Transcription and post-transcriptional modification targeted for MBBS, MD and BDS students.

1. TRANSCRIPTION
RNA METABOLISM
TRANSCRIPTION
Prof. Dr .V P Acharya
MD, PhD Biochemistry

3. Specific features
 Macromolecule, needed for storage and
transmission of information
 Ribozyme discovery- expanded the domain of
enzymes from proteins to RNA
 Has both structural and catalytic role
 3 major RNAs
 Many regulatory RNAs
 Cellular Transcriptome- The sum of all the RNA
molecules produced in a cell under a given set of
 conditions

4. mRNA
Transcription
The Central Dogma
Cell
Polypeptide
(protein)
Translation Ribosome
Reverse
transcription DNA

5.  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

6.  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).

7. 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)

8. Transcription

9. 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

10. 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

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

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

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

15. 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++

16. 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

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

18. Stages of Transcription
 Initiation
 Elongation
 Termination

20. 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

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

23. 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

24. 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

25. ELONGATION

26. Steps of prokaryotic
transcription

27. 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

28. Promoter clearance
 In eukaryotes- a transition phase
 Just before Elongation proper
 After initial synthesis of +3- ~10
nucleotides have been
polymerized, conformational
change occurs in RNAP and it
physically moves away from the
promoter down the transcription
unit
 On many genes σ factor

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

30. Termination

31. 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

32. 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

33. Termination
Rho independent

34. 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

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

36. 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

37. Formation of POL-II Transcription
complex
 PIC= pol II + 6 GTF (TF IIA, B,D, E, F, H)
 GTFs- serve to promote RNAP II transcription
 15 subunit TF II D complex- 15 subunits+ TBP+
13-14- TBP TAFs
 Bind to TATA box promoter through TBP and TAF
 TF II D – capable of specific and high affinity
binding to promoter independently
 Most TFs bind to DNA on major groove
 TBP binds to DNA on minor groove- makes a 1000
kink
 This kink facilitates interaction between complex
and other factors

38. Eukaryotic basal transcription
complex
 To the TF II D – promoter
complex TF IIA and then TF
II B bind giving rise to a
stable multi-protein complex
↓
Attract and tether Pol II and TF
II F to the promoter
↓
TFIIE and TFIIH bind
↓
Formation of PIC (-30 to +30
encompassing TSS +1;

39. 2 mechanisms of PIC formation

40. Nucleosomes regulate PIC
formation??
 If genes lack TATA box then Inr (Initiator
sequence)&/or DPE (downstream promoter element)
serve to position the complex for accurate
transcription
 Nucleosome at times masks the promoter elements
i.e. TATA-Inr-DPE
↓
TFs bind to enhancer upstream and recruit chromatin
remodeling and modifying co-regulatory factors such
as Swi/Snf, SRC-1, p300/CBP, p/CAF etc
↓
Repressing nucleosomes removed
 Epigenetic code of DNA and histone and protein

43. Multiple sites of transcription

44. HN RNA
•Primary mRNA transcript
•Heteronuclear RNA
Formed in Nucleus
Undergoes extensive
editing

45.  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

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

47. 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

49. 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

50. Mechanisms of splicing are many

51. MC mechanism of splicing in
eukaryotes
 There are reasonably conserved sequences at
each of the two exons–intron (splice) junctions
and at the branch site, which is located 20 to
40 nucleotides upstream from the 3′–splice
site
 Spliceosomes= SnRNA+ >60 proteins+
conserved RRM (RNA recognition & SR
(serine–arginine) protein motifs)
 SnRNPs position exon and intron segments

52. 5’ end exon and intron cut
↓
Achieved by nucleophilic attack by an adenylyl residue in the branch
point sequence
located just upstream from the 3′ end of this intron
↓
Free end forms a lariat/ loop
↓
Loop is linked by an unusual 5′–2′
phosphodiester bond to the reactive A in the PyNPyPyPuAPy branch
site
Sequence
↓
The branch site identifies the 3’site and 2nd cut (Transesterification
reaction); 3′ hydroxyl of the upstream exon attacks
the 5′ phosphate at the downstream exon–intron boundary and the lariat
structure containing the intron is released and hydrolyzed
↓
5’ & 3’ ends are ligated

53. Alternate processing of mRNA-
leads to genetic regulation

54. 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

55. 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

57. TREX represents a likely molecular link between
transcription elongation complexes, the RNA splicing
machinery, and
nuclear export

58. 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

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

60. 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)

62. For more ppt on medical Biochemistry please visit my
website www.vpacharya.com