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BIO1140 Textbook Exam Notes 2.docx

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Kathleen Gilmour

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BIO1140 Textbook Exam Notes 2 DNAReplication and Important Background 1. Griffith Experiment: -R is avirulent strain, S is virulent t strain. -Heat killed S strain, the living R strain picked up genetic material of S strain. Mouse Dies. -Transforming Principle: Viruses can pick up genetic material from other virusus. 2.Avery, McCarty, McLeod Experiment: -They took the Griffith experiment, but destroyed either the: Protein, DNA, RNA, and injected them all. Then when the DNAwas destroyed the mouse survived. This was because DNA was the heredity information of the viruse. 3. Hershey and Chase: -Labelled Protein and DNAwith a radio isotope. -When viruse injected its genetic material into the cell, it was the labelled DNAthat turned out to be the genetic matrial. The protein just housed the DNA. 4. Nucleotide vs. Nucleoside: -Nucleotide: Phosphate + Pentose Sugar + Nitrogenous Base (Purine & Pyrimidine) -Nucleoside: Pentose Sugar + Nitrogenous Base (Purine & Pyrimidine) 5. Purine vs. Pyrimidine: -Purines:Adenine & Guanine -Pyrimidines: Cytosine, Uracil, Thymine -20%A—20%T or U. 30%G—Cytosine (Chargaff’s Rule) -URACIL REPLACES THYMINE IN RNA 6. DNAReplication (DNAis read 3’—5’, but layed out 5’—3’) -Helicases unwind DNAto expose template strands for new DNAsynthesis ~The exposed single-stranded segments of DNAbecome coated with SSBP (Stabalizes DNAin single chain form) ~topoisomerase relieves overtwisting and strain of DNAahead of replication fork. (In circular DNA) -RNAprimers provide starting points for DNApolymerase to begin synthesizing a new DNA chain ~DNApolymerase can add nucleotides only to the 3’end of an existing strand ~Primers are put down so the RNAstrand can attach to the unwinded DNA -One new DNAstrand is synthesized continuously ; the other discontinuously ~Leading strand; continuous replication ~Lagging strand; discontinuous replication (short added lengths=Okazaki Fragments) -Multiple enzymes coordinate their activities in DNAreplication ~Helicase, Primase, and DNApolymerase ~DNALigase: Seals nicks left after RNAprimers are replace with DNA -Telomerases solve a specialized replication problem at the ends of linear DNAmolecules ~Abuffer of non-coding DNAat the ends of each eukaryotic chromosome “telomeres” ~Enzyme telomerase maintains buffer by adding telomerase repeats. ~Telomere repeats added to the template strand -DNAreplication Begins at Replication Origins (Sites Recognized by Proteins that bind to DNA) 7. DNAOrganization in Eukaryotes and Prokaryotes: -In Eukaryotes Histones and Non-Histones are associated with DNAstructure and regulation. ~Chromosomal Proteins ~Chromatin are the building blocks of a chromosome. -Histones Pack Eukaryotic DNAat successive levels of organization ~Positively Charged basic Proteins ~5 types of Histones in eukaryotes H1, H2A, H2B, H3, H4 ~Pack DNAmolecules into narrow confines of cell nucleus, and regulation of DNA activity -Histones and DNApacking ~Nucleosome packed by 2 molecules of H2A, H2B, H3, H4 form an octomer -Histones and Chromatin Fibres ~1 H1molecule binds to the entrance/ exit of the core particle and linker DNA ~Solenoid, is a group of approximately 7nucleosomes before it turnes.Also known as the 30nm chromatin fibre ~Nucleosome wrapped into solenoids are better protected ~DNA must unwind entirely from solenoids and nucleosomes when becoming active -Packing at still higher levels: Euchromatin and Heterochromatin ~Loosely packed regions of DNAare known as Euchromatin (eu = regular) ~Densely packed regions of DNAare known as Heterochromatin (hetero = different) -Many Nonhistone Proteins have Key Roles in the Regulation of Gene Expression ~Proteins associated with DNAthat are not histones are Nonhistone proteins -DNAIs Organized More simply in Prokaryotes than in Eukaryotes ~DNAcircle is packed into an irregular shaped mass called the nucleoid 8. The New Model Proposed That Two Polynucleotide Chains Unwind into a DNAhelix -Watson and Crick constructed the Double stranded helix model using Wilkins and Franklins x- ray diffraction data and Chargraffs chemical analysis. -DNA’s Back bone is a Sugar Phosphate backbone. 9. Types of DNA:A,B,C,D,Z -B standard -Z DNAis GC-rich -Natural DNAforms a right handed helix. Gene Structure and Expression 1. The Pathway from Gene to Polypeptide Involves Transcription and translation: -Transcription: DNAis made into a complementary RNAcopy -Translation: RNAis read to assemble amino acids into polypeptide chains -Central Dogma: DNARNAProtein 2. The Genetic Code is written in 3 Letter Words using a 4 Letter Alphabet: -AUG (methionine) is the start codon. 