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FLC BIO 455 - CHAPTER 6 • How Cells Read the Genome

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Chapter 6Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Elongation is Coupled to RNA ProcessingSlide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36Slide 37Slide 38Slide 39Slide 40Slide 41Slide 42Slide 43Slide 44Slide 45Slide 46Slide 47Slide 48Slide 49Slide 50Slide 51Slide 52Slide 53Slide 54Slide 55Slide 56Slide 57Slide 58Slide 59Slide 60Slide 61Slide 62Slide 63Slide 64Chapter 6•How Cells Read the Genome: From DNA to ProteinTest Your Knowledge1. What are two major differences between transcription in prokaryotic and eukaryotic cells?2. RNA polymerase and DNA polymerase enzymes catalyze the “same” reaction, but there are some distinct differences in what is required to make them begin catalysis and end catalysis. What are these differences?3. Which is more accurate, DNA replication or RNA transcription? 4. Explain the proteins and mechanisms involved in the initiation of transcription5. What determines how many copies of a transcript (mRNA) are made?6. How are elongation and termination of the transcript regulated?Video overview of transcription“The Protein Players” - RNA polymerases, transcription factors, initiation factors, enhancers, repressorsProkaryotes?Prokaryotic transcription videoProkaryotic TranscriptionProkaryotes•Promoter – •Pribnow Box (also called the -10 element) – TATAAT•-35 element - TTGACA DNA Sequences Important to TranscriptionEukaryotes•Promoter –(asymmetrical sequence)•Basic core promoter –TATA box (TATAAA(A)); within 50bp upstream of start site; found in unicellular eukaryotes•Core promoter PLUS•Downstream promoters•Proximal promoter elements•Initiator sequences•Regulatory Elements/Response Element - Response elements are the recognition sites of certain transcription factors Most of them are located within 1 kb from the transcriptional start site.D •Enhancer elements -upon binding with transcription factors (activators), can enhance transcription; located either upstream or downstream of the transcriptional initiation site.D•Upstream enhancer elements•Downstream enhancers•Distal enhancer elements•Silencers - upon binding with transcription factors (repressors), can repress transcription. •InsulatorsGene Regulatory Networks – control the number and type of transcripts made by a cell.Simple core promoterUAS = upstream activator sequence RE = regulatory elements INR = initiator sequence DPE = downstream promoter elementsvideoSequences that can act as promoters (TATA is preferred)Proteins Involved in TranscriptionRNA PolymeraseGeneral (or Basal) Transcription Factors:TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH Transcription Factors that Bind to Regulatory ElementsHoloenzyme orInitiation ComplexTranscription Factors Have Common DNA Binding Motifs•Zinc finger•Helix-turn-helix•Leucine zipperRNA PolymeraseRecognizes and binds to TATA box; TBP + 10 TBP associated factors; position setTBP bends DNA ~80TBP bends DNA ~80o o and forces open the and forces open the minor groove.minor groove.Recognizes and binds to TATA box; TBP + 10 TBP associated factorsBinds and stabilizes the TFIID complexRecruits RNA pol II + TFIIF to the locationTwo subunits - RAP38 & RAP74. Rap74 has a helicase activity; RAP38 binds RNAPolII Two subunits - recruits TFIIH to the complex thereby priming the initiation complex for promoter clearance and elongation complex of 9 subunits. One w/ kinase activity; one w/ helicase activity; one is a cyclin (cdk7)Sequential Binding Model for assembly of preinitiation complex-30 +1TATAInrPolymerization of 1st few NTPs and phosphorylation of CTD leads to promoter clearance. TFIIB, TFIIE and TFIIH dissociate, PolII+IIF elongates, and TFIID + TFIIA stays at TATA.IIBEukaryotic RNA polymerase IITFIID}TBPTAFsIIBIIECTD of large subunit of Pol IIPol IIaor TBPIIAIIFhelicaseprotein kinaseIIHTATAInrIIAPol IIaIIFIIEpreinitiation complexTATAInrIIAIIBPol IIaIIFIIEATP hydrolysisinitiation complex, DNA melted at InrIIHIIH= PICActivated PICTranscription initiation in the cell often requires the local recruitment of chromatin-modifying enzymes, including chromatin remodeling complexes and histone acetylases - greater accessibility to the DNA present in chromatinRNA polymerase is also assisted by DNA supercoilingPhosphorylation of the carboxy terminal domain (CTD) of one of the subunits of RNA PolII; RNA polymerase II dissociates from the transcription factors and other protein complexes that were required for assembly and elongation begins Phosphorylation also promotes the accumulation of elongation factors – other proteins that arrest transcription long enough to recruiting RNA processing enzymesElongation is Coupled to RNA Processing•Capping•Splicing•PolyadenylationVideo of transcription and cappingRNA Capping enzymes:•Phosphatase•Guanyl transferase – GMP in 5’ to5’ linkage•methyltransferaseCBC – cap binding complex proteins also associate and protect the cap; Later they will direct transcript in its exit from the nucleusRNA SplicingHow Introns Are Identified:Consensus sequences at (5’ to 3’ direction)•5’ splice site•Lariate loop closure site of the intron sequence •3’ splice siteR=A or G,Y=C or UThe Spliceosome FormssnRNAs (U1, U2, U4, U5 and U6) and associated proteins = snRNPs•U1 binds to the GU sequence at the 5' splice site, along with accessory proteins/enzymes, •U2 binds to the branch site, and ATP is hydrolyzed;•U5/U4/U6 trimer binds, and the U5 binds exons at the 5' site, with U6 binding to U2;•U1 is released, U5 shifts from exon to intron and the U6 binds at the 5' splice site;•U4 is released, U6/U2 catalyzes transesterification, U5 binds exon at 3' splice site, and the 5' site is cleaved, resulting in the formation of the lariat;•U2/U5/U6 remain bound to the lariat, and the 3' site is cleaved and exons are ligated using ATP hydrolysis. The spliced RNA is released and the lariat debranches.Rearrangements that occur during splicing• U1 replaced by U6•BBP (branch binding protein) and U2•U5 complex branch forming enzymes in U6 and U2Allows for “check and recheck” at each splice site.Why is splicing so accurate?Introns are small-large; Exons are about 150bp longExons might be easily identified, while introns probably couldn’t be.As the RNA is being transcribed, SR proteins


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FLC BIO 455 - CHAPTER 6 • How Cells Read the Genome

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