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Midterm Review.docx

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University of Toronto St. George
Kenneth Yip

Lecture 1 Readings Ch.1: pg 1-8 DNA: long un-branched polymer chains with four types of monomers: adenine, thymine, cytosine, and guanine. Nucleotide: deoxyribose sugar, phosphate backbone, and nucleotide base (ATCG) DNA is formed by template from pre-existing DNA Transcription: DNA RNA. RNA: ribose sugar, AUCG nucleotide bases, mass produced and disposable, for protein synthesis and catalysis Protein: long unbranched polymer chains. The monomers are amino acids. Proteins can be functional (specific enzymes) or structural. **Autocatalytic: some proteins catalyze the transcription and translation of DNA into proteins. Codon: three consecutive nucleotides (64 possible codons [4x4x4] ). tRNA: RNA with an amino acid at one end and the anticodon on the other. It reads the codes. Gene: fragment of DNA corresponding to a protein. Noncoding DNA is regulatory. Molecular building blocks: simple sugars, nucleotides, amino acids, ATP Eukaryotes: DNA is in a membrane enclosed compartment (nucleus) Prokaryotes: no distinct nuclear compartment. Live independently or loosely organized communities. Often with a cell wall or flagellum. i. Organotropic: use organic material for food ii. Phototropic: use light energy iii. Lithotropic: eat inorganically and use CO2 and H2S Mutations: random accidents altering nucleotide sequence.  Neutral changes may or may not be perpetuated.  Harmful mutations are rarely perpetuated.  DNA coding for necessary proteins will be conserved Lecture 1 Three primary branches in the tree of life: 1. Eubacteria: prokaryotes 2. Archaeabacteria: prokaryotes – no nucleus but similar to eukaryotes 3. Eukaryotes Two main cell types: 1. Prokaryotic cells: single-celled, lack nucleus and organelles. 2. Eukaryotic Cells: can be EITHER single or multicellular with nuclei and organelles. Prokaryotic Cell:  Approximately 1 micrometer.  Plasma membrane is a selectively permeable filter  Cell wall is present occasionally, it’s a protective coat  The DNA is not enclosed, may be in a nucleoid (compact structure)  Flagellum for locomotion are also present in some  Ribosomes are present throughout the cytosol for protein synthesis Eukaryotic Cell:  Much larger (over 50 micrometers)  Contains microtubules for the cytoskeleton  Peroxisome breaks down hydrogen peroxide  Plasma membrane is selectively permeable.  DNA is enclosed in nucleus.  Lysosomes, ER, mitochondria, and vesicles are present Phagocytosis: bacteria releases chemicals detected by neutrophil which engulfs it Genomes: - All known life form has a genome - Most genomes are made of DNA (viruses have RNA genomes) - Genome expression is the release of biological info stored in genome. Genome Expression:  Accounts for different type of cells with the same genome  Transcriptome: the repertoire of RNA molecules present in a cell at a particular time - Produced by transcription (changing DNA  RNA) - Setting and environment affect gene expression - DNA Microarray: snapshot of the transcriptome (red: a lot)(green: a little)(black: baseline)  Proteome: collection of all proteins in a cell - 2D Gel Electrophoresis: snapshot of proteome (red: present in both cells)(blue: distinct) - Maintained by translation (RNA  protein)  Central dogma: Lecture 2 Transcriptional Regulation  Crucial for: (i) Responses to extracellular stimuli (for both multicellular and unicellular organisms) (ii) Defining Cell types (for multicellular organisms first)  RNA polymerase enzyme transcribes DNA to RNA (moves in 3’  5’ direction, making RNA 5’3’)  Transcription in PROKARYOTES: 1. Promoter region on DNA is the transcription start site. Sigma factor binds to promoter. 2. RNA polymerase holoenzyme: RNA polymerase and transcription factors 3. RNA polymerase unwinds DNA 4. Transcription begins (sometimes promoter is transcribed, sometimes not) 5. Once ~10 nucleotides are synthesized, sigma factor is released 6. Transcription elongation occurs (RNA tends to loop) 7. Transcription Termination, release of RNA and DNA leaving just the RNA polymerase core enzyme.  