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Lectures by Chris Romero, updated by Erin Barley with contributions from Joan Sharp
Chapter17
FromGenetoProtein
TheFlowofGene3cInforma3on
• Centraldogmaofgene3cs
– Onewayflowofinforma3on
• DNA→mRNA→protein
• Informa3oninDNAisheldinthespecificsequencesofnucleo3des
– DNAcodesforspecificproteins
• Linksbetweengenotypeandphenotype
• Geneexpression
– processbywhichDNAdirectsproteinsynthesis
• includestwostages:transcrip3onandtransla3on
EvidencefromtheStudyofMetabolicDefects
• ArchibaldGarrod‐1909
– Bri3shphysician
– Suggestedthatgenesdictatephenotypes
– Thoughtsymptomsofaninheriteddiseasereflectaninabilitytosynthesizeacertainenzyme
• Linkinggenestoenzymesrequiredunderstandingthatcellssynthesizeanddegrademoleculesinaseriesofsteps,ametabolicpathway
Nutri3onalMutantsinNeurospora
• GeorgeBeadleandEdwardTatum
– ExposedbreadmoldtoX‐rays
• Createdmutantsunabletosurviveonminimalmedium
– Resultofinabilitytosynthesizecertainmolecules
• Usingcrosses
– Iden3fiedthreeclassesofarginine‐deficientmutants
• Eachlackingadifferentenzymenecessaryforsynthesizingarginine
• Theydevelopedaonegene–oneenzymehypothesis,whichstatesthateachgenedictatesproduc3onofaspecificenzyme
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Fig. 17-2
RESULTS
EXPERIMENT
CONCLUSION
Growth:Wild-typecells growing and dividing
No growth:Mutant cellscannot grow and divide
Minimal medium
Classes of Neurospora crassaWild type Class I mutants Class II mutants Class III mutants
Minimalmedium(MM)(control)MM +ornithine
MM +citrulline
Con
ditio
n
MM +arginine(control)
Class I mutants(mutation in
gene A)Wild type
Class II mutants(mutation in
gene B)
Class III mutants(mutation in
gene C)
Gene A
Gene B
Gene C
Precursor Precursor Precursor PrecursorEnzyme A Enzyme AEnzyme A Enzyme A
Enzyme B
Ornithine Ornithine Ornithine OrnithineEnzyme B Enzyme B Enzyme B
Citrulline Citrulline Citrulline CitrullineEnzyme C Enzyme C Enzyme C Enzyme C
Arginine Arginine Arginine Arginine
TheProductsofGeneExpression
• onegene–oneprotein
– Someproteinsaren’tenzymes
• Manyproteinsarecomposedofseveralpolypep3des
– eachofwhichhasitsowngene
• Therefore,BeadleandTatum’shypothesisisnowrestatedastheonegene–onepolypep1dehypothesis
• Notethatitiscommontorefertogeneproductsasproteinsratherthanpolypep3des
Protein Synthesis
BasicPrinciplesofTranscrip3onandTransla3on
• Transcrip3on
– synthesisofRNAunderthedirec3onofDNA
• producesmessengerRNA(mRNA)
• Transla3on
– synthesisofapolypep3de
• Ribosomes
– sitesoftransla3on
• Prokaryotes
• mRNAproducedbytranscrip3onisimmediatelytranslatedwithoutmoreprocessing
• Eukaryotes
• Nuclearenvelopeseparatestranscrip3onfromtransla3on
BasicPrinciplesofTranscrip3onandTransla3on
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• Eukaryo3cRNAtranscripts
• modifiedthroughRNAprocessingtoyieldfinishedmRNA
• Primarytranscript
• ini3alRNAtranscriptfromanyeukaryo3cgene
BasicPrinciplesofTranscrip3onandTransla3onFig. 17-3
TRANSCRIPTION
TRANSLATION
DNA
mRNARibosome
Polypeptide
(a) Bacterial cell
Nuclearenvelope
TRANSCRIPTION
RNA PROCESSING Pre-mRNA
DNA
mRNA
TRANSLATION Ribosome
Polypeptide
(b) Eukaryotic cell
TheGene3cCode
• Gene3ccode
– SystemforreadingsequencecodeinmRNA
• 20aminoacids
– butonlyfournucleo3debasesinDNA
TripletsofBases
• tripletcode
– aseriesofnon‐overlapping,three‐nucleo3dewords
– smallestunitsofuniformlengththatcancodeforalltheaminoacids
• Example:AGTonaDNAstrandresultsintheplacementoftheaminoacidserineatthecorrespondingposi3onofthepolypep3detobeproduced
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• Templatestrand
– OneofthetwoDNAstrandsprovidesatemplatefororderingthesequenceofnucleo3desinanRNAtranscriptduringtranscrip3on
• Codon
– mRNAbasetripletsreadinthe5′to3′direc3onduringtransla3on
– specifiestheaminoacidtobeplacedatthecorrespondingposi3onalongapolypep3de
TripletsofBases
• CodonsalonganmRNAmoleculearereadbytransla3onmachineryinthe5′to3′direc3on
• Eachcodonspecifiestheaddi3onofoneof20aminoacids
Codons,TripletsofBases
Fig. 17-4
DNAmolecule
Gene 1
Gene 2
Gene 3
DNAtemplatestrand
TRANSCRIPTION
TRANSLATION
mRNA
Protein
Codon
Amino acid
CrackingtheCode• 64codons
– Decipheredbythemid‐1960s
– 61codeforaminoacids
• 3tripletsare“stop”signalstoendtransla3on
