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Dr. N. Sivaranjani
Asst. Prof.
Definition
Synthesis of RNA using ssDNA as a template by DNA dependent
RNA polymerase enzyme.
Similar to replication in terms of chemical
mechanism, polarity, and use of template
but differs in
-does not require primers
-only a short segment of DNA is
transcribed
DNA
RNA
Protein
TRANSCRIPTION
TRANSLATION
Nucleus
All 3 types of cellular RNA’s are copied during
transcription
Messenger RNAs (mRNAs) encode the amino acid sequence
of one or more polypeptides specified by a gene.
Transfer RNAs (tRNAs) read the information encoded in
the mRNA and transfer the appropriate amino acid to a
growing polypeptide chain during protein synthesis.
Ribosomal RNAs (rRNAs) are constituents of ribosomes,
the intricate cellular machines that synthesize proteins.
Salient features of transcription
Synthesis of ALL types of RNA in Nucleus
 Only ONE STRAND of DNA participates
 Ribonucleotides are used in RNA synthesis.
 RNA Synthesis occurs in 5’-3’ direction , DNA template is read from
3’-5’ direction
 Synthesis follows Waston-Crick base pairing rules – A to U, G to C
 DNA dependent RNA polymerase or RNA polymerase
• ss DNA
Template
•DNA dependent RNA polymerase
Enzyme
• ATP, GTP,CTP,UTPRibonucleoside
triphosphates
Basic Requirements of Transcription
Steps involved in Transcription
INITIATION
ELONGATION
TERMINATION & Release
1.Promoter region: It is the specific region in DNA ,where
transcription is initiated.
2.Transcription unit: It is the region where DNA template is
transcribed. Present in b/w promoter and terminating units.
3.Termination unit: It is the region where transcription terminates.
Template DNA
DNA
Promoter region Transcription unit Termination unit
Initiation
Starts with the recognition of promoter sequence on the DNA coding ( anti-
template ) strand by RNA polymerase
Promoter sequence
Are specific areas on the DNA that act as starting signals for initiation
process recognized by RNAP.
 Two common sequences are present on the upstream side of the start site
of transcription .
 Start site is denoted by +1
Pribnow box or TATAAT box:
It contains 6 nucleotide bases TATAAT located -10 bases
away on the left of origin of transcription.
 -35 sequence / 5`-TTGACA-3`seqence:
it is 35 bp upstream of the transcription unit
Promoters of prokaryotes
TTGACA5` 3`
Pribnow box
RNAP- prokaryotes
RNAP of prokaryotes has 5 sub units-2 alpha units , beta, beta1 and
sigma unit (2α,β,β’,δ).
alpha
Alpha`
betaBeta`
Sigma
factor
Core enzyme
Beta Beta1
Alpha Alpha
 RNA polymerase without
σ subunit is called core
enzyme
 Core enzyme contains the
catalytic activity.
 Core enzyme with sigma
factor is holo-enzyme
Subunit Role
α Binds regulatory proteins
β Forms phosphodiester bond
β’ Fixes RNAP to DNA template
σ Recognizes and binds to promoter region of DNA,
Initiates transcription.
Functions of RNAP
In bacteria, one species of RNAP can synthesize all the RNA molecules
( mRNA, tRNA, rRNA )
RNA polymerase differs from DNA Polymerase in two aspects
1. No primer is required for RNAP.
2. RNAP lacks a separate proofreading 3’ to 5’ exonuclease activity.
The error rate for transcription is higher.
Error rate is 104-105 times more than replication
Errors cause little damage – as they are not transmitted to daughter
cell/ next generation.
Identification of promoter region:
Sigma unit of RNA Polymerase identifies the promoter region on
template DNA .
RNA polymerase binds to the promoter region of the template DNA
Beta unit fixes to the promoter region of the template strand and
initiates transcription.
Transcription bubble
RNA
Sigma factor is released and RNAP in promoter region unwinds DNA
helix.
A Transcription bubble is formed as the DNA unwinds down stream
Transcription bubble contains RNAP,
DNA and the new RNA formed
Supercoils is overcome by Topoisomerase
Elongation
RNA
polymerase
RNA nucleotide
Direction of
transcription
Newly made RNA
Template
strand of DNA
 RNA polymerase elongates an RNA strand
by adding ribonucleotide units to the 3’-
hydroxyl end.