Gene expression is regulated by: Gene Regulatory Proteins: Transcription Factors which bind to Regulatory regions of DNA (cis elements: DNA elements) (i) Activators: gene regulatory proteins that turn genes on (ii) Repressors: gene regulatory proteins that turn genes off E. Coli: - Unicellular prokaryote - DNA: one circular chromosome encoding 4,300 proteins - Transcription regulated by food availability - Operon: a single RNA molecule coding for multiple genes (Prokaryotes only) Tryptophan Operon (Trp Operon) - Five genes needed to make tryptophan in one RNA strand (EDCBA) - Encodes enzymes for tryptophan biosynthesis - Regulated by single promoter. - Trp operon promoter has two protein bound states: 1. Bound by RNA polymerase: Trp gene expression ON 2. Bound by tryptophan repressor protein: Trp gene expression OFF - Operator: specific DNA sequence of promoter which the repressor protein binds to - Trp Operon Promoter binding to Trp repressor: (i) RNA polymerase cannot bind because the promoter access is blocked (ii) Negatively regulates Trp Expression - Regulation of Trp repressor: must have two tryptophan bind to it o Low Trp levels: no trp binding to repressor, promoter region free for polymerase to bind o High Trp levels: 2 trp bind to repressor, repressor binds to operator, promoter access is blocked - Trp Repressor: Helix-turn-Helix DNA binding motif binds in major groove of DNA double helix. When Trp binds, the repressor undergoes a conformational change to be able to fit into this major groove. Lac Operon: - Three genes for transporting lactose and it’s breakdown (encodes -Galactosidase: break down lactose  glucose + galactose - Enables the use of lactose in absence of glucose - Dual regulation: positive and negative 1. Activator: Catabolite activator protein (CAP) promotes Lac in low glucose, high lactose 2. Repressor: Lac Repressor Protein inhibits Lac expression in low lactose conditions 3. Low lactose levels: Lac repressor bound to operator (expression Is off but not completely because we need -Galactosidase) 4. 5. Allolactose binds to Lac Repressor: o Conformational change o Decrease DNA binding activity release from the operator o Release from operator - RNA polymerase binding is inefficient to Lac Promoter, CAP has an helix-turn-heliz DNA binding domain to help RNA polymerase efficiency - CAP binding regulated by low glucose: 1. Low glucose  higher cyclic AMP (cAMP) 2. cAMP binds to CAP: conformational change leads to increased DNA-binding activity so now CAP binds to CAP-binding Site. 3. CAP recruits RNA polymerase Lecture 3 Recap: Prokaryotic Gene Regulation - Ligands can bind to remove or allow the regulatory protein to bind - Negative Regulation: competition between RNA polymerase and repressor for promoter binding… (i) Ligand bind to remove the repressor protein thus turning gene on (ii) Ligand is removed from repressor protein, thus turning gene on. - Positive Regulation: activator protein recruits RNA polymerase to the promoter to activate transcription (i) The ligand binds to the activator protein, removing it, switching the gene off (ii) The ligand is removed from activator protein, protein unbinds, thus – switching gene off Regulatory elements can also be found: - Far upstream of gene - Downstream of gene (eukaryotes) - Within gene (introns, eukaryotes only since prokaryotes don’t have introns)  DNA looping (Lac repressor is a tetramer and can bind to two operators simultaneously. Bacteriophage Lambda: - Virus that infects bacterial cells - It attaches to host cell and injects its own lambda DNA - Positive and negative regulatory mechanisms working together (the two preoteins repress each other’s synthesis) - Two states of existence: 1. Prophage o Favorable growth conditions o Hidden ninja state o All it does is replicate Viral DNA using the host bacteria’s mechanisms 2. Lytic Pathway o Host cell is damaged (induction) o Proteins from the viral DNA are now synthesized o Lambda DNA replicated and is packaged o Cell lysis releases large number of new viruses - Two state1s are regulated by two proteins: Lambda Repressor Protein (cI) and Cro Protein o cI : blocks Cro synthesis, activated own synthesis, little bacteriophage transcription (binds to other gene operators to stop promoters)(in the prophage state) o Cro: blocks cI synthesis, allows own synthesis, extensive bacteriophage DNA transcription (lytic state). Transcriptional Circuits: 1. Positive feedback loop (cI activates it’s own synthesis): creates cell memory that lasts generations caused by transient signal. 