Lecture 3 Post Ts Modification-lecture notes.pdf

Post Ts Modification-lecture notes

1. PosttranscriptionalModifications(occursinEukaryotes)
1.5’Capping
2.RNASplicing
3.3’PolyAtail Dr.ManikandanKathirvel
AssistantProfessor,
DepartmentofLifeSciences,
KristuJayantiCollege(Autonomous),
Bengaluru560077

2. •Replication
•Transcription
•Translation
-Coupled Transcription and Translation
•Replication
•Transcription
•Post transcriptional modification
•Translation
•Post translational Modifications
mRNAprocessedandtransported
outofnucleusfortranslation

3. •Replication
•Transcription
•Post transcriptional modification
•Translation
•Post translational Modifications
In Eukaryotes

4. DNA
Transcription
Pri-RNA
tRNA
4
mRNA
rRNA
MaturedRNAs
Processing
Introduction
Pri-transcript
Post transcriptional
modifications-mRNA
Processing

5. Eukaryoticvs.ProkaryoticTranscription
•Ineukaryotes,transcriptionandtranslation
occurinseparatecompartments.
•Inbacteria,mRNAispolycistronic;
•ineukaryotes,mRNAisusuallymonocistronic.
–Polycistronic:onemRNAcodesformorethanone
polypeptide
–monocistronic:onemRNAcodesforonlyone
polypeptide
•“Processing”ofmRNAisrequiredineukaryotesfor
thematuration
•Noprocessinginprokaryotes(mRNAmatureson
transcription)

6. Structure of a Eukaryotic gene
•Replication
•Transcription
•Post transcriptional modification
•Translation
•Post translational Modifications
In Eukaryotes

7. •Capping(additionofa
5’7-methylguanosine
cap)
•Splicing
intervening
(introns)
toremove
sequences
•Polyadenylation
(additionofapoly-A
tailatthe3’)
Post transcriptional modifications-mRNAProcessing
7

8. 1.) 5’Capping
6

9. CapFunctions
Capprovides:
1.Protection from someribonucleasesdegradation
2.StabilizesmRNA
3.Enhanced translationandsplicing
4.Enhancedtransportfrom nucleustocytoplasm

10. 2) 3’ Poly A tail-Polyadenylation
PolyadenylationComplex
10
Consensussequencefor3’
process

12. PolyadenylationofmRNAatthe3’end
CPSF:cleavageandpolyadenylationspecificityfactor
bindsupstreamAAUAAApoly(A) Signal5’end.
CStF:cleavagestimulatoryfactorFinteractswitha
downstreamGU-sequence&bound withCPSF forminga
loop inRNA
CFI&CFII:cleavagefactorI&II.
PAP:
poly(A)polymerasestimulatescleavageat polyA site
BoundPAPadds ≈12Aresiduesat aslowrateto3’-
OHgroup
PABPII:poly(A)-bindingproteinII.
PABPII(shortpolyAtail)acceleratesrateofaddition
ofAbyPAP
After200–250Aresidueshavebeenadded,PABPII signals
PAPtostoppolymerization
Poly(A)tailcontrolsmRNAstability&influences
translation

13. 1
3
•mRNAiscalledhnRNA(heterogenousnuclearRNA) beforesplicing
occurs
•ThehnRNPproteinstohelpkeepthehnRNAinasingle-strandedform
andto assistinthevariousRNAprocessingreactions
•Exon andintron lengths&numbersvary invariousgenes
•Exon(Expressedsequences)isanysegment of aninterruptedgene
thatisrepresentedinthe matureRNAproduct.
•Intron (intervening sequences )is a segment of DNA that is
transcribed,butremoved fromwithinthetranscriptbysplicing
togetherthe sequences(exons) on eithersideofit.
3.) mRNAsplicing

14. 3.) mRNAsplicing
Example: The sequence of steps in the production
of mature eukaryotic mRNAas shown forthe
chickenovalbumingene.

15. SpliceJunctionConsensusSequence
•GU-AGruledescribesthepresenceoftheseconstantdinucleotidesat thefirst
twoandlasttwopositionsofintronsofnucleargenes.
•Splicesitesarethesequencesimmediatelysurroundingtheexon-intronboundaries
•Splicingjunctionsarerecognizedonlyinthecorrectpairwisecombinations
1
5

16. 16
•SplicingismediatedbyalargeRNPs
(Ribonucleoproteins)complexcalled
"spliceosome"
•Spliceosomecontainsaspecificsetof
baseUracil-richsnRNPs(smallnuclear
RNPs)associatedwithproteins
(snRNAcomplexwithprotein)
FunctionofsnRNPs:
•Recognizingthe5’splicesiteandthe
branchsite.
•Bringingthosesitestogether.
•Catalyzing(orhelpingtocatalyze)the
RNAcleavage.
mRNAsplicing

