Central Dogma PDF

lOMoARcPSD|47538836 o RNA = ribose = prex ribo o Nucleodes linked via phosphodiester bonds; 5’ phosphate reacts with 3’ OH of another LECTURE 6 – DNA REPLICATION  Dene; semiconservave, origin, bidireconal, replicaon fork, Okazaki fragment.  mechanism of leading a...

lOMoARcPSD|47538836 o RNA = ribose = prex ribo o Nucleodes linked via phosphodiester bonds; 5’ phosphate reacts with 3’ OH of another LECTURE 6 – DNA REPLICATION  Dene; semiconservave, origin, bidireconal, replicaon fork, Okazaki fragment.  mechanism of leading and lagging strand replicaon and role of the RNA primer.  funcons of the proteins at the DNA replicaon fork.  List major DNA polymerases of prokaryotes and eukaryotes and their funcons. Chromosome replicaon  Complementary base pairing = semiconservave DNA replicaon  DNA synthesis iniates at origins o Synthesis moves bidireconally away from origin via 2 replicaon forks = replicaon bubble o 5’  3’ o Requires primer Complementary base pairing = accurate DNA replicaon  Each strand of dsDNA molecule = template for synthesis of new complementary strand Semi conservave DNA replicaon  Daughter molecule = parental and new strand  1000 nucleodes per second without error DNA synthesis iniates at Origins  dsDNA pried apart at replicaon origin by DNA helicase; posion idened by ori DNA sequence Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836  protein machine moves along replicaon fork  DNA polymerase adds deoxyribonucleodes to 3’ end of new (primer) strand aached to template (parent) strand o creates phosphodiester bonds o Proofreads = reduce error and catalyses synthesis Bidireconal synthesis from Origins  Circular/short chromosomes of prokaryotes = single origin of replicaon  Long linear chromosomes of eukaryotes = mulple origins of replicaon Replicaon fork structure  daughter strands polymerised 5’  3’  Leading strand synthesised connuously  Lagging strand synthesised disconnuously = short Okazaki fragments o DNA polymerase can only aach to 3’ end Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 DNA primase = primer for DNA synthesis  DNA Primase = enzyme = synthesises short strand of RNA on a DNA template  Okazaki fragments primed by RNA primer at 3’ of lagging strand (synthesised by DNA primase)  DNA ligase joins Okazaki fragments by sugar-phosphate backbone Replicaon machine  Proteins at a replicaon fork cooperate = machine  Single-strand DNA-binding proteins stabilise ssDNA and aid the helicase  Helicase unwinds double helix to form ssDNA for replicaon  Sliding clamp moves behind DNA polymerase across DNA template strands to ensure aachment of polymerase.  Clamp loader assembles the clamp on the DNA using ATP energy  Lagging strand DNA = folded = brings DNA polymerase into a complex with leading strand DNA polymerase. DNA polymerase in prokaryotes  Polymerase 1 o DNA repair o Maturaon of Okazaki fragments (removes RNA primer and lls gaps)  Polymerase 2 o DNA repair  Polymerase 3 o DNA replicaon enzyme (both strands) DNA polymerase in eukaryotes  Alpha α = primase and elongates primer with short length of DNA (mul subunit enzyme)  Beta β = DNA repair  Gamma γ = replicaon of mitochondrial DNA  Delta δ = synthesis of lagging strand  Epsilon ε = synthesis of leading strand Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 LECTURE 7 – MUTATION AND REPAIR OF DNA  Explain mutaon of DNA  Describe the processes that result in the mutaon of DNA  Describe the consequences of depurinaon, deanimaon, thymine dimer formaon and double stranded breaks on DNA replicaon  What transposable DNA elements and infecous agents introduce mutaons into DNA  Explain the 2 mechanisms for DNA repair: O Mismatch repair system O homologous recombinaon Mutaon  permanent and heritable change in the DNA sequence o damaged DNA = replicaon problems o change caused by replicaon errors (rare)  DNA replicaon = High Fidelity (error by polymerase = one in a billion) o base paired structure of DNA o primer requirements of DNA polymerases + proofreading of polymerase (diagram)  3’5’ exonuclease acvity removes incorrect nucleode  restoraon of correct nucleode sequence  possible incorrect N base incorporated Chemical factors Nucleode instability = DNA polymerase randomly assigns nucleodes to match damaged nucleode = change nucleode sequence  xed & inherited  Depurinaon = loss of A/G from DNA backbone  Deaminaon = loss of NH2 from A/G/C (= U) Mutagenic chemical  Alkylaon = electrophiles (carcinogens) add alkyl groups to N bases = stall replicaon  Intercalaon = compound inserts into ds helix = distoron but no