TRANSCRIPTION

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THRISSUR
Definition

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THRISSUR
 TRANSCRIPTION IS DEFINED
AS
SYNTHESIS OF NASCENT RNA FROM DNA

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 FEATURES REPLICATION TRANSCRIPTION
SIMILAR FEATURES
1.GENERAL STEPS INITIATION,ELONGATION AND TERMINATION WITH 5’-3’ POLARITY

2.INITIATION LARGE INITIATION COMPLEX

3.BASE PAIRING ADHERENCE TO WATSON AND CRICK BASE PAIRING

DIFFERING FEATURES
1.NUCLEOTIDES DEOXYRIBONUCLEOTIDES RIBONUCLEOTIDES

2.BASE THYMINE URACIL

3.THE PROCESS a.ENTIRE GENOME COPIED a.ONLY PORTIONS OF GENOME COPIED

b.PRIMER REQUIRED b.NO PRIMER REQUIRED

4.PROOF READING HIGHLY ACTIVE NO PROOF READING

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Terminologies

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 TERMINOLOGY DESCRIPTION

1.TEMPLATE STRAND The strand of DNA transcribed into RNA

2.CODING STRAND The other/ Non-template strand of DNA .It corresponds

exactly to the sequence of mRNA primary transcript

3.TRANSCRIPTION UNIT Region of DNA that includes the signals for transcription

4.PRIMARY TRANSCRIPT The RNA product which is synthesized in 5’-3’

DIRECTION
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ENZYME:RNA
Polymerase

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 Bacterial/Prokaryotic
RNA polymerase
DNA-dependent
RNA polymerase
Eukaryotic RNA
polymerase.

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Bacterial DNA-dependent RNA polymerase
• Multisubunit enzyme

• 2 identical α subunits

• 2 similar but not identical β subunits

• ω subunit

• σ subunit
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CORE ENZYME
ββ’α2ω (E)
HOLOENZYME
Eσ

σ SUBUNIT
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DEPARTMENT OF BIOCHEMISTRY,GOVT MEDICAL COLLEGE
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• The σ factor increases holoenzyme affinity to promoter DNA.

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THRISSUR
Eukaryotic DNA-dependent RNA polymerase
RNA MAJOR PRODUCT SENSITIVITY TO
POLYMERASE AMANTIN

I rRNA Insensitive

II mRNA , miRNA, snRNA High sensitivity

III tRNA Intermediate

sensitivity
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• Eukaryotes : 3 distinct nuclear DNA dependent RNA polymerase

• 2 large subunits and number of small subunits.

• All of them require general transcription factors (GTFs).

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Steps of transcription

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 TERMINATIO
INITIATION ELONGATION
N

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Transcription in
Prokaryotes
Initiation

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Signals for initiation :Promoters
Consensus sequences:

a) -35 : 5’-TGTTGACA-3’

b) -10 : 5’-TATAAT-3’ a.k.a TATA Box or PRIBNOW box

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DEPARTMENT OF BIOCHEMISTRY,GOVT MEDICAL COLLEGE
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DEPARTMENT OF BIOCHEMISTRY,GOVT MEDICAL COLLEGE
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Initiation:The PROCESS.

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 RNAP holoenzyme
Unwinding or melting
binds to a region in The complex of DNA around TSS . The unwinding allows
DNA called undergoes a
PROMOTER with the (PRE-INITIATION active site of Eσ to
temperature
help of σ factor. COMPLEX/OPEN access the template
dependent
PROMOTER strand
(CLOSED PROMOTER conformation change
COMPLEX)
COMPLEX)

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 The first Polymerase
Formation of
nucleotide undergoes
stable
associates with RNAP catalyses conformational
ternary transcri
nucleotide the formation RNAP incorporates change and
ption complex,
binding site on of first nucleotides(3-10) moves away
(enzyme, from the
the beta phosphodieste at which point..
the DNA, and promoter
subunit of r bond
the (PROMOTER
RNAP.
nascent RNA.) CLEARANCE)

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Transcription in
Prokaryotes
Elongation

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 The RNAP Synthesis
Release of σ
proceeds along along 5’-3’
factor from
the template direction
polymerase
DNA @40nt/sec

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TRANSCRIPTION BUBBLE

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DEPARTMENT OF BIOCHEMISTRY,GOVT MEDICAL COLLEGE
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• Successive residues are added to the 3′-OH terminus of the
nascent RNA molecule until a transcription termination signal (T)
is encountered.