3 codons code for termination codons: UAA, UAG, UGA -There is only one correct reading frame for each mRNA 3. DNAReplication vs. Transcription: -In a given gene, only one of the two DNAnucleotide strands acts as a template for synthesis of a complimentary copy, instead of both, as in replication. -Only a relatively small part of a DNAmolecule—the sequence encoding a single gene—serves as a template, rather than all of both strands, as in DNAreplication. -RNApolymerase catalyzes the assembly of nucleotides into an RNAstrand, rather than the DNApolymerase that catalyzes replication -The RNAmolecules resulting transcription are single polynucleotide chains not double polynucleotide chains, as in DNAreplication. 4. DNATranscription: -RNApolymerases work like DNApolymerases but require no primer. -Specific sequences of nucleotides in the DNAindicate where transcription of a gene begins and ends. ~Promoter: RNApolymerase binds to the promoter, where transcription begins ~Transcription Unit: Part of the gene that is to be transcribed into RNA ~Terminators: Where transcription ends. (No terminator sequence in eukaryotes, proteins bind to polyadenylation signal and cleave pre-mRNA). ~TATAbox: Important in transcription initiation. It is a regulatory DNAsequence found in the promoters of many eukaryotic genes transcribed by RNApolymerase 2. 5. RNAPolymerase: -RNApolymerase 1transcribes rRNAin the nucleolus (part of the nucleus) -RNApolymerase 2 transcribes mRNAand most snRNA’s ~RNApolymerase 2 cannot recognize the promoter sequence, instead proteins called transcription factors recognize and bind to the TATAbox, then recruit the polymerase. -RNApolymerase 3 transcribes tRNA, 5S rRNA, some snRNAand scRNA’s 6. Processing of mRNAs in Eukaryotes: -Eukaryotic Protein Coding GenesAre Transcribed into Precursor-mRNAs That Are Modified in the Nucleus -Modifications of Pre-mRNAand mRNAEnds: ~mRNACapping: a 7MeG cap is added to the 5’end (connected to chain by 3 P groups). Cap is the initial attachment site for mRNAs to ribosomes ~Polyadenylation: Poly(A) tail is added to 3’end ~ 50-250 adenine nucleotides AAA..Etc ~Modifications made to protect mRNA from RNA-digesting enzymes -Sequences Interrupting the Protein-Coding Sequence ~Introns (removed during mRNA splicing by spliceosomes) ~Exons (AminoAcid coding sequences) ~Alternative Splicing: The process by which the exons of the RNAproduced by transcription of a gene are re-connected in multiple ways during RNAsplicing. Allows us to produce many more proteins 7. Translation: mRNA-Directed Polypeptide Synthesis: -tRNAs are small RNAs That BringAminoAcids to the Ribosome for addition to the polypeptide chain ~Anticodon:Asequence of three nucleotides in tRNAthat binds to the complementary codon in messenger RNAto specify an amino acid during protein synthesis ~Wobble Hypothesis: First two nucleotides of the anticodon and codon must match exactly. The third nucleotide has more flexibility. Ex) a tRNAreading 3’AAA5’ can read both codons 5’UUU3’& UUC for the same protein ~Codon Bias:Although several codons code for a single amino acid, an organism may have a preferred codon for each amino acid. -Addition of AminoAcids to their Corresponding tRNAs ~Aminoacylation: Addition of an amino acid to a tRNA -Ribosomes are rRNA-Protein Complexes that Work asAutomated ProteinAssembly Machines ~Ribosome: Made of a large & small subunit. Each subunit is made up of rRNAand ribosomal proteins. Large(35S rRNA+50proteins), Small(28S rRNA+31proteins. ~Asite: Incoming aminoacyl-tRNAenter the ribosome ~P site: Where the tRNAcarrying polypeptide chain is bound ~E site: Where an exiting tRNAbinds when leaving the ribosome ~Peptidyl Transferase: Catalyses the movement of polypeptide chain from PA -Multiple Ribosomes Simultaneously Translate a Single mRNA(Polysomes) ~Increases the overall rate of polypeptide synthesis from a single mRNA -Newly Synthesized Polypeptides Are Processed and Folded into Finished Form. ~Removal of one or more AA’s from the protein chain + addition of organic group ~Chaperones: (Helper Proteins)Assist folding process by combining with folding protein 8. 