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BIO120H1 (305)


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University of Toronto St. George
Doug Thomson

BIO130 Final Exam Study Sheet P a g e1| Part 1 Cells and Genomes - All cells: o Store data as DNA o Use RNA as intermediary form of data o Use proteins as catalysts o Translate RNA the same way (via ribosome) o Are enclosed in a plasma membrane - Homologous genes genes that were once the same gene (but was changed) o Orthologs Same gene diverged due to intragentic mutations into 2 different genes Usually results in speciation o Paralogs Duplication and divergence of genes in the SAME organism - Gene transfer between organisms o Bacteriophage viruses can implant DNA into bacterial genomes o Procaryotes can take up random DNA from environment - Model Organisms (M.O.) o Rapid development o Tractability o Small adult (mature stage) o Readily available o Genes understandable Created by Marina Wang © www.notesolution.com BIO130 Final Exam Study Sheet P a g 2 | E. Coli • Used to model prokaryotes and bacteria • Lives in your gut Synechocystis • A phototrophic cyanobacterium Yeast • M.O. for eukaryotes Arabidopsis • M.O. for plants • The common Thale cress Caenorhbditis elegans • Nematode worm • Has exactly 959 body cells Drosophila melanogaster • Fruit fly - Eukaryotes o Entered endosymbiosis with a small energy generating bacteria (later the mitochondria) o Some cells then formed symbiosis with a chloroplast-like bacteria These cells grew cell walls as they no longer had a need to move DNA/RNA - DNA: deoxyribosenucleic acid - RNA: ribosenucleic acid (extra OH group at carbon 2) - Formed from triphosphates Created by Marina Wang © www.notesolution.com BIO130 Final Exam Study Sheet P a g 3 | o Phosphates linked by phosphoanhydride bonds - U replaces T in RNA - U, C, T= Pyrimidines (1 ring) (“C” The Pyramids) - A, G= pyrines (2 rings) - A=T or A=U less stable than G≡ C - DNA double strand has a major and minor grove (easily seen) o Most reactions at major groove - DNA nucleotides linked by phosphodiester bonds o Between 1 phosphate and 1 sugar - Bases are hydrophobic and form internal core of double strand - 10 base pairs is 1 complete twist of DNA Proteins - Made of amino acids (AA) - 4 groups o Basic (Lysine) o Acidic (Glutamic acid) o Polar (Valine) o Non-polar (Serine) - α helix & β sheet o Created by H-bonds in N-H and C=O of polypeptide backbone o α helix is 1 chain of peptides with N-H of each peptide bond H-bonds with C=O 4 peptide bonds away o Amphipathic helixes hydrophobic parts hidden inside helix o β sheet can be parallel or antiparallel Created by Marina Wang © www.notesolution.com BIO130 Final Exam Study Sheet P a g 4 | - Cysteine is an AA that can oxidize it’s SH group to form disulphide bridges - Polypeptide starts with amine group, ends with carboxylic acid group DNA Organization - Bacteria “chromosomes” are NOT REAL CHROMOSOMES o Circular genome o Prokaryotes condense DNA 1000 folds to form nucleoid - Eukaryotes have 2 copies of each chromosome o Are homologous chromosomes (except X/Y) - Organism size does not equal genome size - DNA + proteins = chromatin - Histones form nucleosomes o Organizes DNA o Each nucleosome contains 8 histones Histones contain lysine and arginine (basic AAs) to neutralize DNA o Histone H1 links nucleosomes into a 30nm fiber - Euchromatin o Not very condensed o genes expressed - Heterochromatin o Highly condensed chromatin o Found at centromeres and telomeres and 1 X chromosome in females (barr body) o Genes are not expressed DNA Replication and such Created by Marina Wang © www.notesolution.com BIO130 Final Exam Study Sheet P a g 5 | - Bacteria have 1 replication origin whereas eukaryotes have multiple - Duplication o DNA polymerase builds 5’ to 3’ Uses deoxyrobonucleoside triphosphates o Starts at a place rich in A=T bonds o Yeast uses ARS fragments for plasmids to be opened by promoter proteins - Steps of Replication: 1. Initiator proteins bind at replication origins to form origin recognition complex (ORC) which is activated by protein kinases 2. Helicase and DNA primase bind to create Primosome 3. Helicase opens DNA double strand (uses ATP) at replication origin 4. DNA primase adds an RNA primer on template DNA 5. DNA polymerase starts building on leading strand 6. Single Stranded Binding proteins (SSB) keep lagging strand DNA from forming secondary structures 7. Lagging strand copied in Okazaki