• The pyrophosphate released is degraded into 2 phosphates

making the reaction irreversible.

• As elongation proceeds, DNA unwinding occurs to provide

appropriate base pairing.
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Transcription in
Prokaryotes
Termination

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 Termination
2 modes

Rho Rho
dependent independent
Rho dependent termination
• Rho factor : Protein with RNA dependent ATPase activity.

• Rho factor translocates along nascent RNA till it catches up with RNA
polymerase.

• Disrupts the transcription complex.

• Releasing template DNA and RNA from each other ,also from the
polymerase.
Rho independent termination
G-C rich palindrome preceeds a sequence of 6-7 Uracil
residues in RNA chain

• At the end of transcription , The RNA transcribed from this

region folds back on itself, and the complementary C and G
nucleotides bind together : hairpin/stem-loop structure

• This is followed by the oligo U sequence which weakly

interacts with template DNA.
• Stem-loop structure stalls the RNA
polymerase

• Along with weak interaction ,causes

RNA polymerase to detach,
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Transcription in
Eukaryotes
Initiation

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 Signals for transcription
Sl no: Function of the signal Signals
1 Promoters -32 : TATAAAAG :functional similarity to TATA box
Defines where transcription
starts
-25 to -30 :TATAAA :Goldberg-Hogness box

2 Determines frequency of GC sequence

transcription
CAAT box

3 Regulates the rate of Enhancers

transcription
Repressors

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 Transcription factors
Sl no: CLASSES FACTORS
1
BASAL COMPONENTS RNAP II,TBP, GTFs(TFIIA, TFIIB, TFIID, TFIIE, TFIIF,
TFIIH)

2
COREGULATORS TAFs, Mediators

3
ACTIVATORS SP1,ATF,CTF

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 TBP (TATA binding
protein)
TFIID
Binding to the TATA
box :FIRST step in
initiation
TAF(TBP associated
factors)

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DEPARTMENT OF BIOCHEMISTRY,GOVT MEDICAL COLLEGE
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 PRE-INITIATION COMPLEX

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Phosphorylation of the repetitive C-terminal domain (CTD) of the largest
RNA polymerase (RNAP) II subunit plays a key role in the progression of
RNAP through the transcription cycle. 

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Transcription in
Eukaryotes
Elongation

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• Similar to elongation in prokaryotes.

• RNA polymerase has intrinsic unwindase activity that opens the helix.

• Topoisomerase preceeds and follows the progressing RNA polymerase

to prevent tensions.

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Transcription in
Eukaryotes
Termination

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• Exact termination sequence not known.

• Endonuclease
E
cleaves the transcript 15 bases 3’ to AAUAAA.

• This sequence serves as termination as well as

polyadenylation signal.
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DEPARTMENT OF BIOCHEMISTRY,GOVT MEDICAL COLLEGE
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POST TRANSCRIPTIONAL
MODIFICATIONS/PROCES
SING
REPLICATION TRANSCRIPTION PTM TRANSLATION
Primary transcript

DEFINITION :mRNAs formed and released from DNA template .

SYNONYM : Heteronuclear RNA or hnRNA

Post transcriptional modification
in
Prokaryotes
a. Translation starts even before
transcription ends.

b. Transcription and translation

are coupled.

c. No compartmentalisation as in
eukaryotes.

d. Prokaryotic rRNA and tRNA

requires more processing than
mRNA
Post transcriptional modification
in
Eukaryotes
 mRNA tRNA rRNA

CAPPING AT 5’END CLEAVAGE SINGLE LONG PRECURSOR

RELEASES SMALLER
rRNAS
TAILING AT 3’END 3’ADDITION

REMOVAL OF INTRONS MODIFIED BASES

SPLICING OF EXONS

METHYLATION

RNA EDITING
PTM1 : 5’ Capping at 5’ end
• First processing reaction of mRNA.

