RNA

Synthesis
(Transcription)
 Transcription
Definition:
Transcription is the process of copying a segment
of DNA into RNA. The segments of DNA transcribed
into RNA molecules that can encode proteins are
said to produce messenger RNA. OR
The copying process, during which a DNA strand
serves as a template for the synthesis of RNA, is
called transcription OR
transcription is the synthesis of RNA from DNA.
 The Central Dogma of Life
(Francis Crick,1958)

Replicatio
n
Requirements
• Template = DNA single strand
• Substrates = Ribonucleotides (ATP, GTP, UTP, CTP)
• Initiation factors
• Transcription factors [Tf–II or GTF (General Transcription factors)]
(Tf-II include Tf-IIA, Tf-IIB, Tf-IIC, Tf-IID, Tf-IIE, Tf-IIF, Tf-IIH)

– Tf-IID contains TATA binding protein (TBP) and 14 TAFs

(Transcription activating factors)

• Activators, Co-activators and other components of PIC

(pre-initiation complex)

• ATP & GTP (energy)

• Mg++ or Mn++
 Requirements
• Enzymes = DNA-dependent RNA polymerases
[RNA polymerase (RNAP) I, II, III]
– RNAP–I synthesizes rRNA
– RNAP–II synthesizes mRNA
– RNAP–III synthesizes tRNA, 5S rRNA

• Each RNAP holoenzyme consists of Core-enzyme + one or

more sigma () factors

• Sigma () factors assist the core enzyme to recognize
promoter region
• (A sigma factor is a protein needed for initiation of transcription)
 RNA Synthesis (Transcription)
• A cyclical process, involving RNA chain initiation, elongation &
termination

• Sigma factor(s) helps the core enzyme to recognize & bind to the
promoter region

• RNA polymerase attaches to promoter and initiates transcription

at a distinct site(TSS)

• GTFs (Tf-II) facilitate promoter-specific binding of RNAPs to form

pre-initiation complex (PIC)

• Other proteins (co-activator proteins) regulate the rate of

transcription initiation
 RNA Synthesis (Transcription)Steps
(six steps occurring in the nucleus of eukaryotic cells)
1. Attachment of RNAP to promoter region at DNA ( hydrogen
bonds b/w two strands are broken & DNA melts-----unzips)
2. DNA strands pull apart from each other
3. RNAP finds ribonucleotides (as ATP, UTP etc) and matches
them up with one (template) strand of unzipped DNA
4. RNAP continues to match up ribonucleotides with template
strand of DNA until it reaches a point(sequence) on DNA
which signals “termination”
5. RNA breaks off from DNA & leaves the nucleus
6. Once RNA leaves , the DNA “zips” back together
Chain Initiation
 RNA PRIMER
• DNA polymerases cannot initiate synthesis of a
complementary strand of DNA on a totally single
-stranded template.

• Rather, they require an RNA primer, which is a

short, double -stranded region consisting of RNA
base -paired to the DNA template, with a free
hydroxyl group on the 3'-end of the RNA strand.

• This OH group serves as the first acceptor of a

deoxy nucleotide by action of a DNA
polymerase.
 Primase & Primosome

• A specific RNA polymerase, called primase ,

synthesizes the short stretches of RNA
(approximately ten nucleotides long) that
are complementary and anti parallel to the
DNA template.
• In the resulting hybrid duplex, the U (uracil)
in RNA pairs with A in DNA.
 Primosome
• The addition of primase converts the
prepriming complex of proteins required for
DNA strand separation to a primosome.

• The primosome makes the RNA primer required

for leading strand synthesis and initiates
Okazaki fragment formation in lagging strand
synthesis .
• As with DNA synthesis , the direction of
synthesis of the primer is 5'→3'.
Chain Elongation
 RNA Synthesis (Steps)
Elongation
• After addition of 10 – 20 ribonucleotides, RNAP undergoes a
second structural change
• Grips the template more firmly and moves away from the
“promoter” (elongation phase).
• Progresses along DNA molecule – thereby producing “bubble”
(DNA unwinding) along DNA template as RNA synthesis
proceeds (elongation)
 Elongation is not smooth along DNA molecule; instead RNAP is
made to back-up, proof-read and erase the incorrect
nucleotide(s), before further RNA synthesis
 Chain Elongation
All four substrates (NTP)
-(de oxyadenosine triphosphate [dATP ],
-de oxythymidine triphosphate [dTTP],
-de oxycytidine triphosphate [dCTP] &
-de oxyguanosine triphosphate [dGTP])
must be present for DNA elongation to
occur.
Note: If one of the four is in short supply,
DNA synthesis stops.
 Chain Elongation
• 2. Proofreading of newly synthesized DNA:
• It is highly important for the survival of an
organism that the nucleotide sequence of
DNA be replicated with as few errors as
possible .
• Misreading of the template sequence could
result in deleterious or even lethal mutations.
 Chain Elongation
• 2. Proofreading of newly synthesized DNA:
• To ensure replication fidelity, DNA pol III has
a “proofreading” activity (3'→5'
exonuclease, in addition to its 5'→3'
polymerase activity).
 Chain Elongation
• As each nucleotide is added to the chain,
DNA pol III checks to make certain the
added nucleotide is, in fact, correctly
matched to its complementary base on
the template .
• If it is not, the 3'→5' exonuclease activity
removes the error.
 Example of proof reading
• For example , if the template base is C
and the enzyme mistakenly inserts an A
instead of a G into the new chain, the
3'→5' exonuclease activity hydrolytically
removes the misplaced nucleotide. The
5'→3' polymerase activity then replaces
it with the correct nucleotide containing
G.
• [Note : The proof reading exonuclease
activity requires movement in the 3'→5'
direction, not 5'→3' like the polymerase
activity. This is because the excision must
be done in the reverse direction from that
of synthesis .]
‫‪ - Ayat No 53‬سورة حم السجدہ ‪Surat No 41 :‬‬