3 Stages of Translation: -Initiation: Brings the Ribosomal subunits an mRNA& first aminoacyl tRNAtogether. -Elongation: Polypeptide chain grows during the elongation stage of translation. -Termination: Releases a completed polypeptide from the ribsosome 9. Protein Targeting: -Finished Proteins Contain Sorting Signals that Direct Them to Cellular Locations ~Proteins with no signals remain in the cytoplasm. ~Proteins with signals are directed to specific locations -ER Bound ~Signal peptide emerges from ribosome. SRP binds and translation stops. ~SRP binds to the SRP receptor. Translation resumes. Polypeptide enter the rough ER lumen and binds to signal peptidase ~Signal peptidase cleaves the signal peptide from the growing polypeptide ~Translation of the mRNA is complete; ribosomal subunits are about to dissociate ~Vesicles are used to transport proteins from the ER through he stacks of the Golgi to the plasma membrane. 10. N terminus vs. C terminus -N Terminus: NH th3t is on the end of a polypeptide chain. -C Terminus: COO that is on the end of a polypeptide chain. -Usual Signal Location within a Protein: ~N-Terminal: ER, Mitochondria, Chloroplast ~C-Terminal: Peroxisome, Vacuole ~Internal: Nucleus 11. Base-Pair Mutations CanAffect Protein Structure and Function: -Base-Pair Substitution Mutations: ~Involve a change of one particular base to another  This will change a base in a codon ~Missense Mutation:Amutation alters the codon itself specifying for a different amino acid ~Nonsense Mutation: Changes anAAcoding codon to a termination Codon resulting in a premature stop ~Silent Mutations:Achange in a base that does not result in a coding for a differedAA ~Frameshift Mutation: Starts coding at the wrong base, resulting in a non-functional protein b/c of significant changes to theAAsequence Control of Gene Expression: 1. Regulation of Gene Expression in Prokaryotes: -The Operon is a Unit of Transcription ~Operon: Acluster of genes and the DNAsequence involved in their regulation ~Promoter: Region where RNApolymerase starts transcription. ~Operator: Ashort segment of DNAto which a regulatory protein binds. (Controls the operation of the genes next to it) ~Repressor: Aregulatory protein, when active prevents genes of the Operon from being expressed ~Activator:Aregulatory protein, when active stimulates the expression of genes ~Inducer: Something that will deactivate the repressor. 2. The Lac Operon (Negative Gene Regulation): -The Lac Operon for Lactose Metabolism is Transcribed When a Inducer Inactivates a Repressor ~Lactose metabolism in E. Coli requires three genes Lac Z+Y+A= Operon ~Lac Operon controlled by a regulatory protein termed “Lac Repressor” ~When Lactose is absent from the medium, active Lac repressor binds to the operator, blocking the RNApolymerase from binding to the promoter; as a result transcription doesn’t occur. ~When lactose is added to the medium, the Lac Operon is turned on and all threes enzymes are synthesized rapidly. Excess present lactose is converted to the inducer Allolactose.Allolactose binds to the Lac repressor, inactivating it so that it cannot bind to the operator. This allows RNApolymerase to bind to the promoter, and transcription of the Lac Operon occurs. Translation of the mRNAproduces the three lactose metabolism enzymes. ~i.e. When a substance which needs to be broken down by enzymes enters the system. Some of that substance acts as an inducer to the repressor and inactivates the repressor. This allows the enzyme to be transcribed for. This will create the enzyme to break down the substance that has entered the system. ~Lac Operon is referred to as an inducible Operon 3. The Lac Operon (Positive Gene Regulation): -Transcription of the Lac Operon isAlso Controlled by a Positive Regulatory System. ~CAP is an activator that stimulates gene expression. ~When lactose is present and glucose is low or absent. cAMP binds to the CAP, activating it.Active CAP binds to the CAP site and recruits RNApolymerase to the promoter. Transcription then occurs. ~When lactose is present and glucose is present, cAMP levels are low.As a result, CAP is inactive and cannot bind to the CAP site. RNApolymerase then is unable to bind to the promoter and no transcription occurs. -The repressor is off when lactose enters the system (via allolactose). It is just that cAMP levels are low so the CAP is inactive. RNApolymerase cannot bind to the CAP site because cAMP has not binded to the CAP (cAMP produces the energy for the CAP to bind to the CAP site) 4. The Trp Operon: -Transcription of the trp Operon Genes for tryptophan Biosynthesis is Repressed When TryptophanActivatesARepressor. ~Tryptophan is anAA, if not present in the E. coli, it must be made ~When tryptophan is absent from the medium, the TRP repressor is inactive in binding to
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