Student questions of the dayStudent questions of the day……..Q: Does splicing involve DNA or RNA?A: Splicing refers to processing of the primary RNA. A: Splicing refers to processing of the primary RNA. Q: Is information lost from RNA during splicing?A: Yes. A: Yes. introns introns and some and some exons exons are permanently lost.are permanently lost.Q: Does RNA really break and rejoin during splicing?A: Yes, the process involves cutting and A: Yes, the process involves cutting and religationreligation..7.0133.5.07Molecular Biology III(RNA processing,translation and genetic code)transcriptionRNAsplicingtranslationnucleusexport to cytoplasmgene(dsDNA)Gene expression: can be regulated at any pointprotein trafficking/modification1H. Sive MIT 2007See Purves 14.111. RNA processingSplicing (conclusion)transcriptiongenematuremRNAGene includes exons and intronsIntrons = in primary RNA/ non-coding Exons = in primary and mature mRNA/ protein coding5’ 3’primary (“nascent”)RNASplicing =introns removedexonintron“lariats”cytoplasmSplicingH. Sive MIT 2007See Purves 14.4, 14.105’ 3’16primaryRNASplicing = introns (+/- exons) removed5’ 3’12 3 4exon5’ 3’12 3 4 5’ 3’12 3 4 Alternate splicing events: any join between splice donor and splice acceptor sitesH. Sive MIT 2007175’ 3’1 2 3 4matureRNA5’ 3’ 1 3 45’ 3’1 45’ 3’1 2 3 45’ 3’ 1 3 45’ 3’1 4RNAproteinDifferent protein products from alternately spliced RNAsH. Sive MIT 200718The Dscam gene can yield 38,000 different spliced mRNAs!!Schmucker et al, 200018a1. RNA processingCap/poly Agene(dsDNA)5’3’5’3’ template strandnon-template strandCap/polyA tail addition to mRNAsH. Sive MIT 200719templateis copied5’ 3’5’3’7MeG “cap” added stop site5’3’RNA (primary transcript)5’ 3’RNA cut/polyA addition siteA200capParts of a mRNAUTR = “untranslated region”3’AUG XXX5’ MeG5’ UTRcoding3’UTRPoly A tailCapAAAA…ribosome recognitiontranslation start site = initiation codontranslation stop site = termination codon2H. Sive MIT 2007translationregulationtranslationinhibition(miRNAs)2. TranslationcodeSee Purves 12.5: genetic code3See Purves 12.5: genetic code3DNA template3’ ACCAAACCGAGT 5’From DNA to protein/ the “genetic code”Proteintrp phe gly serNH2 COOHtranslationDirection of translation4RNA5’ UGGUUUGGCUCA 3’codontranscriptionH. Sive MIT 20072. Translationinterpreter is tRNAPurves 12.7: tRNA structure and recognition sitesanticodonAmino acid covalently attaches to 3’ CCAAnticodon (triplet) recognizes complementary triplet on mRNA52. Translationfactory is the ribosometunnel for mRNA/tRNA6Ribosome structure/RNA(gold) and proteins (colors)tunnel for mRNA/tRNA7G A ALeutRNATranslationA U G5’ C U U A G U U A A3’AUG XXX5’ MeGcodingAAAA…ribosome recognitioninitiation codon termination codonpeptide bond formation3’ U A CMettRNAchargedtRNA-Met 3’UAC = anticodon8H. Sive MIT 2007RNASee Purves 12.10, 12.11, 12.12A U G C U U A G U U A A U A CLeu5’NH2MettRNA U C ASertRNApeptide bondTranslation elongation U G CtRNAuncharged tRNA-Met9H. Sive MIT 2007RNAA U G C U U A G U U A A5’LeuNH2Met U C ASertRNATranslation termination U G CtRNAuncharged tRNA-Leu10H. Sive MIT 2007RNAStop codonNo matchingtRNA U C AtRNAuncharged tRNA-SerLeuNH2Met SerCOOHpeptide releasedTranslation animation11PolyA tail brings inititation factors to cap1213E. Miska, U. CamMIT/ Prof. BartelProf. Sharp3. From genotype to phenotypegiantismsickle cell anemia5’ 3’Mutations can change the amount orsequence of protein producedPromoterexonsintronsExon mutations can change protein sequence and function.Promoter mutations (5’, 3’) can change amountof protein14H. Sive MIT 2007Intron mutationscan changeprotein sequenceor amountDNA 5’ATGTGGCTCCTGGATTAAtemplate TACACCGAGGACCTAATT mRNA: 5’AUGUGGCUCCUGGAUUAAprotein: mettrpleuproaspWild type gene, protein sequence15DNA: 5’ATGTGGCTCCTGGATTAAtemplate TACACCGAGGACCTAATTmRNA: 5’AUGUGGCUCCUGGAUUAAprotein: mettrpleuproasp nonsense mutation: truncates proteinDNA: 5’ATGTAGCTCCTGGATTAAtemplate TACATCGAGGACCTAATTmRNA: AUGUAGCUCCUGGAUUAAprotein: met16DNA: 5’ATGTCGCTCCTGGACTAAtemplate TACAGCGAGGACCTGATTmRNA: AUGUAGCUCCUGGACUAAprotein: mettrpleuproaspsilent mutation: does not change protein DNA: 5’ATGTGGCTCCTGGATTAAtemplate TACACCGAGGACCTAATTmRNA: 5’AUGUGGCUCCUGGAUUAAprotein: mettrpleuproasp 17DNA: 5’ATGTCGCTCCTGGTTTAAtemplate TACAGCGAGGACCATATTmRNA: AUGUAGCUCCUGGUUUAAprotein: mettrpleuprovalmissense mutation: changes single amino acid DNA: 5’ATGTGGCTCCTGGATTAAtemplate TACACCGAGGACCTAATTmRNA: 5’AUGUGGCUCCUGGAUUAAprotein: mettrpleuproasp 18frameshift mutation: changes >1 amino acidDNA: ATGTGGACTCCTGGATTAAtemplate TACACCTGAGGACCTAATTmRNA: AUGUAGACUCCUGGUUUAAprotein: mettrpthrprolysglu..insertion DNA: 5’ATGTGGCTCCTGGATTAAtemplate TACACCGAGGACCTAATTmRNA: 5’AUGUGGCUCCUGGAUUAAprotein: mettrpleuproasp
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