‫َا‬ ‫ؕ ‬ ‫ُّق‬ ‫ۡل‬ ‫ا‬ ‫َّن‬‫َا‬ ‫ۡم‬ ‫َل‬ ‫َن‬ ‫َت‬ ‫ی‬ ‫ّٰت‬
‫َس ُنِر ۡی ِہۡم ٰا ٰی ِتَنا ِفی اٰاۡل َفاِق َو ِفۤۡی َاۡن ُفِس ِہۡم َح َی َبَّی ُہ ُہ َح َو‬
‫﴾ َلۡم َیۡک ِف ِبَر ِّبَک َاَّنٗہ َع ٰل ی ُک ِّل َش ۡی ٍء َش ِہۡی ٌد ﴿‪۵۳‬‬

‫ہم عنقریب انہیں اپنی نشانیاں َاطراِف عالم میں اور خود ُان کی‬
‫ذاتوں میں ِد کھا دیں گے یہاں تک کہ ُان پر ظاہر ہو جائے گا کہ وہی‬
‫حق ہے ۔ کیا آپ کا رب ( آپ کی حقانیت کی تصدیق کے لئے )‬
‫کافی نہیں ہے کہ وہی ہر چیز پر گواہ ( بھی ) ہے‬
 DNA LIGASE
• The final phosphodiester linkage between the
5'-phosphate group on the DNA chain
synthesized by DNA pol III and the 3'-hydroxyl
group on the chain made by DNA pol III is
catalyzed by DNA ligase.
• The joining of these two stretches of DNA
requires energy, which in most organisms is
provided by the cleavage of ATP to AMP +
PPi.
Chain Termination
Termination
• Specific sequences or specific signals are recognized
by rho () factor of RNAP (in Prokaryotes)
• Rho factor attaches to DNA; the RNAP cannot move
further,
• Dissociates from DNA and

• Newly synthesized RNA is released.

• The Rho factor is a protein that acts in bacterial cells
to mediate termination of transcription at distinct sites.
 RNA Synthesis (Transcription Steps)

• In eukaryotes, the termination is independent of Rho factor

• RNA endonucleases cleave the RNA at consensus sequence

AAUAAA in RNA script which serves as cleavage signal

• Completed RNA chain and RNAP are released from template

DNA strand

• RNAP holoenzyme reforms, finds another promoter and

synthesis of new RNA starts
RNA Synthesis (Transcription)
 Drugs inhibiting transcription
Actinomycin D (anti-tumor drug)
• Binds to DNA template & blocks the movement
of RNA polymerase

Rifampin (anti-tuberculous drug)

• Binds to β-subunit & inhibits prokaryotic RNA
polymerase

α-Amanitin (present in poisonous mushrooms)

• Tightly binds to eukaryotic RNA polymerase &
inhibits transcription
 Comparison
of
Replication & Transcription
Comparison of Replication & Transcription
S.No Replication Transcription

1 DNA template is required DNA template is required

Whole genome is copied Selective parts of DNA

2 are copied
Initiation & termination Initiation & termination
3 signals are not required signals are required
4 RNA primer is required Primer is not required
Synthesized DNA strand Synthesized RNA does
5 remains attached to not remain attached to
template strand template DNA strand
Deoxy-ribonucleotides are Ribonucleotides are
6 required required
Comparison of Replication & Transcription
S.No Replication Transcription
Synthesis occurs in 5 − Synthesis occurs in 5 −
7
3 direction 3 direction
Phospho-diester bonds Phospho-diester bonds
8 are formed are formed
9 DNA polymerases RNA polymerases
Different functions by Different functions by
10 different enzymes one enzyme
Very much accurate Less accurate
11 (exact)
No changes are required Changes are required
12 after replication after transcription
Comparison of Replication & Transcription
S.No Replication Transcription
Replication bubble Transcription bubble
13 formation after unwinding formation after unwinding

Location- nucleus Location- nucleus

14
Products do not degrade Products gets degraded
after their function. after their function get
15 complete
 Possible Questions(VIVA & SEQs)

• What is transcription?
• What are requirements for transcription.
• Name steps of transcription.(little bit
explanation)
• What is Pre-initiation complex (PIC)
• Comparison of replication and transcription.
• Functions of RNA polymerases.
• How RNA polymerase is different from DNA
polymerase?