 RNAP uses ribonucleoside triphosphates,
forms 3’5’ PDE bond b/w adjacent
ribonucleotide and releases ppi each time
a nucleotide is added to the growing chain
 Each nucleotide in the newly formed RNA
is selected by Watson-Crick base-pairing
interaction. (G to C, A to U)
Rho dependent termination - A hexameric protein called
Rho factor attaches to the DNA strand and RNAP can not move
further and dissociates from DNA strand terminating the
transcription
 Rho independent termination – palindrome like bases
occurs at end sequence of DNA. Due to these sequences, the newly
synthesized RNA folds on itself to form hair pin loop (due to
complemenatry basepairing) which terminates the movement of
RNAP.
 palindrome – word / phrases that read alike backwards and forwards –
MADAM, MALAYALAM
Termination
Termination
Rho- factor
ATP ADP + pi
Rho factor is an ATP dependent RNA-DNA helicases
Recognizes and bind to the termination signals and
disrupts the nascent RNA/DNA complex
Termination
HAIR PIN LOOP
Prokaryotes Eukaroytes
Simple More complex
One RNAP 3 distinct RNAP
Promoter site –
Pribnow box
35 sequence
Promoter site –
TATA box – Hogness box ,
CAAT box
Initiation –
Only requires sigma
factor
Initiation –
6 Transcription factors interact with
eukaryotic promoter region.
POST TRANSCRIPTIONAL
MODIFICATION
Promoters of eukaryotes
Goldberg –hogness box;
In eukaryotes a sequence TATAAA is located at 25-30 bp
upstream to the start point it acts as signal to initiate
the transcription.
CAAT box :
GGCAATCT Sequence is located 70 bp upstream to start point.
TATAAA
RNAP of eukaryotes are of 3 types
RNAP-I synthesizes - rRNA
RNAP-II synthesizes - mRNA
RNAP-III synthesizes - tRNA
Transcription factors binds to DNA
sequences in promoter region
Stimulated by enhancers
6 Transcription factors – TFIID,
TFIIA,TFIIB, TFIIF, TFIIE, TFIIH.
Transcription factors binds to each
other and in turn to enzyme RNAP – form
preinitiation complex or basal
transcription complex
 Enhancer – increases gene expression
by 100 fold
 Enhancers bind to TFs to form
Activators
 They have no Promoter activity of
their own but stimulate the
transcription gene.
 They can be present upstream,
downstream or within a gene
 Silencers – DNA sequences which bind
proteins that act to inhibit the rate of
transcription.
Post transcriptional modifications
The mRNA formed from DNA is called the primary transcript or
hnRNA.
It undergoes extensive modifications to become active and mature
mRNA.
These modifications are called as post transcriptional modifications.
Eukaryotic RNA is processed before leaving the nucleus
hnRNA to mRNA
1.capping at 5`end
2. Poly A tailing at 3’
3.Splicing - removal of introns
4. mRNA EDITING
Post transcriptional modifications
5’ capping
7-methylguanylate attached by a unusual 5’-5’ triphosphate linkage to
the ribose at the 5’-end.
Addition of GTP part of the cap is catalyzed by nuclear enzyme
guanylyltransferase.
Methylation of terminal guanine occurs in the cytosol- SAM is the
source of the methyl group
Catalysed by guanine-7-methyl transferase.
Importance
The cap binds mature mRNA to the ribosome during protein
biosynthesis.
Cap Stabilizes mRNAs against digestion by ribonucleases.
Eukaryotic mRNAs lacking the cap are not translated efficiently.
Addition of poly A tail
Poly A tail added at 3’ end of mRNA
200-300 adenylate residues linked by
PDE bonds
ATP is the donor of adenylate group
It is involved in stabilization of mRNA
Splicing
Process by which introns are removed & exons are joined to form
the functional mRNA .
Requires energy
Small nuclear RNAs associated with specific proteins to form complex
–snRNPs (small nuclear ribonucleic protein particles) or snurps ,
involved in formation of spliceosomes.
Spliceosomes –is a complex containing multiple snRNPs that contain
snRNA that catalyze hnRNA to mRNA, by removing introns and joining
exons.
15% genetic disease – due to splicing defects
Faulty splicing – causes βThalassaemia.