17. SpliceosomeComplex
•SplicingsnRNPs:
•U1:5'-siterecognition
•U2:branchsiterecognition
•U4:formsbasepaired
complex& actswithU6
•U5:3'-junctionbindingof
U4-U6complex
•U6:complexwithU4makes
spliceosometransesterase
spliceosomesrecognizeintronsstartingwith5'-GUandendinginAG-3’
17

18. U1
3′5′
5′splicesite 3′splicesite
Branchsite
A
GU
Exon1 Exon2
U1bindsto5′splicesite.
U2bindstobranchsite.
AG
3′5′
U4/U6andU5trimerbinds.Intronloopsout
andexonsarebroughtclosertogether.
U1snRNP
A
U2snRNP
3′5′
A
U4/U6snRNP
U5snRNP
U2
Intron loops out
andexonsbrought
closertogether
18
MechanismofSpliceosome
SplicingsnRNPs:
U1:5'-siterecognition
U2:branchsiterecognition
U4: formsbase paired complex
& actswithU6
U5: 3'-junction binding of
U4-U6complex
U6:complexwithU4makes
spliceosometransesterase
spliceosomesrecognizeintronsstartingwith5'-GUandendingin
AG-3’

19. U1
U4
5′
3′
5′
5′splicesiteiscut.
5′endofintronisconnectedtothe
Ainthebranchsitetoformalariat.
U1andU4arereleased.
3′splicesiteiscut.
Exon1is connectedto exon2.
Theintron(intheformofalariat)isreleasedalongwith
U2,U5,andU6(intronwillbedegraded).
A
U5U6
U5
U6
U2
A
IntronplusU2,
U5,andU6
3′
Twoconnected
exonsExon1
Exon2
U2
Intronwillbedegraded
and the snRNPs used
again
MechanismofSpliceosome
14
SplicingsnRNPs:
U1:5'-siterecognition
U2:branchsiterecognition
U4: formsbase paired complex
& actswithU6
U5: 3'-junction binding of
U4-U6complex
U6:complexwithU4makes
spliceosometransesterase

20. Steps involved in RNA splicing

21. •5’cap
•5’untranslatedregion
•Startcodon
•Codingsequence
•Stopcodon
•3‘untranslatedregion
•PolyAtail
21
MaturedmRNA

22. pre-mRNAaresplicedinseveraldifferentways,allowingasingle
genetocodeformultipleproteins
ThegenerationofdifferentmaturemRNAsfromaparticulartype
ofgenetranscriptcanoccurbyvaryingtheuseof5’-and3’-splice
sites
Alternativesplicing
SexdeterminationintheDrosophila
22

23. 2.tRNA
TransferRNA/SolubleRNA/supernatantRNA/Adaptor
RNA
•Smallestamong RNAs(75-93nucleotides)
•RecognizescodononmRNA
•Showshighaffinitytoamino acids
•Carryamino acidstothesiteofproteinsynthesis
•tRNAistranscribedbyRNApolymeraseIII
•tRNA genes also occur in repeated copies
throughoutthegenome, andmaycontainintrons.
23

24. 1.Removalofleadersequence&
trailer
2.Replacement of nucleotide
3.Modificationofcertainbases:
•ReplacementofUresiduesatthe
3′endofpre-tRNAwithaCCA
sequence
•Additionofmethyland
isopentenylgroupstothe
heterocyclicringofpurinebases
•Methylationofthe2′-OHgroup
in the ribose of anyresidue;and
conversion of specific uridines to
dihydrouridine(D),pseudouridine(y)
4.Excisionofanintron
ProcessingoftRNA
20

25. Ribozyme
RNAcanactasanEnzymeandcatalysereactionsincludingitsown replication
tRNAPROCESSINGANDMATURATION

26. 26
•Incell>80%ofrRNA
•ServestoreleasemRNAfromDNA
•Actas ribozymesinproteinsynthesis
•RelativelyG:::Crich
•Ribosome
•Prokaryotes–70S(50S&30S)
•Eukaryotes–80S(60S&40S)
•Prokaryotes–In 50S subunits-23S&5S
In30Ssubunits-16S
•Eukaryotes–In60Ssub-units–28S,5.8Sand5S
In40Ssub-units–18S
:31proteins
:21proteins
:50proteins
:33proteins
3.RibosomalRNA(rRNA)

27. 27
ProcessingofribosomalRNA
•Processingof45smoleculesoccursinsidenucleolus
•45smoleculestightlyassociated protein forming(RNPs)
•Fristcleavage:occursatsiteI &remove5’terminal leader
sequence, produces41sintermediate &18s
•Secondcleavage:occurs 41s intermediate at site 3’
generates32s intermediate
•Finalcleavage:separationof32sintermediateinto28s,5.8s
•ProcessedrRNA28s,5.8s&18s

28. 2
nd
24
Cleavage
ProcessingofribosomalRNA

29. ProcessingofribosomalRNA
29

30. Synthesisof5SrRNA
•rDNAcistronfor5SrRNAispresentoutsideNucleolar
organizer
•TranscriptionrequiresRNApol III+TFIIIA,TFIIIB&
TFIIIC
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