Eukaryotic transcription

I. Pre initiation complex

To prepare for transcription, a complete set of general transcription factors and RNA polymerase need to be assembled at the core promoter to form a pre initiation complex.
promoters that contain a TATA box near the TSS (transcription start site), the recognition of “TATA box by the TBP subunit of TFIID, initiates the assembly of a transcription complex.
TFIID, is a complex made up of TATA binding protein (TBP) and at least 12 TBP associated factors (TAF’s).
TBP is a sequence specific protein that binds to DNA via its unusual TBP domain which makes contact with the minor groove in the region of the TATA box.
During transcription, TAFS assists in attachment of TFIID to the TATA box.
After TFIID has attached to the core promoter, the pre-initiation complex (PIC) is formed by attachment of the TFIIA and TFIIB,
which stabilize the DNA-TFIID complex and recruit RNA polymerase II in association with TFIIF and additional transcription factors.

TFIIF serves as a bridge between the TATA bound TBP and polymerase.
The GTF’s, TFIIE and TFIIH are one of the last transcription factors to be recruited to the pre-initiation complex.
TFIIH, which play an important role in promoter melting and escape possess both kinase and helicase activity.
The final step in assembly of the initiation complex is the addition of phosphate groups to the C-terminal domain (CTD) of the largest subunit of RNA polymerase
Activation of TFIIH results in phosphorylation of CTD resulting in the formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region and begin synthesizing RNA.
TFIIH therefore plays an important role in control of transcription elongation.

II. Promoter melting and open complex formation

After formation of preinitiation, complex melting or separation of the two DNA strands and the positioning of the template strand to the active site of the RNA polymerase.
For polymerase II transcribed genes and unlike bacterial RNA polymerase, promoter melting requires hydrolysis of ATP and is mediated by TFIIH. (having both ATPase and protein kinase activities).
While the upstream promoter DNA is held in a fixed position by TFIID, TFIIH pulls downstream double stranded DNA into the cleft of the polymerase driving the separation of DNA strands and the transition of the pre-initiation complex from the closed to open state.
TFIIB aids in open complex formation by binding the melted DNA and stabilizing the transcription bubble.

III. Abortive initiation

Once the initiation complex is open, the first ribonucleotide is brought into the active site to intiate the polymerization reaction in the absence of a primer.
This generates a nascent RNA chain that forms a hetero duplex with the template DNA strand. However, before entering the elongation phase, polymerase may terminate prematurely and release a short transcript.
This process is called abortive initiation.
Many cycles of the abortive initiation may occur before the transcript grows to a sufficient length (-10 nucleotides) to promote polymerase escape from the promoter.
Throughout abortive initiation cycles, RNA polymerase remain bound to the promoter and pulls downstream DNA into its catalytic cleft in a scrunching kind of motion.

IV. Promoter escape

When the transcript attains the threshold length of ten nucleotides, it enters the RNA exit channel.
The polymerase breaks its interactions with the promoter elements and any regulatory proteins associated with the initiation complex that it no longer needs.
Promoter escape in eukaryotes requires ATP hydrolysis and in case of pol II, phosphorylation of the CTD
Meanwhile, the transcription bubble collapses down to 12-14 nucleotides providing kinetic energy required for the escape.

V. Elongation

After escaping the promoter and shedding most of the transcription factors for initiation, the polymerase acquires new factors for the next phase of transcription i.e. elongation.
Transcription elongation is a processive process.
Double stranded DNA that enters from the front of the enzyme is unzipped to avail the template strand for RNA synthesis.
For every DNA base pair, separated by the advancing polymerase one hybrid RNA DNA base pair is immediately formed.
DNA strands and nascent RNA chain exit from separate channels; the two DNA strands unite at the trailing end of the transcription bubble while the single stranded RNA emerges alone.

Elongation factors:

Among the proteins recruited to polymerase are elongation factors thus called because they stimulate transcription elongation.
There are different classes of elongation factors.
Some factors can increase the overall rate of transcription, some can help the polymerase through transient pausing sites, and some can assist the polymerase to transcribe through chromatin.
One of the elongation factors, P-TEFb (Positive transcription elongation factor) is particularly important.
P-TEFb phosphorylates and activates SPT5 which is a universal transcription factor that helps recruit 5′-capping enzyme to pol II with a CTD phosphorylated at Ser-5.
P-TEFb also helps suppress transient pausing of polymerase when it encounters certain sequences immediately following initiation.

VI. Termination

The last stage of transcription is termination which leads to the dissociation of the complete transcript and the release of RNA polymerase from the template DNA.
The process differs for each of the three RNA polymerases.

(a) Factor dependent termination

The termination of transcription of pre-r RNA genes by RNA polymerase I is performed by a system that needs a specific transcription termination factor.
The mechanism bears some resemblance to rho dependent termination in prokaryotes.

