Transcription in Eukaryotes-Complete.ppt

TRANSCRIPTION IN EUKARYOTES
INTRODUCTION
TYPES OF RNA
A STRUCTURAL GENE
EUKARYOTIC RNA POLYMERASES
MECHANISM OF TRANSCRIPTION IN EUKARYOTES:
- INITIATION
- ELONGATION
- TERMINATION
TRANSCRIPTION FACTORS
ACTIVATORS, MEDIATORS & CHROMATIN MODIFYING PROTEINS
RNA SPLICING
DIFFERENCE BETWEEN PROKARYOTIC & EUKARYOTIC TRANSCRIPTION

1. TRANSCRIPTION IN EUKARYOTES
Presented by:
Prashant v c
Dept of zoology
guk
1

2. CONTENTS
•INTRODUCTION
•TYPES OF RNA
•A STRUCTURAL GENE
•EUKARYOTIC RNA POLYMERASES
•MECHANISM OF TRANSCRIPTION IN EUKARYOTES:
-INITIATION
-ELONGATION
-TERMINATION
• TRANSCRIPTION FACTORS
• ACTIVATORS, MEDIATORS & CHROMATIN
MODIFYING PROTEINS
•RNA SPLICING
•DIFFERENCE BETWEEN PROKARYOTIC &
EUKARYOTIC TRANSCRIPTION
2

3. INTRODUCTION
Transcription, orRNA synthesis, is the process of
creating RNA, copy of a sequence of DNA.
BothRNAandDNAarenucleicacids,which
usebasepairsofnucleotidesasacomplementary
languagethatcanbeconvertedfromDNAtoRNAin
thepresenceofthecorrectenzymes.
Duringtranscription,aDNAsequenceisreadbyRNA
polymerase,whichproducesacomplementaryRNA
strand.
3

4. Types of RNA

5. 5
Types of RNAs Produced in Cells
Types of RNAs
Functions
mRNAs (messenger) code for proteins
rRNAs (ribosomal) comprise ribosomes
tRNAs (transfer) adaptors between mRNA and
amino acids in protein synthesis
hnRNAs (heterogeneous nuclear) precursors & intermediates of mature
mRNAs & other RNAs
snRNAs (small nuclear) splicing of pre-mRNAs
snoRNAs (small nucleolar) rRNA processing/maturation/
methylation
scRNAs (small cytoplasmic) signal recognition particle (SRP) /
tRNA processing
miRNAs (Micro) regulatoryRNAs (regulation of
transcription and translation, other??)
siRNAs (small interfering) regulatoryRNAs (regulation of
transcription and translation, other??)
Other non-coding RNAs telomere synthesis, X-chromosome
inactivation, protein transport

6. The Central Dogma
▪Thecentraldogmaofmolecularbiologydescribesthetwo-step
process,transcriptionandtranslation,bywhichtheinformation
ingenesflowsintoproteins.
▪Conventionalconcept(pre-bioinformaticsera)ofcentraldogma
oflife:
Itisanoversimplificationofmolecularbiology.
DNA→RNA→Protein
▪Currentconcept(Bioinformaticsera)ofcentraldogmaoflife:
Withtheadvancesincellbiologyandrapiddevelopments
inbioinformatics,thetermGenome,Transcriptomeand
Proteomeareincurrentusetorepresentthecentraldogmaof
molecularbiology.
Genome→Transcriptome→Proteome

7. The Central Dogma

8. 8
The Central Dogma

9. The Central Dogma

10. ASTRUCTURAL GENE

11. Basic Structure of a Protein-Coding Gene
▪A protein-coding gene consists of a promoter followed by the coding
sequence for the protein and then a terminator.
▪The promoter is a base-pair sequence that specifies where
transcription begins.
▪The coding sequence is a base-pair sequence that includes coding
information for the polypeptide chain specified by the gene.
▪The terminator is a sequence that specifies the end of the mRNA
transcript.

12. DNA and RNA:Nucleotides, Bases and Polynucleotide's

13. •Exons:Exonscodeforaminoacidsand
acidsequenceoftheproteinproduct.Itis
theseportionsofthegenethatare
representedinfinalmaturemRNA
molecule.
•Introns:Intronsareportionsofthegene
thatdonotcodeforaminoacids,andare
removed (spliced)fromthemRNA
moleculebeforetranslation.