• A cap of 7-methyl guanosine is attatched backward at 5’ terminal end

of mRNA

• Bond: 5’-5’ triphosphate linkage

Importance of capping :

a) Guides RNA into cytoplasm.

b) Protection of 5’end from exonuclease activity.

c) Efficient translation initiation

PTM 2 :Addition of poly A tail
• mRNA is cleaved about 20nt downstream from an AAUAA recognition
sequence.

• Poly(A) polymerase adds poly(A) tail which can be extended to as

many as 200 adenine residues.
• Importance:

a) Protection of 3’end from exonuclease activity.

b) Facilitate translation.
PTM 3: Methylation
• Secondary methylation of mRNA molecule occurs after entry
into cytoplasm.

• Usual methylated residues include :

a. N6 mono & di-methyl adenylate
b. Methylation of ribose of terminal nucleotide
PTM 3: Removal of introns
PTM 4: Splicing of exons
EXONS: Genetic sequences in hnRNA which appears in mature
RNA and gets expressed .

INTRONS : Genetic sequences which neither appear in mature

RNA nor contribute to genetic information .

Introns are more heterogenous than exons .

 SPLICING

The process by which introns, the noncoding

regions of genes, are excised out of the primary
messenger RNA transcript, and the exons (i.e.,
coding regions) are joined together to generate
mature messenger RNA.
 Spliceosom
e

SnURPS hnRNA

snRNA Proteins hnRNA

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DEPARTMENT OF BIOCHEMISTRY,GOVT MEDICAL COLLEGE
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PTM 5 : RNA Editing

• Molecular process through which some cells can make

discrete changes to specific nucleotide sequences within an
RNA molecule after it has been generated by RNA
polymerase.

• Coding information can be changed at RNA level.

Example of RNA editing :

Apo B gene Liver : apoB gene is transcribed into mRNA that directs
synthesis of apoB100

Intestine :Same gene directs synthesis cytidine deaminase converts CAA

into UAA forming apoB48
Processing of tRNA
a) Non specific cleavage occurs at 3’ end by RNAase D and at 5’ end by
ribonuclease P.

b) Addition of CCA by tRNA ribonucleotidyl transferase

c) Removal of introns in the anticodon loop.

d) Modification in bases : Dihydrouracil, methylated bases and

Pseudouridine catalysed by specific enzymes.
Processing of rRNA

• Of all rRNAs found in ribosomes(5S,5.8S,18S,28S) ,all except

5S are synthesized from single large rRNA precursor of size
45S.

• Some bases are modified by methylation .

• 5S is transcribed independently.
 mRNA tRNA rRNA

CAPPING AT 5’END CLEAVAGE SINGLE LONG PRECURSOR

RELEASES SMALLER
rRNAS
TAILING AT 3’END 3’ADDITION

REMOVAL OF INTRONS MODIFIED BASES

SPLICING OF EXONS

METHYLATION

RNA EDITING
 INHIBITORS OF
TRANSCRIPTION
 INHIBITOR SOURCE MODE OF ACTION
ACTINOMYCIN-D ANTIBIOTIC FROM STREPTOMYCES INSERTION OF PHENOXAZONE
RING BETWEEN G-C bp OF DNA

RIFAMPICIN SYNTHETIC DERIVATIVE OF RIFAMPIN BINDS TO BETA SUBUNIT OF RNA

POLYMERASE

ALPHA-AMANTIN TOXIN FROM MUSHROON INACTIVATES RNA POLYMERASEII

3’-DEOXY ADENOSINE SYNTHETIC ANALOGUE INCORRECT ENTRY INTO CHAIN

CAUSING CHAIN TERMINATION
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