change in bases o Ethidium bromide Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 Radiaon UV radiaon  Forms thymine dimers Gamma Rays  Aack DNA bonds (single and double stranded breaks) o Produce free electrons = aack backbone o Generate hydroxide free radicals Mobile DNA Infecous agents (viruses and bacteriophages) = Insert/recombine into a target DNA molecule  Recombinaon = breaking & rejoining of DNA molecules = new combinaons o Non-homologous = NO similarity between DNA molecules (bacterial DNA and phage DNA)  targeted recombinaon catalysed by integrases/transposases (enzymes)  phage encoded integrase = promotes recombinaon between aachment sites o Homologous = both donor and acceptor DNA have similar sequences  Retroviruses (HIV) integrate into host DNA = use host replicaon machinery o Lyc (enter and lyse/kill host) o Lysogenic (enter and integrate into host chromosome)  Inseron of foreign DNA = disrupts coding region/gene Transposable elements  Transposons = linear DNA molecules o Move within/between chromosomes o Insert into dierent DNA sequences = disrupt gene o Excision of transposon = small duplicaon of DNA = disrupt gene  Transposase monomers (enzymes) encoded by transposon = aach at short inverted repeat sequences = allow break away from chromosome by forming transpososome loop o Loop integrated into other chromosome at short direct repeats of target DNA sequences  Cut and paste non replicave transposion o Transposase encoded by transposon cut out transposon and insert into new DNA  Replicave transposion o Transposon replicated by DNA replicaon  into target DNA = both donor and target DNA have transposon Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 Repair Mismatch (mis-paired nucleodes)  repairs mutaons in newly synthesised DNA strand = restore to original sequence of template strand  repair proteins recognise and excise strand of DNA containing mismatch o bind to mismatch sequence and excised by nucleases = ssDNA patch o 2nd strand synthesised by repair DNA polymerase using free 3’OH group as primer o Ligaon of DNA backbone by DNA ligase Homologous recombinaon  repairs double stranded breaks in phosphodiester backbone of DNA o = restoraon of original sequence + rearrangements of local regions of DNA sequence  Similar sequence regions align  double strands break = cross over = new combinaons of DNA aer repair  Exonuclease removes nucleodes from 5’  3’ = single stranded 3’ overhang = migrates to other chromosome of homologous sequences  DNA polymerase synthesises complementary strand  Holiday Juncon = crossed strands = rotate  secon of strand joined to another strand = exchange DNA Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 LECTURE 8 – TRANSCRIPTION MECHANISM  Describe the structure of RNA and the dierences to DNA  Dene; transcripon, understand how DNA is transcribed to RNA  dierences in the transcripon processes between eukaryotes and prokaryotes  Describe and understand the funcon of RNA polymerase  Describe the transcripon iniaon and terminaon signals in DNA  Describe the process of capping and polyadenylaon  List the dierent classes of RNA molecules RNA Structure  Ribose sugar  A, C, G, U  Single stranded Transcripon = RNA from DNA template Iniaon  elongaon  terminaon Convenons  Coding/sense strand = 5’  3’  RNA polymerase = transcribes complementary mRNA strand of an-parallel/template strand from P to T = same sequence of coding strand (T replaced with U) o Template strand = 3’  5’ from promotor to terminator Requirements of enzymac synthesis of RNA  4 ribonucleode 5’ triphosphates (5’ATP, 5’GTP, 5’CTP, 5’UTP)  Mg2+  DNA template (no primer)  RNA polymerase (RNAP) Transcripon in prokaryotes  RNA polymerase (holoenzyme) binds to promoter on dsDNA  Sigma factor leaves = core enzyme = begins RNA synthesis (elongaon) = mRNA strand is complementary to template strand from P to T  RNA polymerase reaches terminator = stop = complete mRNA synthesis & (sigma factor rebinds) o Iniaon signal = promoter  Holoenzyme binds to TGTTGA sequence = upstream/promoter region  TATAATG box shows where consensus sequence in coding strand ends and where coding region begins o Terminaon signal = Hair-pin structure Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836  Inverted repeat followed by a stretch of T bases (coding strand of DNA sequence)  protein factor (Rho); makes polymerase fall o hair loop RNA Polymerase  Forms phosphodiester bonds between ribonucleosides  Uses energy stored in ribonucleoside triphosphates for polymerisaon  Unlike DNA polymerase; RNA synthesis starts without a primer