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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

2

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

5

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