DNA
RNA
transcript
with cap
and tail
mRNA
Exon Intron IntronExon Exon
Transcription
Addition of cap and tail
Introns removed
Exons spliced together
Coding sequence
NUCLEUS
CYTOPLASM
Tail
Cap
mRNA editing
0.01% of the mRNAs undergoes editing.
 enzyme mediated alteration of base sequence of RNA (not by splicing )
Ex:- conversion of CAA codon in mRNA (of apoprotein B gene) to UAA
by the enzyme cytidine deaminase .
 Originating from the same gene, the liver synthesizes a 100-kDa
protein (apoB 100) while the intestinal cells synthesize 48-kDa protein
(apoB 48).
This happens due to formation of a termination codon UAA from CAA
in RNA editing.
tRNA
Cleavage of a 5’leader sequence.
Splicing to remove intron.
Replacement of the 3’terminal
UU by CCA &
Modification of several bases –
dihydrouridine, pseudouridine,
Thymine, methylated bases.
 28 s, 18s, 5.8 s are synthesized
as long precursor – Preribosomal
45S RNAs
 This is cleaved and trimmed to
produce mature functional rRNA
 5 S rRNA is produced by
transcription of 5S gene by RNA
polymerase III & modified
separately.
rRNA
Inhibitors Mode of action
Rifampicin antitubercular
drug
Only inhibits
prokaryotes
binds to beta subunit
of RNA polymerase
Actinomycin D synthesized by
Streptomyces
Both Binds with DNA strand
& blocks movements of
all forms of RNA.
a-Amanitin toxin produced
by Mushroom,
Amanita
phalloides
Eukaryotes tightly binds with RNA
polymerase ll & inhibits
transcription
Inhibitors of transcription
Reverse transcription
Generation of DNA from RNA is
reverse transcription
Enzyme catalyzing this is reverse
transcriptase or RNA dependent
DNA polymerase
Usually seen in RNA viruses of
retrovirus group
 Some of the tumor viruses
Eg: HIV causing AIDS is a
retrovirus.
genetic information is
transferred from RNA to DNA
Ribozyme
Enzymes made up of RNA are called ribozymes
Catalytic RNA molecules with sequence specific cleavage activity
exhibit Michaelis-Menten kinetics
Ex:-
Spliceosomes - contains ribozymes as well as protein. Involved in Post
transcriptional modification.
 RNAse-P- generates the ends of tRNAs.
 Peptidyl transferase -used for protein biosynthesis

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RNA Transcription: A Concise Guide

  • 2. Definition Synthesis of RNA using ssDNA as a template by DNA dependent RNA polymerase enzyme. Similar to replication in terms of chemical mechanism, polarity, and use of template but differs in -does not require primers -only a short segment of DNA is transcribed DNA RNA Protein TRANSCRIPTION TRANSLATION Nucleus
  • 3. All 3 types of cellular RNA’s are copied during transcription Messenger RNAs (mRNAs) encode the amino acid sequence of one or more polypeptides specified by a gene. Transfer RNAs (tRNAs) read the information encoded in the mRNA and transfer the appropriate amino acid to a growing polypeptide chain during protein synthesis. Ribosomal RNAs (rRNAs) are constituents of ribosomes, the intricate cellular machines that synthesize proteins.