o Error rate higher than DNA polymerase o Unwinding of DNA template doesn’t require a helicase or ATP. E. coli RNA polymerase  5 SUBUNITS  complex of α2ββ’ ω = core enzyme  σ = promoter recognion (pyridine-rich DNA sequence of >10 bases). o σ + core enzyme = holoenzyme = iniaon  correct iniaon achieved = σ dissociates from holoenzyme = core enzyme connues elongaon of RNA chain. Messenger RNA Prokaryoc messenger RNA  mRNA = copy of coding strand (DNA) = determine amino acid sequence of proteins  DNA segment (coding sequence corresponding to protein) + start and stop signals = cistron. o A single mRNA encoding 1 polypepde = monocistronic mRNA Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 o Polycistronic mRNAs = code for several dierent polypepde chains  mRNA’s have 5’ leaders, 3’ termini and intercistronic regions called spacers (polycistronic)  Prokaryoc mRNA’s have short half-lives = minutes  Primary transcript not processed Eukaryoc messenger RNA  Gene contains coding (exons) and non-coding (introns) regions  RNA is processed (Idenes RNA molecule as an mRNA) o RNA capping (G with methyl group added to 5’ end) o Polyadenylaon ( “A”s added to 3’ end)  Introns removed by a spliceosome = recognises boundaries between introns and exons  Exons stched back together and translated  mRNA molecules eventually degraded LECTURE 9 – TRANSCRIPTION PRODUCTS  Describe the major classes of RNA molecules  Describe the dierences between prokaryoc and eukaryoc mRNA  Describe the structure and funcon of tRNA and funcon of aminoacyl-tRNA synthetases  Describe dierences between prokaryoc and eukaryoc ribosomes  Understand the roles of ribosome binding sites in translaon of mRNA to protein  Dene; polycistronic mRNA, polyribosome, mRNA cap, poly-A tail, codon, ancodon, amino acyl tRNA Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 Major classes of RNA Messenger = mRNA  Copy of DNA – codes for protein  Decoded in sets of 3 nucleodes = codons Transfer = tRNA  Small adaptor molecules which align specic amino acids opposite their triplet codon in the mRNA molecule during translaon of mRNA into protein o Amino acids can’t recognise mRNA codon = requires adaptor (tRNA) Ribosomal = rRNA  Integral part of ribosomal structures – part of protein synthesising machinery RNA transcripts Eukaryotes = Transcripon/translaon = separate – dierent compartments = control Prokaryotes = Transcripon/translaon = coupled – simultaneous Eukaryotes:  dsDNA = gene encoded by exons – separated by introns  transcripon iniated = RNA polymerase binds to template strand (3’  5’) of DNA and synthesises mRNA (copy of coding strand; 5’  3’)  post transcripon modicaons = o splicing = removes introns by spliceosomes o 5’ cap methylated G recognises stop = polymerase drops o  polyadenylaon = poly-A-tail at end of gene = mature mRNA  Mature mRNA released into cytoplasm o No cap or poly-A-tail = mRNA degradaon  Ribosome recognises mRNA start codon (AUG) and protein synthesis beings eukaryoc mRNA = monocistronic Roles of 5’ end cap and 3’ end polyadenylaon o assist the export of mRNA molecules from the nucleus o Protect mRNA from degradaon in cytoplasm and increase its half-life o Allows binding of ribosomes = promote translaon  CAP = 7-methylguanosine  Poly-A tail = many adenosines linked to 3’ end of mRNA (not in DNA sequence) o = binding site for proteins Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 prokaryoc mRNA = polycistronic single prokaryoc mRNA molecule can encode several dierent proteins prokaryoc ribosomes iniate translaon at ribosome-binding sites, located in the interior of an mRNA molecule = prokaryotes synthesize >1 type of protein from 1 polycistronic mRNA tRNA  Small adaptor molecules = align amino acids opposite triplet codon in mRNA molecule during translaon of mRNA into protein o Amino acids can’t recognise mRNA codon = requires adaptor (tRNA)  Bases other than A, G, C and U are present in tRNA due to post-transcriponal modicaon o inosine (modied A), pseudouridine & dihydrouridine Structure  Internal complementary base pairing of RNA gives cloverleaf structure  Ancodon sequence of 3 bases determines mRNA codon binding  Amino acid aachment site is at 3’ end of tRNA  Modied bases o D = dihydrouracil o Ψ = pseudouridine  3D shape determines aachment of correct amino acid (matching codon/ancodon pair) by aminoacyl-tRNA synthetases Adaptors Genec code translated by 2 adaptors  Aminoacyl-tRNA synthetase couples amino acid to corresponding tRNA o For every amino acid = 1 aminoacyl-tRNA synthetase - links amino acid to several tRNAs o Enzymes