2. Negative Feedback loop (Trp binds to repressor to stop Trp synthesis) 3. Flip-Flop Device (Cro/cI switch) 4. Feed-Forward Loop Synthetic Biology - Constructing artificial circuits and examine their behavior in cells - Transcriptional Circuits: combinations of regulatory circuits combine in eukaryotic cells to create complex regulatory networks - Example: creating a simple gene oscillator (on/off) using a delayed negative feedback circuit (repressillator) - Circadian gene regulation o Tim and Per protein form heterodimer o Dimer dissociates in cytosol o Tim and Per enter nucleus. o Per suppresses Tim and Per expression o Per is phosphorylated and degrades o Tim and Per expressed, form heterodimer o Tim also degrades in light Transcription Attenuation - Transcription attenuation is a premature termination of transcription. - A structure interferes with RNA polymerase (regulatory proteins can bind to RNA) - Riboswitches are in PROKARYOTES (some plants and fungi) – they regulate gene expression Riboswitches: - Short RNA sequences - Change conformation when bound by small molecule - Prokaryotic riboswitch regulates purine (AG) biosynthesis o Low guanine levels: purine biosynthesis gene is on o High guanine levels: guanine binds to riboswitch, riboswitch has conformational change, RNA polymerase terminates transcription – synthesis is off Lecture 4 - General transcription factors are proteins which help in transcription initiation (position the RNA polymerase at the eukaryotic promoter site: used by RNA polymerase II) - Eukaryotic Gene Regulation: 1. RNA polymerase II transcribes protein encoding gene, it requires 5 transcription factors: (prokaryotes only need sigma factor) (i) TF II B (ii) TF II D (iii) TF II E (iv) TF II F (v) TF II H 2. Eukaryotic DNA is packaged into chromatin 3. Mediator: intermediate between regulatory proteins and RNA polymerase 4. Gene: regulatory part and the coding part 5. Eukaryotic gene regulation requires many proteins, some of which form protein complexes 6. Coactivators and Corepressors which assemble on the DNA-bound gene regulatory proteins but don’t bind directly to the DNA 7. Activator Proteins: a. DNA binding Domain: recognizes the DNA sequence b. Activation Domain: accelerates rate of transcription 8. Activator proteins attract, position, and modify, General TFs, the mediator, and RNA polymerase II directly or indirectly (modifying chromatin structure) Chromatin - DNA wound around a histone octamer: ( H2A, H2B, H3, H4) x2 - Linker DNA is the strand of DNA between two chromatin structures - Nucleosome: 200 nucleotide pairs and the core histones - Wayts activator proteins alter chromatin: 1. Nucleosome sliding: Uses ATP to unroll it using chromatin remodelling complex 2. Remove histones by chaperone (ATP dependent) 3. Replace histones to unwind (chaperone removes H2A and puts in H2Az – ATP dependent) 4. Recruit histone modifying enzyme to produce specific patterns of histone modification called the histone code: a. Kinase enzyme adds phosphate by phosphorylation b. Acetyltransferase enzyme adds acetyl by acetylation c. Methyltransferase adds methyl by methylation o Writers modify the tails o Readers recognize the modifications and provide meaning to code Histone Code Example: (histone code can spread using reader righter mechanism 1. Histone AcetylTransferase (HAT) is attracted to activator protein on chromatin 2. HAT acetylates K9 of H3 and K8 of H4 3. Histone Kinase is attracted by activator protein 4. HK phosphorylates S10 of H3 (must occur after HAT acetylates K9 of H3) 5. Serine signals for HAT to acetylate K14 of H3 6. Code done - transcription initiation is written 7. TF II D and chromatin remodelling complex bind to acetylated histone tails (initiate transcription) Transcriptional Repression: - Eukaryotic repressor proteins use mechanisms to inhibit transcription (not competition)) 1. Interfere with activator binding (bind on the activator binding site)4 2. Interfere with activator function: binds to activator protein 3. Block assembly of transcription factors 4. Recruit chromatin remodelling complex to rewind the DNA, making TATA inaccessible. 5. Recruit histone deacetylases 6. Recruit histone methyl transferases The spread of the histone code by reader-writer complexes: 1. DNA methylase enzyme attracted by reader, methylates cytosines 2. DNA methyl-binding proteins bind to these methyl groups and stabilize structure (inherited by epigenetic inheritance) Epigenetic Inheritance: - Ability of daughter cell to retain memory of parental gene expression pattern Lecture 5 Transcriptome Analyses provides signature of cell state - Response to extracellular stimuli - Disease states (compare chemically treated HL-60 model-leukemia cells to see if chemical helps – IC50 is the amount of compound needed to lower leukemia by 50%) Post-Transcriptional Gene regulation Eukaryotic RNA processing coupled to transcription: - Covalent modification of RNA ends - Removal of intron sequences RNA Capping - Guanine nucleotide to 5’ end of pre-mRNA - Cap is bound by the Cap-Binding Complex (CBC) - Functions: (i) Helps