RNA synthesis

1. TRANSCRIPTION
Prof. Dr .V P Acharya

3. mRNA
Transcription
The Central Dogma
Cell
Polypeptide
(protein)
Translation Ribosome
Reverse
tanscription DNA

4.  Decoding DNA:
DNA→ RNA → PROTEIN
Two separate processes involved:
 Transcription – DNA used as the template
to make RNA
 Translation – RNA serves as the template
for the sequence of amino
acids in a protein

5.  The first step in expressing a gene
 Occurs in the nucleus
 DNA-directed RNA synthesis
 An RNA copy of DNA is made.
 This RNA serves as a messenger
between the nucleus and the
cytoplasm (mRNA).

6. How big part of human transcribed RNA
results in proteins?
 Of all RNA, transcribed in higher
eukaryotes, 98% are never translated into
proteins
• Of those 98%, about 50-70% are introns
• 4% of total RNA is made of coding RNA
• The rest originate from non-protein genes,
including rRNA, tRNA and a vast number
of other non-coding RNAs (ncRNAs)

7. Transcription

8. Similarities with replication
 Involves general steps of initiation,
elongation and termination
 5’→3’ polarity
 Large, multicomplex initiation
complex
 Adherence to Watson-Crick base
pairing rule

9. Differences between replication and
transcription
 Ribonucleotides
 U in place of T
 Primer not involved
 Very small portion of the genome
is transcribed
 No proofreading function of
transcription
 Doesn’t stop at one cycle

10. Requisites for transcription
 Template
 Substrate– ATP, GTP,CTP &
UTP
 Enzyme– DNA dependent RNA
polymerase/ RNA polymerase
(RNAP)

12. DNA template 3'- ATACTGGAC - 5'
RNA product 5'- UAUGACCUG - 3'
DNA non-template strand
5'- TATGACCTG - 3'

13. Transcription
5’
3’
3’
5’
Template
(antisense) strand
Coding
(sense) strand
5’
RNA
RNA
Pol.

14. Prokaryotic RNAP
 Single RNAP transcribing all 3 RNAs– mRNA,
rRNA & tRNA
 5 subunits-- 2α, β, β’,ω--E
 Sigma (σ)– 5th factor– helps in binding of RNAP
to specific promoter region of DNA template
 E σ- Holoenzyme
 RNAP- Metallozyme– Zn
 β- binds to Mg++

15. Eukaryotic RNAP
 3 RNAP
molecule location product
RNA polymerase I nucleolus 28S, 18S
5.8s rRNA
RNA polymerase II nucleus hnRNA,
mRNA,
some snRNAs
RNA polymerase III nucleus tRNA, 5S rRNA,
some snRNAs

16.  Recognition of the promoter
region
 Melting of DNA (Helicase +
Topoisomerase)
 RNA Priming (Primase)
 RNA Polymerization
 Recognition of terminator
sequence

17. Stages of Transcription
 Initiation
 Elongation
 Termination

19. Initiation (Prokaryotic)
 Promoter region recognised and sigma
factor binds to it
 Proteins called transcription factors
bind to the promoter region of a gene
 If the appropriate transcription factors
are present, RNA polymerase binds to
form an initiation complex
 RNA polymerase melts the DNA at the
transcription start site
 Polymerization of RNA begins

21. Transcription bubble
 Approx. 20 bp area
 Entire complex covers 30-75 bp
 Length depends on confirmation of
RNAP

22. Prokaryotic promoters
 TATA box/ Prinbow box– conserved
sequence on coding strand
 -10 bp– 5’TATAAT’3 sequence
 -35 bp– 5’TGTTGACA3’ sequence
 RNAP binds here to form closed
complexes
 AT rich regions– easily melted

23. Eukaryotic promoters
 Each type of RNAP uses a different
promoter
 Promoters used by RNAP I & II– same as
prokaryotic– upstream
 Promoter used by RNAP III– downstream
 Goldberg- Hogness box-- -25 to -30 bp
TATAAA sequence
 CAAT box-- -70 to -80 bp
 Eukaryotic initiation complex is very
complex

24. ELONGATION

25. Steps of prokaryotic transcription

26. Elongation
 RNAP binds at promoter site– Preinitiation
complex
↓
Conformational change in RNAP
↓
1st nucleotide (almost always a purine)
associates on β-subunit of the enzyme
↓
RNAP catalyses formation of a phosphodiester
bond in presence of appropriate nucleotide
↓
Elongation of RNA in 5’→3’ direction

