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