responsible for aachment of correct amino acid to tRNA:  Aa specied by ancodon of tRNA and aa aachment sequence on the 3’ end o correct amino acid aached to tRNA = tRNA is charged/acylated  ATP hydrolysis required  mischarging = incorrect amino acid aachment  tRNA = ancodon forms base pairs with codon of mRNA Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 rRNA  integral part of ribosomal structures + important parts of the protein synthesising machinery o mRNA message decoded on ribosomes Ribosome = large mul-domain complex  scans along mRNA and captures correct aminoacyl tRNA o Correct base-pairing = covalent linking of aa onto growing polypepde chain  Small subunit matches tRNA to the codon on the mRNA  Large subunit catalyses the formaon of the pepde bonds that link aa together = polypepde  Each ribosome has 1 binding site for mRNA and 3 binding sites for tRNA o tRNA binding sites:  A = new tRNA enters ribosomal complex  P = tRNA aached to the polypepde chain  E = empty tRNA exits ribosomal complex Prokaryoc Ribosome structure Large ribosomal subunit = 5S, 23S rRNA Small ribosomal subunit = 16S rRNA Eukaryoc Ribosome structure Large ribosomal subunit = 5S, 5.8S & 28S rRNA Small ribosomal subunit = 18S rRNA Elongaon of polypepde 1. Aminoacyl-tRNA binds to A-site, spent tRNA leaves from E-site. 2. New pepde bond formed 3. Large subunit moves to next codon 4. Small subunit moves to next codon, A- site now empty, ready for next aminoacyl-tRNA 5. Cycle 1 to 4 repeats Polyribosomes large assembly of ribosomes - 80 nts apart on a single mRNA translate proteins o ribosomes can simultaneously translate the same mRNA molecule = increases producon capacity of a protein Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 LECTURE 10 – TRANSLATION Describe the genec code Explain the history and experimental design associated with the discovery of the genec code Explain the redundancy in the genec code at the mRNA level Explain the redundancy in the recognion of the genec code by tRNAs using wobble hypothesis Genec code = redundant/degenerate Transcripon = DNA to RNA 4 bases matching 4 bases, copy Translaon = RNA  Protein 4 bases and 20 amino acids, translate DNA/RNA language = 4 bases (GATC/ GAUC). Max number of codons in groups of three: 64 possible codons Code = triplet - each codon consists of a unique combinaon of 3 nucleodes code has punctuaons at end of the coding region in mRNA: start and Stop codons codon code = redundant; amino acids specied by more than one codon o methionine and tryptophan are specied by a single codon code = consistent - each codon species a single amino acid code = universal - viruses, bacteria, plants, and animals use the same code o some minor variaons in mitochondria and a few fungi Unique codons AUG = Methionine = Iniaon signal for translaon Prokaryotes = formyl-methionine Eukaryotes = methionine 3 codons not recognised by tRNAs STOP codons =Terminaon signal for translaon UAA, UAG & UGA History 4 bases + codon + 20 aa arcial RNA composed of uracil, poly(U) = synthesize protein of poly-phenylalanine. cell-free system o E. coli cytosol fracon o destroyed nave mRNA o added radiolabelled amino acids Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 Repeated with poly(A) mRNA = poly-Lysine & poly(C) mRNA to create a poly-Proline RNA strands of repeang di- tri- and tetra-nucleode sequences made through transcripon of DNA into RNA with RNA polymerase. Poly UG repeated and poly AG trap trinucleodes (size of a codon) with the ribosome = trap corresponding charged aminoacyl-tRNA (radiolabelled) decipher all the remaining codon sequences = what amino acid they code for. Wobble Hypothesis Degeneracy in tRNA recognion of the genec code o Some tRNA's can bind at >1 codon = "wobble”. o inosine (modied adenosine) – 1st posion in the ancodon of the tRNA = bind to U, A, or C in the 3rd posion of the codon = 1 tRNA recognizes 3 dierent codons. codon and an-codon = an-parallel. o 1st posion of the codon = 5' end of codon (mRNA) and binds to the 3 rd posion (3‘ end) of the an-codon (tRNA) LECTURE 11 – TRANSLATION PROCESS Describe the 3 stages of protein synthesis Understand translaonal reading frames Describe and know the dierences in iniaon of translaon in eukaryotes and prokaryotes Describe the iniaon signals in eukaryotes and prokaryotes Describe the dierence between the iniaon methionine in eukaryotes and prokaryotes Describe the processes of elongaon and terminaon of translaon Describe why certain anbiocs can inhibit bacterial translaon and not eukaryoc translaon Stages of protein synthesis Iniaon = mRNA + ribosomes + iniang tRNA Elongaon = pepde bonds + movement Terminaon = dissociaon of ribosomes and pepdes Translaonal reading frames Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 Translaon iniaon signals translaon machinery picks the right AUG as the iniaon signal Needs other iniaon signals; ribosome binding site mof in the mRNA Eukaryotes = ribosomes - scanning ability to nd the right site Prokaryotes = small subunit of ribosome binds to Shine Dalgarno sequence (5’AGGAGGU3’) located upstream of AUG codon Iniaon in eukaryotes Iniaon site = denes the correct open reading frame. 