14. Control regions
•Start site:-A start site for transcription.
•Promoter:-Aregionafewhundred
nucleotides'upstream'ofthegene(toward
the5'end).ItisnottranscribedintomRNA,
butplaysaroleincontrollingthe
transcriptionofthegene.Transcription
factorsbindtospecificnucleotide
sequencesinthepromoterregionandassist
inthebindingofRNApolymerases.

15. •Enhancers:Sometranscriptionfactors
(calledactivators)bindtoregionscalled
'enhancers'thatincreasetherateof
transcription.Some enhancers are
conditionalandonlyworkinthepresenceof
otherfactorsaswellastranscription
factors.
•Silencers: Sometranscriptionfactors
(calledrepressors)bindtoregionscalled
'silencers'thatdepresstherateof
transcription.

16. Eukaryotic RNA Polymerases (RNAPs)
Inbacteria(prokaryote),allmRNAismadefromthe
sameRNApolymerase (singleRNAP).However,in
eukaryotes,therearethreedifferentRNApolymerases
inanimalsandfourinplants.
1.RNA Polymerase I: synthesizes rRNA
2. RNA Polymerase II: synthesizes all Protein
coding genes & mostly mRNA.
3.RNA polymerase III: synthesizes tRNAsand
also snRNAs(small nuclear RNAs) and
scRNAs(small cellular RNAs).
16

17. 17
Four RNA Polymerases of Eukaryotic Cells
Type of Polymerase Genes Transcribed
RNA pol I rRNAgenes (5.8S, 18S, and 28S)
RNA pol II mRNAgenes (protein coding genes),
snoRNA genes, some snRNA genes,
microRNAs genes
RNA pol III tRNAgenes, 5S rRNA genes
some snRNA genes, genes
for other small RNAs
RNA pol IV plants only; small interfering
RNAs (siRNAs)

18. Eukaryotic Transcription
Initiation:
•In eukaryotes, the initiation of transcription,
requires the presence of a
corepromotersequence in the DNA. Promoters
are regions of DNA which promote transcription
and are found around -10 to -35 base pairs
upstream from the start site of transcription. Core
promoters are sequences within the promoter
which are essential for transcription initiation.
RNA polymerase is able to bind to core promoters
in the presence of various specifictranscription
factors.

19. •The most common type of core promoter in eukaryotes is a
short DNA sequence known as aTATA box(Hogness box). The
TATA box, as a core promoter, is the binding site for a
transcription factor known asTATA binding protein(TBP),
which is itself a subunit of another transcription factor, called
Transcription Factor II D(TFIID).
•One transcription factor, DNA helicase, hashelicaseactivity
and so is involved in the separating of opposing strands of
double-stranded DNA to provide access to a single-stranded
DNA template.
19

20. Eukaryotic transcription
ELONGATION:
•Ineukaryotes,theRNAisprocessedatbothends
beforeitisspliced.
•Atthe5‘end,acapisaddedconsistingofa
modifiedGTP(guanosinetriphosphate).Thisoccurs
atthebeginningoftranscription.The5'capisused
asarecognitionsignalforribosomestobindtothe
mRNA.
•Atthe3'end,apoly(A)tailof150ormoreadenine
nucleotidesisadded.Thetailplaysaroleinthe
stabilityofthemRNA.
20

21. • The Transcription Process
▪RNA synthesis involves separation of the DNA strands and
synthesis of RNA molecule in the 5' to 3' direction by RNA
polymerase, using one of the DNA strands as a template.
▪In complementary base pairing, A, T, G, and C on the template DNA
strand specify U, A, C, and G, respectively, on the RNA strand being
synthesized.

22. EUKARYOTIC TRANSCRIPTION
TERMINATION:
•Transcription termination in eukaryotes is less
understood but involves cleavageof the new
transcript followed by template-independent
addition ofAs at its new 3' end, in a process
calledpolyadenylation.