in RNA processing and export from nucleus (ii) Important in translation of mRNA in cytosol (iii) Protects mRNA from degradation RNA Splicing: - Eukaryotic genes are made up of exons (coding) and introns (non-coding( - Untranslated Regions (UTR) are in exons but aren’t coded - Introns are removed by RNA splicing - Alternative splicing is when an RNA transcript is spliced differently to make different proteins from the same gene - Splicosome: enzyme complex made up of RNAs and Proteins which splices RNA - Exon Junction Complexes (EJCs) are bound to the site of splicing as a marker Alternative Splicing in Drosophilia - Female X:A ratio = 1.0 - Male X:A ratio: 0.5 - Three genes: (i) Sex-lethal Splicing repressor (ii) Transformer: Splicing activator (iii) Doublesex: Sex gene expression regulator - Males (X:A=0.5) o Sex-Lethal (Sxl) has no regulated splicing but has a non-functional splice product o Transformer (Tra) has no regulated splicing but has a non-functional splice product o Doublesex (Dsx) is spliced but not regulated with a functional protein – Dsx represses female gene expression - Female (X:A=1.0) o Transient signal produces Sxl protein o Sxl protein represses splicing of Sxl and Tra – the nonspliced proteins are functional o Tra splicing is repressed by Sxl and so functional nonspliced protein is made (activates Dsx splicing) o Dsx protein is spliced and functional: represses male gene expression 3’ Polyadenylation: - More complex than transcription termination in prokaryotes (guanine riboswitch) - Signals encoded in genome - Aid in RNA export and translation - RNA polymerase transfers protein complexes to RNA: o CstF – cleavage stimulating factor o CPSF cleavage and polyadenylation specificity factor - Poly-A Polymerase (PAP) adds ~200 A nucleotides at the 3’ end of RNA from ATP (not in the genome). This tail is bound by poly-A binding proteins RNA Processsing and Transcription - During transcription elongation, C-Terminal Domain (CTD) of RNA polymerase binds RNA processing proteins and transfers them to RNA – this is regulated by the phosphorylation of RNA polymerase RNA Transport from Nucleus: only 1/20 get exported - Mature mRNA must have: o Cap Binding Complex – CBC o Exon Junction Complexes – EJC o Poly-A Binding Proteins - Immature mRNA has snRNPs (RNA splicing proteins) – degraded in nucleus by exosome HIV virus - Reverse transcriptase changed the virus RNA and integrates it into host cell DNA - HIV proteins are expressed and virus multiplies (30 different mRNAs because of alternative splicing from HIV – those that retain introns aren’t exported) Rev Protein: synthesis from HIV virus - mRNA has no introns - Binds to Rev Responsiv Element (RRE) in unspliced RNA - Interacts with nuclear export receptor - Directs export of unspliced mRNA Lecture 6 Eukaryotic mRNA quality control - Incomplete or damaged mRNA is degraded to prevent aberrant toxic proteins - Translation initiation machinery recognizes 5’-cap and poly-A tail by eukaryotic initiation factors: o eIF4E binds to 5’-cap (recruit small ribosomal complex to initiate translation at the first AUG downstream f 5’ cap) o eIF4G binds to the poly-A binding protein - mRNA is scanned for nonsense STOP codons (wrong places) - EJCs bind to where introns should be removed, Ribosome breaks the EJCs off. If the intron left with a STOP, translation doesn’t occur, EJC stays in place – signalling for exosome to breakdown the mRNA (mediated by Upf proteins) - Degradation is regulated by two mechanisms involving gradual Poly-A Tail Shortening (exonuclease deadenylase chops off the adenylase at the 3’ end – like the fuse for dynamite, when poly-A tail is down to ~25 nucleotides in humans, two things can happen: 1. Decapping followed by rapid 5’-3’ degradation 2. Rapid 3’ to 5’ degradation (no decapping) - Cytoplasmic poly-A elongation can stabilize mRNA, aelso proteins like aconitase interfere with poly-A shortening Translation Recap - tRNAs match amino acids to codons (a codon is 3 consecutive nucleotides) in the mRNA genetic code - mRNA genetic code is read by ribosomes (made up of 50+ proteins and RNA molecules) - Amino acids are added to the C-terminal of the growing polypeptide chain (Proteins are made in the N  C terminal direction) - Initiator tRNA with GTP and EIF2 searches for the first AUG in the UTR Prokaryotic mRNA quality control - Ribosome stalls on broken and incomplete mRNAs – cannot release - tmRNA is recruited to A site with an alanine amino acid - tmRNA acts as a new mRNA – releasing the old stuck one - Alanine is added on polypeptide by the t-part of the tmRNA - 10 codons of tmRNA