1. Iniaon Met-tRNAi loaded into “P” site of small ribosomal subunit with iniaon factors (eIF2-GTP) 2. loaded small ribosomal subunit aaches to 5’end of mRNA (assisted by 5’end cap) 3. scans along mRNA (5’ to 3’ direcon) - idenes 1st AUG codon, surrounded by a long consensus sequence (Kozac sequence) Kozac sequence = degenerate = ribosome iniate translaon from mulple start codons = short polypepdes 4. iniaon factors detach and large subunit binds = complete ribosomal complex 1st amino acid in ‘P’ site = ready for chain elongaon Iniaon in prokaryotes 1. 30S (small subunit) interacts with iniaon factor (IF) - complex binds Shine Dalgarno sequence upstream of AUG start codon 2. Iniator tRNA binds (fMet-tRNAi) aligns with start codon (AUG) in mRNA 3. 50S associates with 30S, releasing IF = 70S complex tRNA occupies P-site of 50S subunit (A site = empty) Iniaon tRNA-Met Prokaryotes: Iniaon of translaon requires iniator fMet-tRNAi. iniator tRNA in bacteria = formylated methionine (N-formylmethionine tRNA or f Met-tRNAi) Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 Eukaryotes - Iniaon of translaon requires iniator Met-tRNAi iniator tRNA = Met-tRNAi (not formylated) Elongaon tRNA-Met Prokaryotes AND eukaryotes = Met codon inside coding region = elongaon Met-tRNA used. Met-tRNA has a dierent stem loop structure = preferenally binds elongaon co-factors Met appears inside an open reading frame = Met-tRNA used  binds elongaon co-factors. Elongaon Use ATP to move ribosome 5’  3’, inserng tRNAs above codons and catalysing pepde bond formaon between pepdes 1. aminoacyl-tRNA molecule binds to vacant A-site on the ribosome 2. new pepde bond formed between amino acids aached to the tRNAs in the P and A sites = tRNA-pepde in the A-site 3. mRNA moves a distance of 3 nucleodes (codon) through small subunit (uses ATP  ADP+PPi) o de-acylated tRNA molecule ejected from E site o A-site-tRNA-pepde  P site o A-site = empty and located over the downstream codon = ready to receive another aminoacyl-tRNA molecule = base pairs with codon posion at which the growing pepde chain is aached to a tRNA does not change during elongaon: always linked to tRNA present at P-site of the large subunit Ribosomes = rRNA and polypepdes Polypepdes = structural rRNA forms structured pocket of H networks + catalyc properes ribosomes = ribozymes o rRNA performs condensaon of carboxy-terminus of amino acid on tRNA in P site with the amino-terminus of the amino acid in tRNA in the A-site = pepde bond o pepde-tRNA in A-site  P-site as ribosome moves down mRNA in the 3’ direcon. Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 Terminaon Terminaon iniated by stop codons: UAA, UAG, UGA = don’t match tRNA Releasing factors bind to a stop codon that reaches ‘A’ on ribosome = alters acvity of pepdyl transferase (which forms pepde bond in large subunit) o adds water molecule to pepde and releases it ribosome releases mRNA  2 subunits dissociate and can bind to same/another mRNA at 5’end Terminaon = ribosome encounters stop codon in A-site = hydrolysis reacon  release pepde from nal tRNA = dissociaon of ribosome from mRNA. Anbacterial acon of anbiocs = inhibing translaon. Streptomycin/neomycin bind 30S subunit (small) = prevents transion from iniaon to elongaon Tetracyclines/puromycin mimic structure of charged tRNA’s and block ‘A’ site of prokaryoc ribosome Chloramphenicol blocks pepdyl transferase reacon Erythromycin blocks translocaon reacon Eukaryoc ribosomes usually not inhibited (except for ribosomes in mitochondria - like prokaryotes) Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 LECTURE 15 – PROTEIN STRUCTURE describe the composion and characteriscs of amino acids classify amino acids according to their side chains characteriscs (basic, acidic, uncharged polar, nonpolar)  determine net charge of a polypepde memorise and use the three-leer amino acid code describe the formaon and characteriscs of pepde bonds describe noncovalent and covalent interacons that play a role in determining the conformaon of a polypepde describe primary, secondary, terary and quaternary protein structures, -helices, -sheets and random coils, polypepde domains, homo- and heteromeric protein Amino acids α-carbon = asymmetric = 2 mirror images = stereoisomers L and D Proteins contain L-amino acids o Glycine = not a stereoisomer = neither L/D pH7.0 = amino and carboxyl groups ionised charge = pH dependent amino and carboxy groups = uncharged if part of the pepde bond of a protein. o only N-terminal amino group and C-terminal carboxy group = charged at neutral pH. Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 Polypepdes Pepde bonds form backbone of polypepde chain Condensaon reacon of a carboxylic acid and amino group oligopepde ≈ ten amino acids polypepde ≥ ten amino acids Flexible Molecules Resonance of amide structure = bond between the carbonyl carbon and nitrogen to have paral double bond character o = restricts rotaon around pepde bond ➔ planar, rigid structure o Impacts 3D pepde structure Non-covalent interacons restrict conformaon of polypepdes  Ionic/electrostac interacons  Hydrophobic bonds  Hydrogen bonds  Van der Waals Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 Covalent interacons Disulphide bonds = stabilise conformaon (cys-cys) o Formed in oxidang condions Denaturaon and Renaturaon of Proteins Denature highly concentrated urea = loses structural conformaon by ‘breaking’ non-covalent interacons ➔ protein = inacve reducing environment = break disulde bond Renature Remove urea/protein in oxidizing (cooling) environment = protein refold into nave conguraon ➔ amino acid sequence = ‘memory’ o Not every protein can do this Protein structure Primary (1) = order (sequence) of amino acids Secondary (2) = local folded structures (folding mofs in proteins) Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 β-sheets rigid cores formed by β-sheets carbonyl oxygens on polypepde backbone in -strand form hydrogen bonds with hydrogen on nitrogen group of another - strand to form a –sheet. o H-bonds keep -strands together R-group sck outwards from sheets = not involved in holding sheets together β-sheets can form with β-strands in same protein/polypepde or between β-strands in dierent polypepde chains An-parallel β-sheet neighbouring β-strands run in opposite orientaon (NC terminus & CN terminus) Parallel β-sheet neighbouring β-strands run in same orientaon (both NC terminus/CN terminus) α -sheets H-bonds between carbonyl oxygen of pepde bond and amide hydrogen of amino acid 4 residues away = stabilise helical structure o R group sck out Transmembrane proteins contain α-helical regions o transmembrane domains = formed by α-helices o hydrophobic side chains sck out into hydrophobic lipid bilayer membrane Random coil unstructured units DON’T form regular secondary structure and NOT characterized by regular H-bonding paern found in 2 locaons in proteins: Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 o Terminal ends - both N-terminus and C-terminus o Loops - between regular secondary structure elements (α– helices & β–sheets) Protein domain/mof region of a polypepde that can fold independently into a compact, stable structure o domain of 4 alpha helices o domain of alpha helices and beta strands o domain of beta strands = beta sheet occur in dierent proteins with dierent amino acids similar domains in proteins with similar funcons in evoluonarily distant organisms o increased organism complexity increases number of domains Terary (3) = full 3-dimensional conformaon ALL -helices, -sheets, random coils, loops of a polypepde chain Quaternary (4) = 3-dimensional relaonship of polypepdes in a protein made up of >1 protein. Mulsubunit proteins (Each protein = a subunit) Homodimer: made up of two idencal protein subunits. Heterodimer: made up of two dierent protein subunits. o Haemoglobin tetramer = heterotetramer LECTURE 16 – PROTEIN FUNCTION amino acid sequences show evoluonary relaonships of proteins and determine, shape, exibility and funcon of proteins Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 explain that all proteins bind other molecules named ligands via non-covalent interacons dene dierent types of proteins and describe their features explain how enzymes work and how they are regulated explain how motor proteins move cargos in cells Amino acid sequence = determines shape, exibility and funcon of proteins Proteins Bind Other Molecules – ligands Ligand = Ion (Mg2+), Small Molecules (ATP) or Macromolecules (proteins, DNA) Binding to binding sites is specic due to