23. Terminationoftranscriptionineukaryotes:
additionofpoly(A)tails
•Ineukaryotes,terminationoftranscriptionoccurs
bydifferentprocesses,dependingupontheexact
polymeraseutilized.ForpolIgenes,transcription
isstoppedusingaterminationfactor,througha
mechanismsimilartorho-dependenttermination
inbacteria.TranscriptionofpolIIIgenesends
aftertranscribingaterminationsequencethat
includesapolyuracilstretch,byamechanism
resembling rho-independent prokaryotic
termination.TerminationofpolIItranscripts,
however,ismorecomplex.

24. Terminationoftranscriptionineukaryotes:
additionofpoly(A)tails
•TranscriptionofpolIIgenescancontinueforhundredsor
eventhousandsofnucleotidesbeyondtheendofacoding
sequence.TheRNAstrandisthencleavedbyacomplex
thatappearstoassociatewiththepolymerase.Cleavage
seemstobecoupledwithterminationoftranscriptionand
occursataconsensussequence(TTATTToncoding
regionoftemplatestrandofDNAandconsequently
AAUAAA sequenceonpre-mRNA).Thepre-mRNA,
carryingthissignalasAAUAAA,isthencleavedbya
specialendonucleasethatrecognizesthesignalandcuts
atasite11to30residuestoits3'side.MaturepolII
mRNAsarepolyadenylatedatthe3′-end,resultinginapoly
(A)tail(Template-independent);thisprocessfollows
cleavageandisalsocoordinatedwithtermination.

25. Termination of transcription in eukaryotes:
addition of poly(A) tails

26. TRANSCRIPTION FACTORS

27. Transcription factors
•Transcriptionalcontrolisorchestratedbyalargenumberof
protein,called“transcriptionfactor”.
•About10%geneinthehumangenome encodes
transcriptionfactors.
•RNA-poldoesnotbindthepromoterdirectly.
•RNA-polIIassociateswithsixtranscriptionfactors-TFIIA,
TFIIB,TFIID,TFIIE,TFIIF,TFIIH.
•Thesefactors,positionpolymerase moleculesat
transcriptionstartsitesandhelptomelttheDNAstrandsso
thatthetemplatestrandcanentertheactivesiteofthe
enzyme.

28. Types
•Thegeneralfactors:RequiredfortheinitiationofRNAsynthesisat
allpromoters.Theydeterminethesiteofinitiation;thiscomplex
constitutethebasaltranscriptionapparatus.
•Theupstreamfactors:DNA-bindingproteinsthatrecognizespecific
shortconsensuselementslocatedupstreamthetranscriptionstart
point(e.g.Sp1,whichbindstheGCbox).Theyincreasethe
efficiencyofinitiation.
•Theinduciblefactors:Functioninthesamegeneralwayasthe
upstreamfactors,buthavearegulatoryrole.Theyaresynthesizedor
activatedatspecifictimesandinspecifictissues.

29. Structure of transcription factors
•IT HAS 2 DOMAINS :
1.DNA binding domain. 2.Activation
domain.
•GAL4 and GCN4 are yeast
transcription activators.
•The glucocorticoid receptor (GR)
promotes transcription of target
genes.
•SP1 binds to GC-rich promoter
elements in a large number of
mammalian genes.

30. Factor Mass ( kD) Function
TFIIA 69 Stabilize TBP & TAF binding
TFIIB 35 Stabilize TBP binding,
recognize BRE element
TFIID TBP 38 Recognizes TATA box
TAF >960 Regulates DNA binding by
TBP
TFIIE 165 Regulates helicase activity of
TFIIH
TFIIF 87 Binding of TFIIE & TFIIH
TFIIH 470 Unwinds DNA at the
transcription start point

31. Importance of transcription factors

32. •TBP and TFII Dbinds TATA
•TFII Aand TFII Bbind TFII D
•TFIIF-RNA-pol complex binds TFII B
•TFII Fand TFII Eopen the dsDNA (helicase and
ATPase)
•TFII H: completion of PIC
Pre-initiation complex (PIC)

33. Pre-initiation complex (PIC)RNA pol II
TF II F
TBP TFIIDTATA
DNA TF II
A TF II
B TF II E
TF II H

34. •TFII His of protein kinaseactivity to
phosphorylateCTDof RNA-pol. (CTDis the
C-terminal domain of RNA-pol)
•Only the P-RNA-polcan move toward the
downstream, starting the elongation phase.
•Most of the TFsfall off from PICduring the
elongation phase.
Phosphorylationof RNA-polymerase
P-RNA-pol

