of molecular biology (DNA → RNA → Protein) is evolutionarily significant as it explains how genetic information is transferred and expressed. This process allows for the diversity of proteins, enabling adaptation and evolution in organisms, ultimately driving complexity in life forms." A laboratory technician analyzes the biochemical composition of E. coli cultures. The results show that 50% of the dry weight is protein, with significant amounts of RNA and smaller proportions of carbohydrates and DNA. The technician notes the roles of different RNA types in protein synthesis. How does the protein content of E. coli compare to its RNA content? Options:A. They are nearly equal in their proportions.B. Protein is more than twice the amount of RNA.C. Carbohydrates are more abundant than both protein and RNA.D. RNA is greater in proportion than protein. "Protein is more than twice the amount of RNA." A researcher is studying the biochemical composition of E. coli for a project on prokaryotic cellular structures. They discover that in terms of dry weight, E. coli consists of approximately 50% protein, 20% RNA, 10% carbohydrates, and 3% DNA. The researcher notes that ribosomal RNA (rRNA) plays a significant role in protein synthesis and accounts for a large portion of the total RNA content. Given that rRNA is crucial for protein synthesis in E. coli, what can be inferred about its relative abundance compared to other types of RNA? "Ribosomal RNA (rRNA) is the most abundant type of RNA in E. coli, indicating its primary role in forming ribosomes and facilitating translation." During a lab experiment, students are tasked with analyzing the transcription process in prokaryotic cells. They observe that RNA polymerase binds to a specific promoter region on the DNA strand and begins synthesizing RNA in the 5' to 3' direction. The students note the presence of various types of RNA being produced, including mRNA, tRNA, and rRNA. However, they also introduce a transcription inhibitor into the reaction mixture, which halts RNA synthesis midway through the process. What role does RNA polymerase play in the transcription process observed by the students? "RNA polymerase is responsible for binding to the promoter region of DNA and catalyzing the synthesis of RNA from ribonucleoside triphosphates in the 5' to 3' direction." How does the presence of specific sequences signal the termination of transcription? "Transcription termination is signaled by specific sequences: - **Rho-independent**: A run of U's causes RNA polymerase to pause, forming a GC-rich hairpin that triggers termination. - **Rho-dependent**: Rho protein binds to rut sites, catches up to RNA polymerase, and pulls the RNA out, causing termination." How do mutations in promoter regions affect gene expression? "Mutations in promoter regions can affect gene expression by altering promoter strength. - Strong promoters enhance transcription, leading to higher gene expression. - Weak promoters reduce transcription, resulting in lower gene expression. Changes in the -10 and -35 sequences impact RNA polymerase binding and activity." What is the significance of the -10 and -35 regions in bacterial promoters? Options:A. They are involved in the termination of transcription.B. They serve as binding sites for ribosomes during translation.C. They facilitate RNA polymerase binding and transcription initiation.D. They are responsible for DNA replication initiation. "They facilitate RNA polymerase binding and transcription initiation." Describe the process of transcription initiation in prokaryotes. "Transcription initiation in prokaryotes occurs at a promoter. Key steps include: - RNA polymerase (RNAP) binds to the promoter with the help of sigma factors. - The promoter has specific sequences at -10 and -35 positions. - The strength of the promoter affects transcription efficiency. - RNAP starts synthesizing RNA at the transcription start site." What is the main component of transfer RNA (tRNA)? Options:A. Amino acidsB. ProteinsC. Ribosomal RNA (rRNA)D. Nucleotides "Nucleotides" What is the purpose of the 5' triphosphate group in the initiating nucleotide? Options:A. It provides energy for RNA synthesis.B. It serves as a binding site for RNA polymerase.C. It helps in the recognition of the promoter region.D. It acts as a primer for RNA synthesis. "It provides energy for RNA synthesis." A bacterial cell is undergoing transcription to synthesize RNA. The RNA polymerase binds to the promoter region of the DNA, unwinding the double helix and synthesizing a complementary RNA strand in the 5' to 3' direction. Various inhibitors are present that could affect this process. How does RNA polymerase initiate transcription in prokaryotes? Options:A. By binding to the promoter region of the DNAB. By unwinding the entire DNA molecule firstC. By recruiting ribosomes to the siteD. By synthesizing RNA from existing RNA strands "By binding to the promoter region of the DNA" What challenges do inhibitors like Actinomycin D pose to transcription