shape complementarity o Binding = via non-covalent interacons Binding of ligand = protein folds to provide close t o Non ng ligands fall out of binding; sum of non-covalent interacons = weak ➔ unwanted associaons prevented o Correct ligand binds ➔ many non-covalent interacons; ght t into binding site Anbodies Bind Ligands – Angens Anbodies: produced by immune system against foreign molecules/angens = ligands 2 heavy and 2 light chains, variable and constant domains bind angens with angen binding site = angen destrucon Variable domains have variable loops ➔ to changing length & amino acid sequence ➔ specicity of anbody to angen ➔ potenally billions produced by an immune system Structural proteins = Fibrous Keran protein = α-helical regions Keran monomers assemble into dimers  Dimers form staggered tetramers  8 tetramers form intermediate laments Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 Genec disorders characterised by blistering of skin from mutaons in keran genes (clumping & disrupon of keran network in basal cells) Relaonships and funconality Relaonships = similar/dierent amino acid sequence → similar sequences cluster (subfamily of proteins) Funconality reected in clustering Enzymes Proteins that speed up reacons by reducing acvaon energy required for reacon to occur o Enzyme (E) binds specic ligand/substrate (S) = enzyme substrate complex (ES) o Enzyme catalyses a change (cleavage) of substrate = enzyme product complex (EP) o EP dissociates = release product (P) from enzyme (E)  Binding = noncovalent interacons in acve site  Organizaon of atoms in the acve site is opmized for catalysis Encourage reacon by; o Bind to 2 S molecules and orients them for reacon o Bind S to E = rearranges electrons in S = delta charges = favour reacon o Enzyme deforms bound S  transion state (S breaks and released) Not consumed during reacon = available to bind new substrates and catalyse reacon repeatedly Substrate specic: o pepdase break pepdase-specic pepde bonds in target proteins Regulated: o by binding other molecules/by phosphorylaon o gene expression and compartmentalise enzymes o regulate enzyme degradaon Posive regulaon = increase number of acve enzymes by binding a dierent molecule to the substrate. Posive eector molecule = X = binds to regulatory site of protein Frequency of enzyme molecule binding to glucose increase = higher metabolism of glucose Negave regulaon = feedback inhibion Negave eector molecule = binds to regulatory site of protein o Product made late in pathway = X o Inhibits enzyme catalysing a reacon early in pathway Regulates connected metabolic pathways o P of 1 enzyme-catalysed reacon = S for another → metabolic pathways o Feedback inhibion at mulple points regulates connected metabolic pathways Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 Allosteric regulaon = Enzymes regulated by eector molecules = Allosteric Proteins o >2 dierent conformaons; acvity regulated by switching conformaons Regulatory and acve sites “communicate” o eector bound to regulatory site = changes shape of enzyme o Binding of posive regulator = allosteric acvator changes shape of acve site = S binds beer o Binding of negave regulator = allosteric inhibitor changes shape of acve site = S doesn’t bind Phosphorylaon regulaon Protein kinases = catalyse phosphorylaon of amino acids with OH (serine, threonine and tyrosine) o Proteins = acvated/inacvated by phosphorylaon Protein phosphatase removes phosphate groups from amino acid Motor proteins Kinesin & dynein o move cytoplasmic components (cargo) along microtubules (cytoskeleton) in opposite direcons ATP hydrolysis (ATP ADP) occurs at head regions of motor proteins Cargo bound at tail regions of motor proteins Move by conformaonal changes Movement of motor proteins = coupled with hydrolysis of ATP o ATP bound = conformaonal changes in motor protein o Hydrolysis of ATP (ATP ADP)= another conformaonal change conformaonal changes = motor protein to move; through cell/along other proteins LECTURE 12 – CLONING Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 dene; recombinant DNA molecule, DNA cloning, restricon enzyme, palindromic sequence, blunt and scky (cohesive) ends, DNA ligase, plasmid vector, agarose gel electrophoresis, large- scale protein producon, protein expression, pepde tag understand the enzymes, DNA sequences, DNA products and experimental steps used in DNA cloning recognise that restricon enzymes are part of a bacterial defence system describe the characteriscs of plasmid vectors and how they can be used to clone DNA fragments and to produce