Latraduzione

ØtRNAsØAminoacil-tRNA sintetasiØRibosoma

Key-componentsduring translation

Introduction

ØIniziazioneØAllungamentoØTerminazione

Theprocessoftranslation

Ilcodicegenetico

“ILDOGMACENTRALE”DNAàRNAàPROTEIN

Crick,F(1970)."Centraldogmaofmolecularbiology.".Nature227(5258):Thecentraldogmaofdemolecularbiologydealswiththedetailedresidue-by-

residuetransferofsequentialinformation.Itstatesthatsuchinformationcannotbetransferredbackfromproteintoeitherproteinornucleicacid.

FLOWOFG

ENETICIN

FROMATIO

NMultiplem

echanisms

ofgeneexpression

DNAReplication(DNAPol)

RNAReplication(virus,RNA

dependentRNApol.

rev.transcription

????????

Come il codice genetico venne decodificato (1960ies)1.Fosforilasi polinucleotidica: degradesRNA,butathighnucleosid diphosphate concentrations

polynucleotidephosphorylase reactionreverses:synthesisofRNAmolecules

Fosforilasi polinucleotidica+UàUUUUUUUUU

Fosforilasi polinucleotidica+Aà AAAAAAAAA

Fosforilasi polinucleotidica+Cà CCCCCCCCC

Isotopelabeledamminoacids+

Isotope-labeled

Isotope-labeled

Isotope-labeled

2.: mixing nucleotides: Inoltre sintesi di poliribonucleotidi a sequenza casuale di due monomeri in proporzione diversa, ad es. G e U in rapporto 3:1, GGUGGGGUGUGGUGGGGGUUGGGUUUGGGGGUGUGGGUGGGGGGGUGGUUGGUUGGGUGGGGG...il codone più frequente era GGG e l’amminoacido più abbondante nel polipeptide era la glicina, dunque GGG codificava per la glicina, ecc..3.STEP: controlled synthesis of oligo-ribonucleotides: verification of all 64 codons

Ilcodicegeneticoedegenerato

• Ilcodiceèbasatosutriplette=64combinazioni.• Èdegenerato– 1 aa canbe encoded by morethen one type ofcodon• SINONIMI/codonsynonym:condos that encode for same aminoacid

Human(2013):497tRNA genes324tRNA pseudogenes

Ilcodicegeneticoedegenerato

mRNA

Tentennamentodell’anticodon“wobbling”ofthe3° positionofcodon-anticodon

FrancisCrick1966

Observation:à 1tRNA anticodon canrecognizemorethenonecodon

ànucleotideinanticodon canbeINOSINEàUandIallowadditionalcombinationofbasepairing

Tentennamentodell’anticodon“wobbling”ofthe3° positionofcodon-anticodon

allthreecodonsencodeforGly

bothcodonsencodeforArg

Ilcodicegeneticoedegenerato

SERINE:6codonsandmin.3tRNAsLEUCINE andARGININE:morethenonetRNA

Ilcodicegenetico

• Icodoniconpirimidine(U/C)in2°posizioneà aaidrofobici

• Icodoniconpurine(G/A)in2°posizioneà aapolari

second

first

third

3 basic rules ofthegenetic code

1. mRNAcodonsarereadfrom5’à 3’orientationà ThisresultsaproteininNH2-aa-aa-aa-COOHorientation

2. Codonsdonotoverlap,;therearenotinterruptions

3. EachORFisadefinedsequence ofcodonsdefinedbythestartcodon

3 basic mutations ofthegenetic code

1. MISSENSE:mutationofcodon results inanotheraaExample:UUU=Phe;UCU=Ser

2. FRAMESHIFT: insertionordeletionofoneortwonucleotidesInsideanORFcausesashiftinthereadingframeExample:UUU/UCU=Phe/Ser;UUU/UUC/U=Phe/Phe/etc

2. NONSENSEorSTOPMUTATION:insertionordeletionofNucleotides thatresultinaprematurestop-codonExample:UUU/UCU=Phe/Ser;UUU/UAA/U=Phe/STOP

3.

Suppressor mutations canrescuemutant ORFs

INTRAGENICSUPPRESSORMUTANT

Deletion=event1Causesframeshift,leadingtotheusageofapremature

Stopcodonà Proteinisnotfunctional!

Insertion=event2Causesanotherframeshift,thatPreventstheuseofthepremature

stopcodonà Proteinfunction(partially)rescued

INTERGENICSUPPRESSORMUTANTs

ProteinAProteinB

ProteinAmut

ProteinB

ProteinAmut

ProteinBmut

functionalcomplex

nocomplex

suppressormutantrescues

complexformation

Different codonusage inmitochondria

Pro/Eucaryotes Mitochondria

MitochondriahavetransferredapartoftheirtRNA genestothenucleusoftheeukaryoticcell

THEORYGOESINTOPRACTICE

Yourtaskinaproject:

DESIGN:1- siRNAsthattargetRAD512- designPCRprimerstoPCRamplifyRAD51forthepurposeof

generatingaRAD51overexpressionvectorinpcDNA3.1(+)3- designPCRprimerstomeasureRAD51mRNAindiversetumorsamples

byreal-timePCR

FIRSTWENEEDTOSTUDYTHERAD51mRNASEQEUNCE!

