From Gene To Phenotype Lecture Outline 11/4/05

From Gene to PhenotypeDNAmolecule The central dogma:Gene 1Gene 3DNA strand(template)3′A5′CCA AACC GAGT5′ Transcription mRNA processingU GGUUU G GCUCA3′TRANSLATIONTrp– Introns and exons Other types of RNACodonProtein– DNA- RNA- protein– Control of gene expression in prokaryotes vseukaryotes– Initiation, Elongation,TerminationTRANSCRIPTIONmRNALecture Outline 11/4/05Gene 2PheGlySerAmino acidIn EukaryotesIn prokaryotes– Transcription and translation NSLATION– RNA transcripts aremodified beforebecoming true mRNA– Transcription andtranslation occur inseparate compartmentsof the cellNuclearenvelopeDNATRANSCRIPTIONPre-mRNARNA ptideFigure 17.3aFigure 17.3b1

“One Gene - One Enzyme”One Gene - One EnzymeBeadle and Tatum studied mutants of the bread moldNeurospora crassa and showed that each gene specified aparticular enzymeWild typeClass IMutantsClass IIMutantsClass ursorMM OrnithineMM ne is an essentialamino acid, required forgrowthEach mutant blocked a particular step of thepathway“One Gene - One Enzyme”PrecursorOrnithineSpecific enzymes (arrows)catalyze each stepMM CitrullineMutant 2 cangrow only ifsupplementedwith citrulline orarginineNormal cells can synthesiszearginine from precursors inthe minimal mediumWhich mutants can grow with whichsupplement?Mut 1 Mut 2 Mut 3none000Ornithine100Citrulline110Arginine111Which mutants can grow withwhich supplement?Mut 1Mut 2Mut 3none000Ornithine 100Citrulline 110Arginine 111CitrullineArginineOne strand of DNA is copiedto make messenger RNAThis is the “sense” or“coding” strand, becauseit reads the same as themRNAThis is the strand that isactually copied5’ . . . ATGAATGTC . . . 3’3’ . . . TACTTACAG . . . 5’5’ . . . augaauguc- . . 3’codingtemplateRNA copyTherefore mutant 2 must not make theenzyme to convert Ornithine to Citrulline2

RNA PolymeraseSynthesis of an RNA TranscriptPromoterTranscription unit5′3′Start point– Initiation3′5′DNARNA polymerase5′3′UnwoundRNA polymerasebinds to a promotersequence3′5′Template strand ofRNA DNAtranscriptRNAC5′3′3′5′5′Figure 17.7ACompleted RNAtranscriptAATTUEGCAAATTermination sequencecauses transcription tostopC3′ endURNAtranscriptCA5′– TerminationTCT5′3′5′3′A3′AGGTTDirection of transcription(“downstream)5′GmRNA copy of gene issynthesized 5’ to 3’Rewound5′3′RNA nucleotidesRNApolymeraseG– ElongationDNANon-templatestrand of DNAElongationGCATemplatestrand of DNA3′Newly madeRNARNA synthesis RNA synthesis is similar to DNA synthesis except:– Does NOT need a primer– No proofreading Why not? mRNA has high turnover. An error in one moleculewill not be inherited Both strands of DNA can serve as the template– Some genes are on one strand, other genes are on the other3

Initiation in eukaryotesSee an animation fromwww.dnai.org1 Eukaryotic promotersDNATRANSCRIPTIONPre-mRNARNA PROCESSINGmRNARibosomeTRANSLATIONTATA boxPolypeptidePromoter5′3′3′5′T AT A A AAATAT T T TTATA boxStart pointTemplateDNA strand2 Several transcriptionfactorsTranscriptionfactorsSeveral transcriptionfactors must bind topromoter sequencesupstream of the gene5′3′3′5′3 Additional transcriptionfactorsRNA polymerase II5′3′Then RNApolymerase can bindRNA processing in eukaryotesA modified guanine nucleotideadded to the 5′ endTRANSCRIPTIONDNAmRNA5′Protein-coding segmentG P P P5′ CapPolyadenylation signalAAUAAARibosome5′ UTRStart codon Stop codon3′ UTRTRANSCRIPTIONTranscription initiation complexDNAPre-mRNA5′ Exon IntronPre-mRNA 5′ Cap30131AAA AAAPoly-A tideA modifiedGTP is added,backwards, onthe 5’ endRNA transcript3. Splice out intronsRNA PROCESSINGPre-mRNARNA PROCESSINGTRANSLATION50 to 250 adenine nucleotidesadded to the 3′ end3′5′5′RNA processing in eukaryotes2. Add poly Atail to 3’ end1. Add 5’ capTranscription factorsExon3′Poly-A tail104105146Introns cut out andexons spliced togetherTRANSLATIONPolypeptidemRNA5′ Cap15′ UTRPoly-A tail1463′ UTRAbout 200 A’sadded at 3’ end4

SpliceosomesPre- mRNA5′1IntronExon 1Exon 2ProteinExample: Red/Green colorblindnessOther proteinssnRNASpecial “smallnuclear RNA”molecules dothe splicingsnRNPsSpliceosome2Most Eukaryotic genes haveintronsRNA transcript (pre-mRNA)5′13 kb3 exons2 intronsExample: beta globinSpliceosomecomponents3Mature mRNA5′mRNAExon 16 exons5 intronsCut-outintronExon 2Alternative splicingMake different mRNAs (and proteins) fromsame gene by splicing out certain exons1.6 kbSome have many introns andalternative formsHuman Dystrophin gene260 kb intron2.4 MbCell-type specific RNA splicing5

Correspondence between exons and proteindomainsFour types of RNAGeneDNAExon 1 Intron Exon 2 Intron Exon 3 mRNATranscription– Messenger RNA, encodes the amino acid sequence of apolypeptideRNA processing rRNATranslation– Ribosomal RNA, forms complexes with protein called ribosomes,which translate mRNA to proteinDomain 3 tRNA– Transfer RNA, transports amino acids to ribosomes during proteinsynthesisDomain 2 snRNADomain 1– Small nuclear RNA, forms complexes with proteins used ineukaryotic RNA processingPolypeptideRNA will fold to specificshapesSummary3′ACCA 5′C GG CC GU GU AA UA UUC*AG *C A C A G UA* CU CG*UGUGG*CC G AGC* *AUGG** GAG CG C HydrogenU A bonds* GAA*C*UA GA Because RNA is single-stranded,parts of the molecule can base pairwith other parts of the samemolecule, causing it to fold intodefined shapes. Some RNA molecules can even actas enzymes (ribozymes)Anticodon6

Transcription RNA processing Translation Domain 3 Domain 1 Domain 2 Polypeptide Four types of RNA mRNA –Messenger RNA, encodes the amino acid sequence of a polypeptide rRNA –Ribosomal RNA, forms complexes with protein called ribosomes, which translate mRNA to protein tRNA –Transfer RNA