  • 4. Salient features of transcription Synthesis of ALL types of RNA in Nucleus  Only ONE STRAND of DNA participates  Ribonucleotides are used in RNA synthesis.  RNA Synthesis occurs in 5’-3’ direction , DNA template is read from 3’-5’ direction  Synthesis follows Waston-Crick base pairing rules – A to U, G to C  DNA dependent RNA polymerase or RNA polymerase
  • 5. • ss DNA Template •DNA dependent RNA polymerase Enzyme • ATP, GTP,CTP,UTPRibonucleoside triphosphates Basic Requirements of Transcription
  • 6. Steps involved in Transcription INITIATION ELONGATION TERMINATION & Release
  • 7. 1.Promoter region: It is the specific region in DNA ,where transcription is initiated. 2.Transcription unit: It is the region where DNA template is transcribed. Present in b/w promoter and terminating units. 3.Termination unit: It is the region where transcription terminates. Template DNA DNA Promoter region Transcription unit Termination unit
  • 8. Initiation Starts with the recognition of promoter sequence on the DNA coding ( anti- template ) strand by RNA polymerase Promoter sequence Are specific areas on the DNA that act as starting signals for initiation process recognized by RNAP.  Two common sequences are present on the upstream side of the start site of transcription .  Start site is denoted by +1
  • 9. Pribnow box or TATAAT box: It contains 6 nucleotide bases TATAAT located -10 bases away on the left of origin of transcription.  -35 sequence / 5`-TTGACA-3`seqence: it is 35 bp upstream of the transcription unit Promoters of prokaryotes TTGACA5` 3`
  • 11. RNAP- prokaryotes RNAP of prokaryotes has 5 sub units-2 alpha units , beta, beta1 and sigma unit (2α,β,β’,δ). alpha Alpha` betaBeta` Sigma factor Core enzyme Beta Beta1 Alpha Alpha  RNA polymerase without σ subunit is called core enzyme  Core enzyme contains the catalytic activity.  Core enzyme with sigma factor is holo-enzyme
  • 12. Subunit Role α Binds regulatory proteins β Forms phosphodiester bond β’ Fixes RNAP to DNA template σ Recognizes and binds to promoter region of DNA, Initiates transcription. Functions of RNAP In bacteria, one species of RNAP can synthesize all the RNA molecules ( mRNA, tRNA, rRNA )
  • 13. RNA polymerase differs from DNA Polymerase in two aspects 1. No primer is required for RNAP. 2. RNAP lacks a separate proofreading 3’ to 5’ exonuclease activity. The error rate for transcription is higher. Error rate is 104-105 times more than replication Errors cause little damage – as they are not transmitted to daughter cell/ next generation.
  • 14. Identification of promoter region: Sigma unit of RNA Polymerase identifies the promoter region on template DNA . RNA polymerase binds to the promoter region of the template DNA Beta unit fixes to the promoter region of the template strand and initiates transcription.
  • 15. Transcription bubble RNA Sigma factor is released and RNAP in promoter region unwinds DNA helix. A Transcription bubble is formed as the DNA unwinds down stream Transcription bubble contains RNAP, DNA and the new RNA formed Supercoils is overcome by Topoisomerase
  • 16. Elongation RNA polymerase RNA nucleotide Direction of transcription Newly made RNA Template strand of DNA  RNA polymerase elongates an RNA strand by adding ribonucleotide units to the 3’- hydroxyl end.  RNAP uses ribonucleoside triphosphates, forms 3’5’ PDE bond b/w adjacent ribonucleotide and releases ppi each time a nucleotide is added to the growing chain  Each nucleotide in the newly formed RNA is selected by Watson-Crick base-pairing interaction. (G to C, A to U)
  • 17. Rho dependent termination - A hexameric protein called Rho factor attaches to the DNA strand and RNAP can not move further and dissociates from DNA strand terminating the transcription  Rho independent termination – palindrome like bases occurs at end sequence of DNA. Due to these sequences, the newly synthesized RNA folds on itself to form hair pin loop (due to complemenatry basepairing) which terminates the movement of RNAP.  palindrome – word / phrases that read alike backwards and forwards – MADAM, MALAYALAM Termination
  • 18. Termination Rho- factor ATP ADP + pi Rho factor is an ATP dependent RNA-DNA helicases Recognizes and bind to the termination signals and disrupts the nascent RNA/DNA complex
  • 20.
  • 21.
  • 22. Prokaryotes Eukaroytes Simple More complex One RNAP 3 distinct RNAP Promoter site – Pribnow box 35 sequence Promoter site – TATA box – Hogness box , CAAT box Initiation – Only requires sigma factor Initiation – 6 Transcription factors interact with eukaryotic promoter region. POST TRANSCRIPTIONAL MODIFICATION
  • 23. Promoters of eukaryotes Goldberg –hogness box; In eukaryotes a sequence TATAAA is located at 25-30 bp upstream to the start point it acts as signal to initiate the transcription. CAAT box : GGCAATCT Sequence is located 70 bp upstream to start point. TATAAA
  • 24.
  • 25. RNAP of eukaryotes are of 3 types RNAP-I synthesizes - rRNA RNAP-II synthesizes - mRNA RNAP-III synthesizes - tRNA
  • 26. Transcription factors binds to DNA sequences in promoter region Stimulated by enhancers 6 Transcription factors – TFIID, TFIIA,TFIIB, TFIIF, TFIIE, TFIIH. Transcription factors binds to each other and in turn to enzyme RNAP – form preinitiation complex or basal transcription complex
  • 27.  Enhancer – increases gene expression by 100 fold  Enhancers bind to TFs to form Activators  They have no Promoter activity of their own but stimulate the transcription gene.  They can be present upstream, downstream or within a gene  Silencers – DNA sequences which bind proteins that act to inhibit the rate of transcription.