35. RNAPII pre-initiation complex

36. Activators, Mediators
&
Chromatin Modifying Proteins

37. CONTENTS
ACTIVATORS
DEFINITION
STRUCTURE
ROLE IN TRANSCRIPTIONAL REGULATION
FUNCTION
MEDIATORS
DEFINITION
STRUCTURE
ROLE IN TRANSCRIPTIONAL REGULATION
FUNCTION
CHROMATIN MODIFYING PROTEINS

38. ACTIVATORS

39. Definition
“ An activator is a DNA-binding protein that
regulates one or more genes by increasing the
rate of transcription.”
They stimulate transcription by two
mechanism:
They interact with mediators proteins and
general transcription factors to facilitate the
assembly of a transcription complex and
stimulate transcription.
They interact with co-activators that facilitate
transcription by modifying chromatin structure.

40. Structure

41. Role In Transcriptional Regulation
How does an activator stimulate transcription?
•The recruitment model argues that its sole effect is to increase the
binding of RNA polymerase to the promoter.
•An alternate model is to suppose that it induces some change in the
conformation of the enzyme.

42. Function
• Activators bind short sequence
elements.
•Activators interact with basal
apparatus

43. •Response
elements are
recognized by
activators.
•Activators help in
the chromatin
modification.
•Recruits
transcription
machinery.

44. MEDIATORS

45. Definition
“Mediator is a multi protein complex
that functions as a transcriptional
co-activator.”

46. Structure
Mediator is a large
complex of 21
polypeptides with a
combined weight of
1MDa.
Single particle
electron microscopy
images reveals that it
had an elongated,
roughly conical
shape,400 A
o
in
length.

47. Role In Transcriptional Regulation
•Assist in the
assembly of Pol II
pre initiation
complexes.
•Also some
mediators have
histone acetylase
activity.

48. Function
•Stimulation of the basal transcription
•Its activity as a co-repressor

49. CHROMATIN MODIFYING
PROTEINS

50. Definition
The general process of
inducing changes in the
chromatin structure is called
chromatin remodeling or
chromatin modification.
And hence the proteins
involved in the modification
of the chromatin structure
so that the transcription
factor and the RNA
Polymerase can get an
access to the promoter
DNA and make the gene
transcribable.

51. Chromatin Remodeling Enzymes
These includes
acetylases,
deacetylases,
methylases, etc.
Changes in the
chromatin structure
are initiated by
modifying the N-
terminus tail of the
histones, especially
H3 & H4.

52. Histone acetyl transferases
•Enzymes that can acetylate
histones are called Histone
acetyl transferases.
•Acetylate conserved lysine
amino acids on histone
protein by transferring an
acetyl group from acetyl CoA
to form e-N-acetyl lysine.
•Histone acetylation
neutralizes the positive charge
which renders DNA
accessible to transcription
factor & hence linked with
transcriptional activation.

53. Histone deacetylases
These are a class of
enzymes that remove
acetyl group from e-N-
acetyl lysine on a histone.
Its action is opposite to the
histone acetyl transferases.
They remove those acetyl
groups increasing the
positive charge of histones
and encouraging high-
affinity binding between the
histones and the DNA
backbone.
Increased DNA binding
condenses DNA structure,
preventing transcription.

54. Histone methylases
Histones methylases are
enzymes that catalyze the
transfer of one to three
methyl groups from the
cofactor S-Adenosyl
methionineto lysine and
arginineresidues of
histone.
Methylatedhistones bind
more tightly, which inhibits
transcription.
Deacetylationallows
methylationto occur,
which causes formation of
a heterochromatic
complex.

55. •Transcription factors bind
to specific sequences.
•Remodeling complex binds
via factor.
•Factor is released.
•Remodeling changes the
nucleosomal organization.
•Acetylasecomplex binds
via remodeling complex.
•Histones are modified.

56. RNA SPLICING

57. DEFINATION OF RNA SPLICING
• The process of cutting the pre-RNA to
remove the introns and joining together of the
exons is called splicing.
• This process is done on RNA strands so it
is known as RNA SPLICING.
• In Eukaryotes it takes place in the nucleus
before the mature RNA can be exported to the
cytoplasm.