ENSEMBL – acollection ofannotated genes/transcripts

SearchforhumanRAD51

ENSEMBL – acollection ofannotated genes/transcripts

PICKTHECORRECTENTRY

ENSEMBL – acollection ofannotated genes/transcripts

PAGEOPENSWITHALLKNOWNRAD51TRANSCRIPTsPart1:annotatedtranscripts---- WHICHTYPESOFRNAsCANYOUIDENTIFY??

ENSEMBL – acollection ofannotated genes/transcripts

PAGEOPENSWITHALLKNOWNRAD51TRANSCRIPTsPart2:mappingtranscriptsagainstgenomicDNA

---- WHICHTYPESOFRNAsCANYOUIDENTIFY??

ENSEMBL – acollection ofannotated genes/transcriptsPICKRAD51transcript009

ENSEMBL – acollection ofannotated genes/transcripts

1. cDNAsequencenote:The7meGcapisNotpartofthecDNASeqeunce!!!!

2.Proteinsequence

ENSEMBL – acollection ofannotated genes/transcripts

3.EXONS

ENSEMBL – acollection ofannotated genes/transcripts

3.EXONS

ENSEMBL – acollection ofannotated genes/transcripts

5’END

3’END

ENSEMBL – acollection ofannotated genes/transcripts

DESIGN:2.PCRoligostosubclone RAD51intoamammalianexpressionvector

RAD51

OVEREXPRESSIONINCELLS

DESIGN:2.PCRoligostosubclone RAD51intoamammalianexpressionvector

RAD51preRNA

RAD51preRNA splicing

RAD51mRNA(RNAPREPARATIONFROMCELLS

AMPLIFYORFWITHPRIMERS

ANDDNAPOLYMERASE

RAD51:5’end: 5’-TTTGTATTTTTCAGTAATGGCAATGCAGATGC-3’Oligo: 5’-GTAATGGCAATGCAGATGC-3’

3’end: 5’ AAAGACUGAATCATTGGGTTTTTCCT 3’Oligo: 3’-ACTTAGTAACCCAAAAAGGA-5’

KOZAK sequence:5’-G/ANNAUGG-3

ReversetranscribemRNA

cDNADESIGNPRIMERSFORAMPLIFATIONOFRAD51ORF

DESIGN:2.PCRoligostosubclone RAD51intoamammalianexpressionvector

RAD51

OVEREXPRESSIONINCELLS

GTAATGGCAATGCAGATGCAGCTTGAAGCAAATGCAGATACTTCAGTGGAAGAAGAAAGCTTTGGCCCACAACCCATTTCACGGTTAGAGCAGTGTGGCATAAATGCCAACGATGTGAAGAAATTGGAAGAAGCTGGATTCCATACTGTGGAGGCTGTTGCCTATGCGCCAAAGAAGGAGCTAATAAATATTAAGGGAATTAGTGAAGCCAAAGCTGATAAAATTCTGACGGAGTCTCGCTCTGTTGCCAGGCTGGAGTGCAATAGCGTGATCTTGGTCTACTGCACCCTCCGCCTCTCAGGTTCAAGTGATTCTCCTGCCTCAGCCTCCCGAGTAGTTGGGACTACAGGTGGAATTGAGACTGGATCTATCACAGAAATGTTTGGAGAATTCCGAACTGGGAAGACCCAGATCTGTCATACGCTAGCTGTCACCTGCCAGCTTCCCATTGACCGGGGTGGAGGTGAAGGAAAGGCCATGTACATTGACACTGAGGGTACCTTTAGGCCAGAACGGCTGCTGGCAGTGGCTGAGAGGTATGGTCTCTCTGGCAGTGATGTCCTGGATAATGTAGCATATGCTCGAGCGTTCAACACAGACCACCAGACCCAGCTCCTTTATCAAGCATCAGCCATGATGGTAGAATCTAGGTATGCACTGCTTATTGTAGACAGTGCCACCGCCCTTTACAGAACAGACTACTCGGGTCGAGGTGAGCTTTCAGCCAGGCAGATGCACTTGGCCAGGTTTCTGCGGATGCTTCTGCGACTCGCTGATGAGTTTGGTGTAGCAGTGGTAATCACTAATCAGGTGGTAGCTCAAGTGGATGGAGCAGCGATGTTTGCTGCTGATCCCAAAAAACCTATTGGAGGAAATATCATCGCCCATGCATCAACAACCAGATTGTATCTGAGGAAAGGAAGAGGGGAAACCAGAATCTGCAAAATCTACGACTCTCCCTGTCTTCCTGAAGCTGAAGCTATGTTCGCCATTAATGCAGATGGAGTGGGAGATGCCAAAGACTGAATCATTGGGTTTTTCCT