27. Promoter clearance
 In eukaryotes- a transition phase
 Just before Elongation proper
 After initial synthesis of 10-20
nucleotides have been
polymerized, RNAP physically
moves away from the promoter
down the transcription unit

28. simultaneous DNA unwinding occurs
20bp / RNAP molecule
↓
Transcription bubble

29. Termination

30. Termination
 Rho dependent requires a protein called Rho,
that binds to and slides along the RNA transcript.
The terminator sequence slows down the
elongation complex, Rho catches up and knocks it
off the DNA
 Bacterial RNAP sometimes recognizes the DNA
encoded termination signals and dislodges
 Rho independent termination
 2nd GC-rich region that likes to form stem loop
structure
 Stem loop forms, pulling mRNA from template

31. Rho – ATP-dependent RNA stimulated helicase that
disrupts the nascent RNA-DNA complex
Terminator
RNA
Pol.
5’
RNA
r
RNA
Pol.
5’
RNA
Help, rho
hit me!
r
RNA
Pol.
5’
RNA

32. Termination
Rho independent

33. Eukaryotic termination
 Less well defined
 May be similar to Rho-independent
type
 RNA processing, termination and
polyadenylation proteins appear to
load onto RNAP-II soon after initiation

34. Differences between eukaryotic and
prokaryotic transcription
 Eukaryotes– different
compartments for transcription
and translation.
 Prokaryotes– translation starts
without undergoing processing
 Promoter sites

35. Eukaryotic transcription
 TATA box is bound by 34kDa protein TATA
binding protein (TBP)
 TBP + TAF (TBP associated factor)
↓
TF II D
↓
1st step of formation of transcription
complex
↓
Other factors attach
 Enhancers and silencers

36. Multiple sites of transcription

37. HN RNA
Primary mRNA transcript
Formed in Nucleus
Undergoes extensive
editing

38.  Endonuclease cleavage
 Poly-A tailing (20-250 A)
 5’ capping– 7-methyl GTP
 Methylation- Methylations of N6 of
Adenine residue and 2’-OH group
of ribose- done in cytoplasm
 Removal of introns
 Splicing of exons

39.  Prokaryotes- translation starts
even before mRNA is completely
synthesised
 t-RNA and r-RNA also undergo
post-transcriptional modification

40. Removal of introns
 Exons– expressed regions
 hn-RNA– M.W. 107 ; mature RNA 1-
2×106
 Introns removed and exons are
spliced (joined) together
 Energy requiring process
 Takes place in nucleus

42. SnRNA (Small nuclear)
 90-300 nucleotides
 U1,U2,U4,U5,U6 &U7
 Uracil-rich
 Present in nucleus
 SnRNA+ specific proteins=SNURP
(small nuclear ribonuclear protein
particles)
 Form spliceosomes (SnRNP +hnRNA at
exon -intron junction)
 Spliceosomes contain Ribozymes

43. Mechanism of splicing

44. Alternative splicing leads to differential
expression and certain diseases
 Beta thalassaemia- a mutation in
an intron- exon junction- absent
beta chain synthesis
 Glucokinase is expressed
differently in liver and pancreas
due to different promoters-
Differential splicing

45. Alternate editing
 ApoB gene generates ApoB100 in liver
and in intestine ApoB48
 In intestine same primary transcript is
formed but a cytidine deaminase
converts a CAA codon into UAA-
produces a 49kDa protein- ApoB48

47. Inhibitors of Transcription
Inhibitor Source Mode of action
Actinomycin-D Antibiotics from
streptomyces
Insertion of
phenoxazone ring
between two G-C bp
of DNA
Rifampin Rifamycin Binds to β-subunit
of RNAP
α-Amanitin Mushroom RNAP II
inactivated
3’-deoxyadenosine Synthetic analogue Incorrect entry
into chain causing
chain termination

48. mRNA
Transcription
Reverse transcription
Cell
Polypeptide
(protein)
Translation Ribosome
Reverse
tanscription DNA

49. mi-RNA
 Derived from large primary transcripts
through specific nucleolytic processing
 Transcribed by RNAP-II
 Genes located independently or within the
intronic DNA
 Comes out to cytoplasm- acted upon by a
dicer nuclease and gets incorporated into
RISC (RNA induced silencing complex)