Lecture 9 – DNA SupercoilingDNA SupercoilingMathematical Treatment of DNA SupercoilingLinking NumberEukaryote DNA Has Negative SupercoilingDNA Supercoiling – Main PointsSupercoiling Affects Electrophoretic MobilityRelaxing DNA – TopoisomerasesFunctions of TopoisomerasesType I TopoisomerasesMechanism of Type I TopoisomerasesType II TopoisomerasesFunction of Type II TopoisomerasesDNA GyraseMechanism of DNA GyraseLecture 10 – Chromosome StructureBacterial vs Eukaryote Chromosome StructureThe Compaction ProblemChromatinEukaryotic Chromosomes & the Cell CycleChromatin Compaction VariesLevels of Chromatin StructureNucleosomesHistones Bend the DNA StructureChromatinRegulation of Chromatin StructureLecture 11 – DNA Replication in BacteriaDNA Replication in BacteriaChemistry of DNA ReplicationAccuracy of DNA ReplicationLeading to Mistakes and Correcting ThemInitiation of DNA Replication in E. coliProteins Required for ReplicationReplisomeInitiation of ReplicationLecture 12—DNA Replication in BacteriaElongation of DNA ReplicationE. Coli DNA Polymerase IIIDNA Pol III CoreY Subunit: The Clamp LoaderRNAse H—Removal of RNA PrimerE. coli DNA Polymerase I (Pol I)DNA LigationTermination of Replication in E. coliControl of DNA Replication in E. coliDNA Replication in EukaryotesEukaryotic Mitotic Cell CycleTelomeres and TelomeraseLecture 13—Transcription in BacteriaTranscription of DNA into RNAWhat is a gene?Types Of RNARNA vs DNADenaturation of RNARNA StabilityRNA Secondary and Tertiary StructureRNA StructureStructure of tRNAStructure of 16S rRNAStructure of Catalytic RNA—RibozymesLecture 14 – TranscriptionTranscription in BacteriaConvention and NomenclatureE. coli DNA-Dependent RNA PolymeraseThe Chemistry of RNA SynthesisReplication vs. Transcription in E. coliThe promoterPromoter StrengthInitiation of TranscriptionTransition to Open ComplexAbortive Transcripts and ScrunchingTranscription ElongationEditing Function of RNA PolymeraseTranscription Termination*Transcription and Translation are coupled*Lecture 15—Transcription in EukaryotesBacteria vs Eukaryote TranscriptionTranscription in EukaryotesConserved Subunits of RNA PolymerasesBacterial vs Eukaryotic RNA PolymerasesThe C-terminal Tail Domain (CTD) of Pol IIPol II-Mediated TranscriptionRNA Pol II Core PromoterFormation of the Pre-Initiation ComplexCore Promoter—Pre-initiation ComplexTransition to the Open ComplexTranscribing Through NucleosomesThe Mediator ComplexmRNA Processing in Eukaryotes5’ Cap of Eukaryotic mRNAsCapping Enzymes are Associated with CTD3’-Poly-AdenylationMature (Translatable) mRNA in EukaryotesSplicing of Eukaryotic TranscriptsRNA splicing in Higher EukaryotesLecture 16—RNA Splicing and Processing (Translation)Consensus Sequences for Splice SitesErrors in Splicing Lead to MutationsThe Splicing ReactionStructure of the Excised Intron (Lariat)The SpliceosomeThe Spliceosome Active SiteCTD Coordinates Splicing During TranscriptionTransport of Mature mRNA to the Cytosol4 Basic Types of Alternative SplicingBacteria Transcription—OperonBacterial Transcription-- rRNA and tRNAEukaryotic Transcription—rRNA and tRNAAntiterminationLecture 19—The Genetic Code / Translation in BacteriaThe Reading FrameTranslation – Aminoacyl tRNATranslation—OverviewThe RibosomeThe Genetic CodeBacterial TranslationBacterial Translation InitiationPeptidyl Transferase ReactionRibosome = RibozymeElongation FactorsFidelity of the RibosomeTranslation TerminationLecture 18—Translation in EukaryotesTranslation in EukaryotesTranslation Initiation in EukaryotesBacterial vs. Eukaryotic TranslationThe Second Genetic CodeAminoacyl-tRNA SynthetasesReverse TranscriptionViral ReplicationReverse TranscriptaseRetroviral Infection of a Eukaryotic CellHIV-1 Reverse TranscriptaseReverse Transcription for Cloning GenesLecture 19—Mutation and RepairDefinitions:Point MutationsInsertion/Deletion Mutations (Indels)Spontaneous Mutation—FidelityTautomerism Causes MispairingSlipped Strand MispairingStrand Slippage Due to MisalignmentSpontaneous Deamination of CytosineSpontaneous Hydrolysis of PurinesOxidative DNA damageInduced Mutations—MutagensUltraviolet Light Causes Thymine DimersChemical Modification of DNAIonizing RadiationMutations via DNA Intercalating CompoundsIncorporation of Nucleotide AnalogsDNA repair in BacteriaMismatch Repair—MutHLSMethyl-directed Mismatch Repair (MMR)Mismatch RepairBase Excision Repair—Udg/UNGBase Excision Repair—DNA GlycosylasesNucleotide Excision Repair (NER)DNA Photolyase Repair of Thymine DimersThe SOS ResponseTranslesion SynthesisMCB 250 Exam 2Lecture 9 – DNA Supercoiling DNA Supercoiling- DNA is compacted through supercoiling- The more supercoiling  greater the impaction- Bacterial cells DNA contain a covalently closed circle- Chromosomal DNA is maintained in a NEGATIVELY supercoiled conformation by the protein DNA Gyrase, but since this puts topological constraints (cannot be altered) on the DNA, the cell regulates this- Under normal conditions, the E. coli chromosome is heavily supercoiled, but can be partially or completely relaxed - Supercoiling is a universal property of how helical objects respond to twisting Mathematical Treatment of DNA Supercoiling- Linking number: the total # of times B-DNA strands cross one anothero Used to describe the topological state of covalently closed, circular DNA (cccDNA)o Invariant property of cccDNA o Composed of 2 parameters, twist (Tw) and writhe (Wr)Lk = Tw + WrTw = # of helical termsWr = # of times the helix crosses itself o Determined by dividing the total base pairs of the molecule by the relaxed bp/turn, which in B-DNA is 10.4Lk = bp/10.4 ~ bp/10Linking Number- Lk0 = Twist (Tw), in its relaxed state- Topoisomerases: enzymes that change the linking number of the cccDNA molecule by altering the twist.o Then the conformation is not energetically stable and the DNA twists into supercoils --- Writhing (Wr)o Decrease in Lk  negative Supercoiling Relaxed B-DNA  Supercoiled DNA  Underwound DNA Eukaryote DNA Has Negative Supercoiling- Experiences a low level of negative supercoiling- Wrapping DNA (right handed helix) around nucleosomes in a left-handed spiral decreases Lk and stores potential energy for strand separation- Because eukaryotic chromosomes are very large, segments in the middle may act as if their ends are anchoredDNA Supercoiling – Main Points- DNA molecules can exist in different topological states that are distinguished