58. The intronis also present in the RNA copy of the gene and
must be removed by a process called “RNA splicing”
protein
translation
mRNA
RNA splicing
pre-mRNA
intron

59. RNA SPLICING
•MostintronsstartfromthesequenceGUandendwith
thesequenceAG(inthe5'to3'direction).Theyare
referredtoasthesplicedonorandsplice
acceptorsite,respectively.However,thesequences
atthetwositesarenotsufficienttosignalthepresence
ofanintron.Anotherimportantsequenceiscalled
thebranchsitelocated20-50basesupstreamofthe
acceptorsite.Theconsensussequenceofthebranch
siteis"CU(A/G)A(C/U)",whereAisconservedinall
genes.
•Inover60%ofcases,theexonsequenceis(A/C)AGat
thedonorsite,andGattheacceptorsite.
59

60. RNA SPLICING
60

61. MECHANISM OF SPLICING
RNA splicing mechanism by Spliceosomes
1. Self or Cis-splicing mechanism
-Splicing in single RNA
-Lariat shape
-Common
2. Trans-splicing mechanism
-Splicing in two different RNAs
-Y-shape
-Rare (e.g. C. elegance and higher
eukaryotes

62. Spliceosome Complex Formation
A spliceosome is a complex of specialized
RNA and PROTEIN subunits.
Composed of five snRNPs (U1, U2, U4, U5
and U6),other splicing factorsand the pre-
mRNAbeing assembled.
U1 binds to the 5’ splice site, and U2 to the
branch point, after that the tri-snRNP
complex of U4, U5 and U6.
As a result, the intron is looped out and the
5’ and 3’ exon are brought into close
proximity.
U2 and U6 snRNP are able to catalyze the
splicing reaction.

63. BBP =
Branchpoint
binding
protein
snRNP=Small
Ribo Nucleotide
Protein
U2AF=Artificial
Factor
ASSEMBLY OF
SPLICEOSOME
RNA SPLICING MECHANISM

65. Self-Splicing mechanism-
Group-I intron sequences
1. Guanine in introns
initiate attack on 5’
splice sites.
2. 3’OH of upstream
exons reacts with
downstream exons.
3. Exons are joined and
lariat is released.

66. “hand”
Self-Splicing mechanism-
Group-II intron sequences
1. Adenine in introns
initiates attack on
5’splice sites.
2. 3’-OH of upstream
exons reacts with
downstream exons.
3. Exons is joined and
lariat is released.

67. TRANS -Splicing MECHANISM
SL RNA=SPLICED LEADER RNA

68. SUMMARY
RNA PROCESSING
3 steps are involved
Addition -5’ cap on pre mRNA
Addition -3’poly A tail on pre mRNA
RNA Splicing

69. Addition of 5’cap and 3’poly(A) tail
Add acapand apoly(A)tail to pre -
mRNA

70. m-RNA splicing
•Eukaryotic mRNAs are spliced by
complexes of small nuclear Ribonucleo-
Proteins (snRNPs).
70

71. Splicing mechanism

72. TRANSCRIPTION IN EUKARYOTES
72

73. 73
COMPLETE PROCESS -Transcription and Translation

74. DIFFERENCE BETWEEN PROKARYOTIC & EUKARYOTIC TRANSCRIPTION
PROKARYOTES
1. Single core DNA dependent
RNA Polymerase
2. RNA Polymerase do not require
additional protein (i.e.
Transcription factor) for
initiation and regulation of
transcription.
3. Transcription takes place on
free DNA
4. Promoter sequences-
TATpuATpu (Pribnow box)
located -10 bp of upstream.&
TTGACA Located -35 bp
upstream
EUKARYOTES
1. Multiple different DNA
dependent RNA Polymerases-
I, II, III
2.. RNA Polymerase requires a
variety of additional proteins
(i.e. Transcription factors) for
initiation and regulation of
transcription.
3. Transcription takes place on
chromatin rather than on free
DNA (so chromatin structure
is an important factor).
4. Promoter sequences-TATAbox
(Hogness box)Located -30bp
upstream & CAATboxLocated
-70 to -80bp upstream
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75. THANK YOU