processes? "Inhibitors like Actinomycin D pose challenges to transcription by: - Intercalating into DNA - Preventing initiation and elongation - Blocking RNA polymerase's path during transcription - Interfering with replication processes, affecting overall gene expression." What is the difference between Rho-dependent and Rho-independent termination of transcription? Options:A. Rho-independent termination involves the Rho protein binding to RNAB. Rho-dependent termination occurs only in eukaryotesC. Rho-dependent requires Rho protein; Rho-independent relies on hairpinD. Rho-dependent termination requires a hairpin structure to occur "Rho-dependent requires Rho protein; Rho-independent relies on hairpin" During a molecular biology lab, students are studying the transcription mechanisms of prokaryotic cells. They observe that core RNA polymerase binds to DNA at specific promoter regions, initiating the synthesis of RNA. The team also discusses how termination can occur through both Rho-dependent and Rho-independent mechanisms. They note the importance of understanding these processes for genetic engineering applications. In the context of transcription initiation, what role does core RNA polymerase play in recognizing promoter regions? "Core RNA polymerase binds to specific sequences in the promoter region, facilitating the unwinding of DNA and the initiation of RNA synthesis." Explain how RNA polymerase recognizes the promoter region of a gene. "RNA polymerase recognizes the promoter region by binding to specific DNA sequences, typically at -10 and -35 positions relative to the transcription start site. It requires a sigma factor for initiation, which helps it identify strong or weak promoters, ensuring correct strand copying and directionality." A molecular biologist is studying the role of RNA in protein synthesis. During her experiments, she observes that certain RNA molecules can catalyze reactions similar to enzymes. She notes that these RNA structures often contain hairpins and bulges, which contribute to their stability and function. Additionally, she focuses on how base pairing between adenine and uracil plays a critical role in maintaining the integrity of these structures. What type of RNA structure is primarily responsible for the catalytic activity observed in the study? "RNA tertiary structures, as they allow for complex folding necessary for enzymatic functions." What is the primary function of RNA polymerase in prokaryotes? Options:A. To synthesize proteins from amino acidsB. To synthesize RNA from the DNA templateC. To replicate DNA for cell divisionD. To translate mRNA into proteins "To synthesize RNA from the DNA template" How does Rifampicin affect transcription in bacteria? Options:A. It prevents initiation of transcription.B. It binds to the α subunit of bacterial RNA polymerases.C. It prevents both initiation and elongation of transcription.D. It enhances the elongation of RNA transcripts. "It prevents initiation of transcription." How does DNA methylation affect gene expression? Options:A. DNA methylation enhances gene expression by promoting transcriptionB. DNA methylation has no effect on gene expressionC. DNA methylation reduces gene expression by inhibiting transcriptionD. DNA methylation increases the stability of mRNA transcripts "DNA methylation reduces gene expression by inhibiting transcription" Which process involves the synthesis of RNA from a DNA template? Options:A. TranslationB. Reverse transcriptionC. DNA replicationD. Transcription "Transcription" Which enzyme is primarily responsible for DNA replication in prokaryotes? Options:A. HelicaseB. DNA polymeraseC. LigaseD. RNA polymerase "DNA polymerase" How can knowledge of nucleotide sequences be applied in biotechnology and medicine? "Knowledge of nucleotide sequences can be applied in biotechnology and medicine by: - Developing gene therapies - Creating genetic tests for diseases - Producing recombinant proteins - Designing targeted drugs - Enhancing crop traits in agriculture These applications leverage the understanding of DNA and RNA functions." What is the central dogma of molecular biology, and how does it relate to gene expression? "The central dogma of molecular biology describes the flow of genetic information: DNA → RNA → Protein. It involves: - **Replication**: DNA copies itself - **Transcription**: DNA is transcribed into RNA - **Translation**: RNA is translated into proteins This process is essential for gene expression and protein synthesis." How do mutations in DNA bases impact protein synthesis? "Mutations in DNA bases can lead to changes in the mRNA sequence during transcription, which may result in altered amino acids in proteins during translation. This can affect protein function, potentially causing diseases or malfunctions in cellular processes." What type of bond links nucleotides together in a nucleic acid chain? Options:A. Hydrogen

Year 1 Biochemistry Cellular Composition PDF

Document Details

Tags

Related

Summary

Full Transcript

Upgrade to continue