fragments See Fig. 5-19 on pg. 276 in textbook - Telomeres o For lagging strand o Telomerase 5’ to 3’ direction RNA template A reverse transcriptase (uses RNA template) o Telomere single strand longer than helix Created by Marina Wang © www.notesolution.com BIO130 Final Exam Study Sheet P a g 6 | Folds back to create T-shape • Protects against exonuclease DNA Proof reading - DNA polymerase proofreads before adding o Correct nucleotide more energetically favoured o Correct geometry causes polymerase to tighten allowing bond formation - Exonuleolytic proofreading o After incorrect nucleotide placing o DNA polymerase will use a different catalytic site to remove o Proofreading occurs in a 3’ to 5’ direction o This proofreading mechanism is due to the fact that DNA polymerase requires the 3’ OH to continue adding on nucleotides - Strand-directed mismatch repair o Detects misfits in base pairs after replication o Repair system must have a way of recognizing newly synthesized strand o Methylation of A residue in sequence GATC after a while of being synthesized in prokaryotes o Use of ‘nicks’ in DNA of eukaryotes (from Okazaki fragments) DNA Repair - DNA damages: o Depurination via deoxyribose hydrolysis ~100 Cytosine to Uracil per day o Sunlight creates covalent links between pyrimidines - Base excision repair Created by Marina Wang © www.notesolution.com BIO130 Final Exam Study Sheet P a g 7 | o DNA glycosylases Recognizes altered bases Catalyses hydrolytic removal Missing base area recognized by AP endonuclease Repaired by polymerase - Nucleotide excision repair o Fixes bulky lesions o Abnormal strand detached o Fixed by polymerase and Ligase - Double strand damage o Nonhomologous end-joining Just cut out all of the bad DNA and stick the other ends together So little DNA actually codes so it’s not a big problem o Homologous recombination Use sister chromatid as template Occurs in G2 and S phase Transcription - RNA polymerase o Uses ribonucleotides o Can unwind DNA w/o helicase NO ATP required o Can start building without primer - In bacteria: o σ-factor binds RNA polymerase Created by Marina Wang © www.notesolution.com BIO130 Final Exam Study Sheet P a g 8 | Holoenzyme o σ-factor then binds to promoter of DNA o σ-factor releases RNA polymerase o Terminator code in DNA causes a hairpin loop in mRNA Code rich in weak A=T bonds - In eukaryotes o RNA polymerase I, II and III II transcribes most protein genes o Instead of σ-factor, transcription requires general transcription factors TFII for transcription factors for polymerase II Refer to Pg. 341 in Textbook 1. TATA binding protein (TBP) (a subunit of TFIID) binds to TATA box (usually 25 nucleotides of A and Ts upstream from actual start site) 2. Large distortion in DNA signals the completion of the transcription initiation complex 3. TFIIH has a helicase subunit (transcription requires ATP) 4. RNA polymerase II will start slowly creating RNA until activator protein tells the mediator protein to release RNA polymerase II 5. At this point TTIIH adds phosphate groups to the C-terminal domain (CTD) of the RNA polymerase II to allow it to undergo the conformational change 6. Pre-mRNA splicing is performed by spliceosome (snRNA and proteins) creating a lariat structure 7. Poly-A-Polymerase adds the Poly A tail 8. 5’ of mRNA is methylated Translation Created by Marina Wang © www.notesolution.com BIO130 Final Exam Study Sheet P a g 9 | - 3 nucleotides=1 codon - 3 possible reading frames - tRNA has AA at 3’ end o attached by aminoacyl-tRNA synthetase (requires ATP) o added to either –OH group on ribose sugar - Cloverleaf fold vs L-shape o L-shape used in translation - Several tRNA per AA - Eukaryote tRNA made by RNA polymerase III - tRNA can be modified o guanine deaminated to inosine o inosine and wobble effect allows the tRNA to recognize more condons - Synthesis of protein o AA added to C-term end of polypeptide o Ribosome Is a ribozyme (has proteins and rRNA) Large and small subunit Has sites E, P, A (EPA!) Peptide bonds formed by peptidyl transferase in large subunit o Elongation factors EF-Tu and EF-G in bacteria EFI and EF2 in eukaryotes • EF-Tu checks to see if base pair is correct and does a hydrolysis of GTP (attached) Created by Marina Wang © www.notesolution.com BIO130 Final Exam Study Sheet P a g 10| • EF-G allows small subunit to catch up - Initiation of Translation o Start codon: AUG Methionine o In eukaryotes tRNA-methionine complex (Met-tRNAi) loaded into P