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, or RNA synthesis, is the process of
creating RNA , copy of a sequence of DNA.
 Both RNA and DNA are nucleic acids, which
use base pairs of nucleotides as a complementary
language that can be converted from DNA to RNA in
the presence of the correct enzymes.
 During transcription, a DNA sequence is read by RNA
polymerase, which produces a complementary RNA
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) regulatory RNAs (regulation of
transcription and translation, other??)
siRNAs (small interfering) regulatory RNAs (regulation of
transcription and translation, other??)
Other non-coding RNAs telomere synthesis, X-chromosome
inactivation, protein transport

6. The Central Dogma
▪ The central dogma of molecular biology describes the two-step
process, transcription and translation, by which the information
in genes flows into proteins.
▪ Conventional concept (pre-bioinformatics era) of central dogma
of life:
It is an over simplification of molecular biology.
DNA → RNA → Protein
▪ Current concept (Bioinformatics era) of central dogma of life:
With the advances in cell biology and rapid developments
in bioinformatics, the term Genome, Transcriptome and
Proteome are in current use to represent the central dogma of
molecular biology.
Genome → Transcriptome → Proteome

7. The Central Dogma

8. 8
The Central Dogma

9. The Central Dogma

10. A STRUCTURAL 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: Exons code for amino acids and
acid sequence of the protein product. It is
these portions of the gene that are
represented in final mature mRNA
molecule.
• Introns: Introns are portions of the gene
that do not code for amino acids, and are
removed (spliced) from the mRNA
molecule before translation.

14. Control regions
• Start site:- A start site for transcription.
• Promoter:- A region a few hundred
nucleotides 'upstream' of the gene (toward
the 5' end). It is not transcribed into mRNA,
but plays a role in controlling the
transcription of the gene. Transcription
factors bind to specific nucleotide
sequences in the promoter region and assist
in the binding of RNA polymerases.

15. • Enhancers: Some transcription factors
(called activators) bind to regions called
'enhancers' that increase the rate of
transcription. Some enhancers are
conditional and only work in the presence of
other factors as well as transcription
factors.
• Silencers: Some transcription factors
(called repressors) bind to regions called
'silencers' that depress the rate of
transcription.

16. Eukaryotic RNA Polymerases (RNAPs)
 In bacteria (prokaryote), all mRNA is made from the
same RNA polymerase (single RNAP). However, in
eukaryotes, there are three different RNA polymerases
in animals and four in plants.
1. RNA Polymerase I: synthesizes rRNA
2. RNA Polymerase II: synthesizes all Protein
coding genes & mostly mRNA.
3. RNA polymerase III: synthesizes tRNAs and
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 rRNA genes (5.8S, 18S, and 28S)
RNA pol II mRNA genes (protein coding genes),
snoRNA genes, some snRNA genes,
microRNAs genes
RNA pol III tRNA genes, 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
core promoter sequence 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 specific transcription
factors.

19. • The most common type of core promoter in eukaryotes is a
short DNA sequence known as a TATA box (Hogness box). The
TATA box, as a core promoter, is the binding site for a
transcription factor known as TATA binding protein (TBP),
which is itself a subunit of another transcription factor, called
Transcription Factor II D (TFIID).
• One transcription factor, DNA helicase, has helicase activity
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:
• In eukaryotes, the RNA is processed at both ends
before it is spliced.
• At the 5‘ end, a cap is added consisting of a
modified GTP (guanosine triphosphate). This occurs
at the beginning of transcription. The 5' cap is used
as a recognition signal for ribosomes to bind to the
mRNA.
• At the 3' end, a poly(A) tail of 150 or more adenine
nucleotides is added. The tail plays a role in the
stability of the mRNA.
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 cleavage of the new
transcript followed by template-independent
addition of As at its new 3' end, in a process
called polyadenylation.

23. Termination of transcription in eukaryotes:
addition of poly(A) tails
• In eukaryotes, termination of transcription occurs
by different processes, depending upon the exact
polymerase utilized. For pol I genes, transcription
is stopped using a termination factor, through a
mechanism similar to rho-dependent termination
in bacteria. Transcription of pol III genes ends
after transcribing a termination sequence that
includes a polyuracil stretch, by a mechanism
resembling rho-independent prokaryotic
termination. Termination of pol II transcripts,
however, is more complex.

24. Termination of transcription in eukaryotes:
addition of poly(A) tails
• Transcription of pol II genes can continue for hundreds or
even thousands of nucleotides beyond the end of a coding
sequence. The RNA strand is then cleaved by a complex
that appears to associate with the polymerase. Cleavage
seems to be coupled with termination of transcription and
occurs at a consensus sequence (TTATTT on coding
region of template strand of DNA and consequently
AAUAAA sequence on pre-mRNA). The pre-mRNA,
carrying this signal as AAUAAA, is then cleaved by a
special endonuclease that recognizes the signal and cuts
at a site 11 to 30 residues to its 3' side. Mature pol II
mRNAs are polyadenylated at the 3′-end, resulting in a poly
(A) tail (Template-independent); this process follows
cleavage and is also coordinated with termination.

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

26. TRANSCRIPTION FACTORS

27. Transcription factors
• Transcriptional control is orchestrated by a large number of
protein ,called “transcription factor”.
• About 10% gene in the human genome encodes
transcription factors.
• RNA-pol does not bind the promoter directly.
• RNA-pol II associates with six transcription factors- TFII A,
TFIIB, TFIID, TFIIE, TFIIF, TFII H.
• These factors, position polymerase molecules at
transcription start sites and help to melt the DNA strands so
that the template strand can enter the active site of the
enzyme.

28. Types
• The general factors : Required for the initiation of RNA synthesis at
all promoters. They determine the site of initiation ; this complex
constitute the basal transcription apparatus.
• The upstream factors : DNA-binding proteins that recognize specific
short consensus elements located upstream the transcription start
point (e.g. Sp1, which binds the GC box). They increase the
efficiency of initiation.
• The inducible factors : Function in the same general way as the
upstream factors, but have a regulatory role. They are synthesized or
activated at specific times and in specific tissues.

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 D binds TATA
• TFII A and TFII B bind TFII D
• TFII F-RNA-pol complex binds TFII B
• TFII F and TFII E open 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 TFIID
TATA
DNA
TF II
A
TF II
B
TF II E
TF II H

34. • TF II H is of protein kinase activity to
phosphorylate CTD of RNA-pol. (CTD is the
C-terminal domain of RNA-pol)
• Only the P-RNA-pol can move toward the
downstream, starting the elongation phase.
• Most of the TFs fall off from PIC during the
elongation phase.
Phosphorylation of 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 Ao 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
methionine to lysine and
arginine residues of
histone.
 Methylated histones bind
more tightly, which inhibits
transcription.
 Deacetylation allows
methylation to 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.
• Acetylase complex binds
via remodeling complex.
• Histones are modified.

56. RNA SPLICING

57. DEFINATIONOF 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 intron is 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
• Most introns start from the sequence GU and end with
the sequence AG (in the 5' to 3' direction). They are
referred to as the splice donor and splice
acceptor site, respectively. However, the sequences
at the two sites are not sufficient to signal the presence
of an intron. Another important sequence is called
the branch site located 20 - 50 bases upstream of the
acceptor site. The consensus sequence of the branch
site is "CU(A/G)A(C/U)", where A is conserved in all
genes.
• In over 60% of cases, the exon sequence is (A/C)AG at
the donor site, and G at the acceptor site.
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 factors and the pre-
mRNA being 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 a cap and a poly(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 & CAATbox Located
-70 to -80bp upstream
74

75. THANK YOU