  • 28.
  • 29.
  • 30.
  • 31. Post transcriptional modifications The mRNA formed from DNA is called the primary transcript or hnRNA. It undergoes extensive modifications to become active and mature mRNA. These modifications are called as post transcriptional modifications. Eukaryotic RNA is processed before leaving the nucleus
  • 32. hnRNA to mRNA 1.capping at 5`end 2. Poly A tailing at 3’ 3.Splicing - removal of introns 4. mRNA EDITING
  • 33. Post transcriptional modifications 5’ capping 7-methylguanylate attached by a unusual 5’-5’ triphosphate linkage to the ribose at the 5’-end. Addition of GTP part of the cap is catalyzed by nuclear enzyme guanylyltransferase. Methylation of terminal guanine occurs in the cytosol- SAM is the source of the methyl group Catalysed by guanine-7-methyl transferase.
  • 34. Importance The cap binds mature mRNA to the ribosome during protein biosynthesis. Cap Stabilizes mRNAs against digestion by ribonucleases. Eukaryotic mRNAs lacking the cap are not translated efficiently.
  • 35. Addition of poly A tail Poly A tail added at 3’ end of mRNA 200-300 adenylate residues linked by PDE bonds ATP is the donor of adenylate group It is involved in stabilization of mRNA
  • 36. Splicing Process by which introns are removed & exons are joined to form the functional mRNA . Requires energy Small nuclear RNAs associated with specific proteins to form complex –snRNPs (small nuclear ribonucleic protein particles) or snurps , involved in formation of spliceosomes. Spliceosomes –is a complex containing multiple snRNPs that contain snRNA that catalyze hnRNA to mRNA, by removing introns and joining exons. 15% genetic disease – due to splicing defects Faulty splicing – causes βThalassaemia.
  • 37. DNA RNA transcript with cap and tail mRNA Exon Intron IntronExon Exon Transcription Addition of cap and tail Introns removed Exons spliced together Coding sequence NUCLEUS CYTOPLASM Tail Cap
  • 38.
  • 39. mRNA editing 0.01% of the mRNAs undergoes editing.  enzyme mediated alteration of base sequence of RNA (not by splicing ) Ex:- conversion of CAA codon in mRNA (of apoprotein B gene) to UAA by the enzyme cytidine deaminase .  Originating from the same gene, the liver synthesizes a 100-kDa protein (apoB 100) while the intestinal cells synthesize 48-kDa protein (apoB 48). This happens due to formation of a termination codon UAA from CAA in RNA editing.
  • 40. tRNA Cleavage of a 5’leader sequence. Splicing to remove intron. Replacement of the 3’terminal UU by CCA & Modification of several bases – dihydrouridine, pseudouridine, Thymine, methylated bases.
  • 41.  28 s, 18s, 5.8 s are synthesized as long precursor – Preribosomal 45S RNAs  This is cleaved and trimmed to produce mature functional rRNA  5 S rRNA is produced by transcription of 5S gene by RNA polymerase III & modified separately. rRNA
  • 42. Inhibitors Mode of action Rifampicin antitubercular drug Only inhibits prokaryotes binds to beta subunit of RNA polymerase Actinomycin D synthesized by Streptomyces Both Binds with DNA strand & blocks movements of all forms of RNA. a-Amanitin toxin produced by Mushroom, Amanita phalloides Eukaryotes tightly binds with RNA polymerase ll & inhibits transcription Inhibitors of transcription
  • 43. Reverse transcription Generation of DNA from RNA is reverse transcription Enzyme catalyzing this is reverse transcriptase or RNA dependent DNA polymerase Usually seen in RNA viruses of retrovirus group  Some of the tumor viruses Eg: HIV causing AIDS is a retrovirus. genetic information is transferred from RNA to DNA
  • 44. Ribozyme Enzymes made up of RNA are called ribozymes Catalytic RNA molecules with sequence specific cleavage activity exhibit Michaelis-Menten kinetics Ex:- Spliceosomes - contains ribozymes as well as protein. Involved in Post transcriptional modification.  RNAse-P- generates the ends of tRNAs.  Peptidyl transferase -used for protein biosynthesis