site of small subunit This complex is the only one that can fit w/o the large subunit present o Small subunit then binds to mRNA and scans for AUG Found the mRNA via 5’cap and the proteins attached eIF4E and eIF4G • eIFs dissociates Leaky scanning means cells sometimes start translation at second AUG o Helicase is present since mRNA can form secondary structures o In bacteria instead of finding the 5’cap small subunit searches for the Shine-Dalgarno region (5’-AGGAGGU-3’) - Stop codons o UAA o UAG o UGA Causes release factor protein (molecular mimicry to look like tRNA) to bind to A site - Protien Chaperones o Hsp and Hsp Heat shock proteins to help proteins fold in increased thermal stress Created by Marina Wang © www.notesolution.com BIO130 Final Exam Study Sheet P a g 11| Part 2 Membrane Structure - Made of phosphoglyceride molecule (5nm) - Cytoplasm o Includes organelles - Cytosol o Does not include organelles - Lumen o Contained within organelles - Phospholipids o Polar head and non polar tail o Diffuses laterally, flipped by translocator protein (aka Flipase) o Synthesized in cytosolic leaflet of endoplasmic reticulum o Steroids help increase fluidity of membrane - Glycolipids o Created on luminal face of golgi apparatus o Contain sugar groups with charge o Found on apical side of epithelia where there are harsher conditions - Decreasing fluidity: o Low temperature o Saturated/longer fatty acid chains - Intermembrane Proteins o Are amphiphilic Alpha-helices vs beta-barrels Created by Marina Wang © www.notesolution.com BIO130 Final Exam Study Sheet P a g 12| o Anchored proteins Anchored by other proteins By glycosylphosphatidylinositol (GPT) Lipid anchor • Myristate • Palmitate - SDS breaks down membrane for protein extraction - Fluorescence Recovery After Photobleaching (FRAP) o Proteins fused to green fluorescence protein (GFP) o Small area bleached with laser o Graph the recovery of fluorescence based on lateral movement of proteins Membrane Transport - Lipid Bilayer permeable to o Hydrophobic molecules o Small uncharged polar molecules - Lipid bilayer impermeable to o Large uncharged polar molecules o Ions - Passive Transport o Simple diffusion down concentration gradient o Channel-mediated Has hydrophilic pore Usually selective for molecules Non-gated Eg K+ channel Created by Marina Wang © www.notesolution.com BIO130 Final Exam Study Sheet P a g e13 Gated • Voltage-gated (membrane potential) • Mechanically-gated (membrane stretching) Ligand-gated ion channel • Seen in synaptic cells and neurotransmitters o Transporter-mediated Molecule binds to integral protein, causing conformational change Uniporter eg. GLUT uniporters for glucose - Active Transport o Move against concentration gradient o Coupled transporter One molecule goes down concentration gradient while the other goes up its concentration gradient Symporter • Two molecules go the same direction • Eg. Sodium/glucose symporter o Moves into cell Antiporter • Two molecules go in opposite directions • Eg. Sodium/hydrogen exchanger o Hydrogen into cell o ATP-driven pumps (ATPases) Uses ATP hydrolysis to move molecules against their concentration gradients Created by Marina Wang © www.notesolution.com BIO130 Final Exam Study Sheet P a g 14| P-type pump • Pumps ions o Eg. Sodium Potassium pump 3Na out, 2K in ATP phosphorlates protein to cause conformational change when Na binds F-type and V-type pumps both involve proton flow and can reverse directions, named according to their usual pumping direction F-type pumps • Uses proton gradient to create ATP • Works like turbine V-type pumps • Uses ATP to pump protons against gradient • Found in lysosomes ABC transporter • Has 2 ATPase domains • Pumps molecules - Resting Membrane potential o Net positive outside, net negative inside o Motive force for charged molecule depends on other charged molecules o Na/K pump contributes net 1 positive charge (10% membrane potential) o Outside cell: Na+ and Cl- vs cytosol: K+ and Cl- K+ diffuses out causing more cations in the extracellular matrix o -20mV to -200mV Created by Marina Wang © www.notesolution.com BIO130 Final Exam Study Sheet P a g 15| Intracellular Compartments - Smooth ER has not ribosomes and create phospholipids - Endomembrane system o ER, Golgi, Endosomes, Lysosomes and maybe peroxisomes - Protein sorting o Proteins move between cytosol and nucleus through nuclear pore complex (NPC) gated transpo
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