• Redundancyofthegene3ccode
– Nocodonspecifiesmorethanoneaminoacid
• butnotambiguous
– Oneaminoacidmaybecodedbymorethanonecodon
• Readingframe
– Codonsmustbereadinthecorrectgroupingsinorderforthespecifiedpolypep3detobeproduced
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Fig. 17-5Second mRNA base
Firs
t mR
NA
bas
e (5′ e
nd o
f cod
on)
Third
mR
NA
bas
e (3′ e
nd o
f cod
on)
Evolu3onoftheGene3cCode
• Gene3ccode
– nearlyuniversal,sharedbythesimplestbacteriatothemostcomplexanimals
• Genescanbetranscribedandtranslateda`erbeingtransplantedfromonespeciestoanother
Fig.17‐6
(a) Tobacco plant expressing a firefly gene
(b) Pig expressing a jellyfish gene
MolecularComponentsofTranscrip3on
• RNApolymerase
– CatalyzesRNAsynthesis
– AddsnewRNAnucleo3des
• followsthesamebase‐pairingrulesasDNA
– excepturacilsubs3tutesforthymine
• Promoter
– DNAsequenceforRNApolymeraseaaachmentinbacteria
• Terminator
– Sequencesignalingtheendoftranscrip3on
• Transcrip3onunit
– ThestretchofDNAthatistranscribed
MolecularComponentsofTranscrip3on
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Fig. 17-7
Promoter Transcription unit
Start point DNA
RNA polymerase
5′5′3′3′
Initiation1
2
3
5′5′3′3′
UnwoundDNA
RNAtranscript
Template strandof DNA
Elongation
RewoundDNA
5′
5′5′
5′
5′
3′3′3′
3′
RNAtranscript Termination
5′5′3′3′
3′5′Completed RNA transcript
Newly madeRNA
Templatestrand of DNA
Direction oftranscription(“downstream”)
3′ end
RNApolymerase
RNA nucleotides
Nontemplatestrand of DNA
Elongation
SynthesisofanRNATranscript
• Thethreestagesoftranscrip3on:
– Ini3a3on
– Elonga3on
– Termina3on
Ini3a3onofTranscrip3on
• Transcrip3onfactors
– mediatethebindingofRNApolymeraseandtheini3a3onoftranscrip3on
• Transcrip3onini3a3oncomplex
– Thecompletedassemblyoftranscrip3onfactorsandRNApolymeraseIIboundtoapromoter
• TATAbox
– Aeukaryo3cpromotercrucialinformingtheini3a3oncomplex
Fig. 17-8A eukaryotic promoterincludes a TATA box
3′
1
2
3
Promoter
TATA box Start point
Template
TemplateDNA strand
5′3′5′
Transcriptionfactors
Several transcription factors mustbind to the DNA before RNApolymerase II can do so.
5′5′3′3′
Additional transcription factors bind tothe DNA along with RNA polymerase II,forming the transcription initiation complex.
RNA polymerase IITranscription factors
5′5′ 5′3′
3′
RNA transcript
Transcription initiation complex
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Elonga3on
• RNApolymerase
– TravelsdownDNA
– Untwiststhedoublehelix,10to20basesata3me
– progressesatarateof40nucleo3despersecondineukaryotes
• AgenecanbetranscribedsimultaneouslybyseveralRNApolymerases
Termina3onofTranscrip3on• Themechanismsoftermina3onaredifferentinbacteria
andeukaryotes
• Bacterialtermina3on
– Thepolymerasestopstranscrip3onattheendoftheterminator
• Eukaryo3ctermina3on
– RNAPolymerasecon3nuesthroughthepolyadenyla3onsignal
• TTATTT(transcribedtoAAUAAA)
– Con3nuestranscrip3ona`erthepre‐mRNAiscleavedfromthegrowingRNAchain
• A`er10‐35nucleo3des
• PolymeraseeventuallyfallsofftheDNA
Altera3onofmRNAEnds
• Eachendofapre‐mRNAmoleculeismodifiedinapar3cularway:
– The5′endreceivesamodifiednucleo3de5′cap
• Methylatedguaninenucleo3de
– The3′endgetsapoly‐Atail
• ~50‐250
• Provideseveralfunc3ons:
– TheyseemtofacilitatetheexportofmRNA
– TheyprotectmRNAfromhydroly3cenzymes
– Theyhelpribosomesaaachtothe5′end
Fig. 17-9
Protein-coding segment Polyadenylation signal3′
3′ UTR5′ UTR
5′
5′ Cap Start codon Stop codon Poly-A tail
G P PP AAUAAA AAA AAA…
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SplitGenesandRNASplicing
• PrimaryTranscript
• Ini3aleukaryo3cRNAbeforeprocessing
• Introns
• Noncodingregionsoftranscript
• Exons
• Regionsoftranscripttranslatedintoaminoacidsequences
• RNAsplicing
• Removesintronsandjoinsexons,crea3nganmRNAmoleculewithacon3nuouscodingsequence
Fig. 17-10
Pre-mRNA
mRNA
Codingsegment
Introns cut out andexons spliced together
5′ CapExon Intron5′
1 30 31 104
Exon Intron
105
Exon
146
3′Poly-A tail
Poly-A tail5′ Cap
5′ UTR 3′ UTR1 146
• Insomecases,RNAsplicingiscarriedoutbyspliceosomes
• Spliceosomes
– consistofavarietyofproteinsandseveralsmallnuclearribonucleoproteins(snRNPs)thatrecognizethesplicesites
• Shortsequencesof
nucleo3desattheend
ofeachintron
RNAProcessing