are translated - The 11 amino acid (alanine + 10) is recognized by proteases which breakdown the protein - Exonucleases degrade most mRNAs Transferrin: - Imports iron into cell - Needed when cellular iron is low - mRNA is stabilized by cytosolic aconitase which binds to the 3’ UTR - Aconitase binds iron causing a conformational change - mRNA is released – 3’ UTR endonucleolytic cleavage site is exposed, polyA is removed, mRNA is degraded Competition between mRNA translation and degradation - Deadenylase shortens poly-A tail but first binding to the 5’ cap - eIFs bind 5’ cap for translation - micro RNA (miRNA) regulate mRNA stability by base-paring with mRNA. It undergoes 5’ cap and poly-A, synthesized by RNA polymerase II, associates with protein complex called: RNA-induced silencing Complex (RISC) – binds with complementary nucleotide sequences with the help of Argonaute protein 1. Cropping: cleavage of mRNA 2. Dicing by dicer enzyme (removing loop) o Argonaute finds an extensive match for RISC, binds to it on mRNA, slices it, Uses ATP to cut in half, RISC is released, mRNA is rapidly degraded from the cut ends o Less extensive match found, RISC binds to it and blocks other ribosomes – translation blocked and reduced, mRNA transferred to P-bodies and degraded RNAI - RNA intereference (RNAi) is found in fungi, plants, and worms – defense against foreign RNA molecules - Many viruses use double-stranded RNA - RNAi destroys double stranded RNA - Dicer protein complex cuts the double stranded RNA - siRNA (small interfering RNAs) interact with Argonaute and RISC, follow miRNA route to destroy the double-stranded RNA - siRNA can regulate transcription by interacting with argonaute and RNA-Induced Transcriptional Silencing Complex (RITS) o interacts with newly transcribed RNA (binds to it) o recruits chromatin modifying enzymes (histone methylation for transcriptional repression) Lecture 7 Both eukaryotes and prokaryotes use translational control mechanisms in to regulate protein expression. Shine-Dalgarno: ribosomal binding site on prokaryotic mRNA which is 6 bases long recruits ribosome to the mRNA to initiate protein synthesis by aligning it with the start codon Prokaryotes: - six nucleotide Shine-Delgarno sequence upstream of AUG start codon - correctly positions AUG in ribosome + provides translational control mechanisms - Mechanism 1: RNA binding protein blocks access to the Shine-Dalgarno sequence - Mechanism 2: temperature related RNA structure (stem loop melts, exposes SD, translation occurs) - Mechanism 3: Riboswitch – small molecule (adenosyl methionine) causes structural rearrangement of RNA, thus blocking SD. - Mechanism 4: antisense RNA produced elsewhere base-pairs with mRNA blocking SD Eukaryotes: - No Shine-Dalgarno sequence - Translational repressors can bind near AUG initiator and inhibit translation - Example: Iron and aconitase o Ferritin: binds and releases iron in a controlled manner, only needed when iron levels are high. o Iron Starvation: Aconitase binds to the ferritin mRNA, blocking translation o Excess Iron: Iron can bind to aconitase, causing a conformational change so it releases the ferritin mRNA, then translation is allowed and Ferritin is made. - Repressor proteins can interfere with 5’ cap and 3’ poly-A tail interactions (inhibits translation) - Small RNA molecules can regulate translation (miRNAs) - Regulation of eukaryotic initiation factors (eIFs) o eIF2 is crutial in translation initiation – in a complex with GTP, it recruits initiatior methionyl tRNA to small ribosomal subunit – this subunit binds to 5’ end of mRNA and scans for AUG o AUG found, eIF2 cuts the P from GTP making GDP (hydrolysis) causing a conformational change in eIF2, thus releasing it from subunit (eIF2 bound to GDP is inactive) o eIF2 requires eIF2B to exchange GDP to GTP (Guanine Nucleotide Exchange GEF) – regulated by phosphorylation o phosphorylated eIF2 turns eIF2B to an inactive complex (can’t change GDP to GTP) by sticking to it o eIF2 > eIF2B – all eIF2Bs are inactive and translation is reduced Protein Regulation - Proteins must fold properly - They are covalently modified with chemical groups - The proteins interact with other proteins and small molecules (cofactors) - Folding can occur as translation goes on. The hydrophobic ones get buried in the interior core - Chaperons (special proteins) for protein folding) are called heat-shock protens (hsp) since they are made to high amounts at high temperatures. - Chaperones are there normally, but are there in higher amounts when high temperatures
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