large-scale amounts of proteins Recombinant DNA molecule = molecule of DNA originated from 2 or more DNA fragments that aren’t found together in nature DNA Cloning = Producon of idencal copies of a parcular DNA molecule Isolaon of a parcular piece of DNA from the rest of a cell’s DNA o = copies of DNA fragments  produce proteins/study gene mutaons Use restricon enzymes = enzymacally insert DNA fragment/plasmids into plasmid vector by cung (DNA/plasmid) at sequence specic sites = recombinant plasmid Restricon enzymes = Endonucleases Digest dsDNA at internal phosphodiester bonds Cut DNA at specic sites = complimentary ends O dened by nucleode (recognion) sequences = restricon sites/sequences Palindromes (Palindromic DNA sequences) = same sequence forward & backwards (5’ 3’ & 3’5’) Restricon enzymes recognise and bind to specic palindromic sequences and cut them O 4-8 nucleodes Leave 3’-OH & 5’-phosphate groups on both cut strands DNA fragments with complementary scky ends can anneal Blunt (DNA) ends Restricon enzyme cut both strands at same posion Scky (cohesive) ends cut makes 5’ or 3’ overhang Name of restricon enzyme reects organism of origin 1st leer from Genus (Escherichia) and the next 2 from the Species (coli) = EcoRI Funcon of restricon enzymes in nature = Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 O Protect cells from invaders (virus = not methylated) O Acts together with a methylase = protects own (methylated) DNA ligase Covalently links 3’-hydroxyl and 5’- phosphate groups Uses ATP o E.g; DNA ligases join Okazaki fragments during DNA replicaon Plasmids Small, extrachromosomal, double-stranded, circular DNA molecules (found in bacterial cells) Carry genes benecial for survival of the bacterium under certain condions Disnct from the bacterial chromosome - replicates independently from chromosomal DNA “Vectors” = move DNA from one place to another Promoter: expression of a gene cloned downstream of the promoter start of making RNA (acvate transcripon) Restricon enzyme sites: cloning of DNA of interest. (inserted cDNA) many unique restricon enzyme sites = mulple cloning site or polylinker Origin of replicaon: Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 replicaon of the plasmid = ensure it’s passed on to daughter cells during bacterial division. = several copies of a plasmid are found in a bacterial cell. Selecon marker: select bacteria that have the plasmid. (grows = contains vector) o marker = anbioc resistance gene, then bacteria without marker will die in the presence of the anbioc. Plasmid (vector) map 1. mulple cloning site or polylinker ➔ insert DNA of interest 2. origin of replicaon ➔ starng point of plasmid DNA replicaon in bacteria 3. anbioc resistance gene – selectable marker  Makes bacteria resistant to ampicillin Transformaon of bacterial cells Heat shock = 42 degrees o Opens up cell wall = looser membrane = DNA into cell Electroporaon o Current applied to cells = bacterial cell wall/membrane permeable to DNA Agarose Gell red algal carbohydrate boil in buer = dissolve  into tray containing comb = solid gel with loading pockets (cooled down) Pull out combs, transfer tray with gel into an electrophoresis tank and overlay with a buer Agarose Gel Electrophoresis Agarose gel placed in an electrophoresis chamber with a posive and negave electrode voltage ➔ DNA (negavely charged) migrates to posive electrode (smallest move fastest) o DNA fragments separated by size Ethidium bromide (uorescent dyes) binds DNA (& RNA) and uoresces when viewed with ultraviolet light Downloaded by stoopid stoopid ([email protected])  lOMoARcPSD|47538836 Large scale producon of proteins Host cells = bacteria/yeast cells/mammalian cells/insect cells Expression vector encodes o highly acve promoter region Pepde tag for isolaon of expressed protein o nucleode sequence coding for a cluster of hisdine residues (His-tagged) cluster of His residues can bind to metal ions o His-tagged, overexpressed proteins o eluted from column by reducing the pH of the solvent LECTURE 13 – COPYING DNA AND RNA IN VITRO list the components of a PCR and explain their roles in a PCR reaction. describe the steps in a PCR cycle and relate this to amplification of DNA. explain the binding of a PCR primer pair to a complementary double-stranded template and extension of the primers by DNA polymerases, including Taq DNA polymerase. describe cDNA synthesis, including the properties of the enzymes used. explain using examples the differences between PCR, RT-PCR and qRT-PCR and when each is used Downloaded by stoopid stoopid ([email protected])