For RNA pol-II termination, as pol II reaches the end of a gene, two protein complexes carried by the CTD, CPSF (Cleavage and polyadenylation specificity factor) and CSTF (cleavage stimulation factor) recognize the poly A signal in the transcribed RNA
Poly A bound CPSF and CSTF recruit other proteins to carry out RNA cleavage and then polyadenylation.
Poly A polymerase adds approximately 200 adenines to the cleaved 3′ end of the RNA without a template.
The long poly (A) tail is unique to transcripts made by pol II.

(b) Factor independent termination

RNA polymerase III can terminate transcription efficiently without involvement of additional factors.
The pol III termination signal consists of a stretch of thymines (on the non template strand) located within 40 bp downstream from the 3′ end of mature RNAs.
The poly-T termination signal pauses Pol III and causes it to backtrack to the nearest RNA hairpin to become a “dead-end” complex .
The RNA hairpin allosterically opens pol III and causes the elongation complex to disintegrate.

Section I: Pre-initiation Complex & General Transcription Factors

Which subunit of TFIID recognizes the TATA box?
a) TAFs
b) TBP ✅
c) TFIIH
d) TFIIF
(PYQ – CSIR NET 2020)
Which transcription factor has helicase activity in eukaryotic transcription?
a) TFIIA
b) TFIIB
c) TFIIE
d) TFIIH ✅
(PYQ – GATE Life Sciences 2021)
The core promoter region usually contains which sequence element?
a) GC box
b) TATA box ✅
c) CAAT box
d) Enhancer
TAFs are associated with which transcription factor?
a) TFIIH
b) TFIID ✅
c) TFIIF
d) TFIIE
Which factor serves as a bridge between TBP and RNA polymerase II?
a) TFIIH
b) TFIIF ✅
c) TFIIE
d) TAF
What is the role of TFIIA in transcription initiation?
a) Helicase activity
b) CTD phosphorylation
c) Stabilizes DNA-TFIID complex ✅
d) DNA repair
CTD phosphorylation is carried out by:
a) TFIIA
b) TFIIH ✅
c) TFIIF
d) TFIID
How many TAFs are typically associated with TBP in TFIID?
a) 2
b) 5
c) 12 ✅
d) 20
Which general transcription factor is the last to join the PIC?
a) TFIIA
b) TFIIH ✅
c) TFIID
d) TFIIB
TFIIH is essential for which step?
a) DNA repair
b) Promoter clearance
c) Promoter melting ✅
d) Splicing

Section II: Promoter Melting and Open Complex Formation

Promoter melting in eukaryotes requires:
a) GTP
b) ATP ✅
c) UTP
d) None
Which factor stabilizes the transcription bubble?
a) TFIIA
b) TFIIB ✅
c) TFIID
d) TFIIE
The open complex is characterized by:
a) Double-stranded DNA
b) DNA-RNA hybrid ✅
c) Protein-RNA duplex
d) None
Which factor pulls downstream DNA into polymerase cleft?
a) TFIIB
b) TFIIH ✅
c) TFIIF
d) TAFs
What is the role of ATP in promoter melting?
a) Phosphorylation
b) Energy for strand separation ✅
c) mRNA splicing
d) DNA replication

Section III: Abortive Initiation

Abortive initiation occurs when polymerase:
a) Fails to bind DNA
b) Releases short RNA transcripts prematurely ✅
c) Melts DNA strands
d) Initiates elongation
Abortive transcripts are usually:
a) >100 nucleotides
b) <10 nucleotides ✅
c) Full-length mRNA
d) DNA-RNA hybrids
During abortive initiation, polymerase performs:
a) Sliding
b) Scrunching ✅
c) Looping
d) Relaxation
RNA polymerase during abortive cycles remains:
a) Free in cytosol
b) Bound to promoter ✅
c) Bound to enhancer
d) Inactive
Abortive initiation is overcome when RNA reaches:
a) 3 nt
b) 5 nt
c) 10 nt ✅
d) 15 nt

Section IV: Promoter Escape

Promoter escape requires:
a) GTP hydrolysis
b) ATP hydrolysis and CTD phosphorylation ✅
c) Helicase activity only
d) RNA capping
What collapses during promoter escape?
a) Entire transcription bubble
b) Transcription bubble to 12–14 nucleotides ✅
c) RNA-DNA hybrid
d) Enhancer region
CTD phosphorylation during promoter escape occurs at:
a) Serine-2
b) Serine-5 ✅
c) Serine-7
d) Threonine
Promoter escape allows:
a) Initiation to restart
b) RNA capping
c) Polymerase to enter elongation phase ✅
d) Chromatin opening

Section V: Elongation

Which factor suppresses RNA polymerase pausing?
a) TFIIB
b) P-TEFb ✅
c) TAFs
d) TFIIA
P-TEFb activates:
a) TBP
b) SPT5 ✅
c) TFIID
d) TFIIH
The nascent RNA exits from:
a) DNA channel
b) RNA exit tunnel ✅
c) Active site
d) CTD
SPT5 is involved in:
a) Enhancer activation
b) 5’ capping ✅
c) RNA splicing
d) CTD dephosphorylation
DNA strands re-anneal at:
a) Transcription start site
b) Leading edge
c) Trailing end of bubble ✅
d) RNA exit channel
Elongation is described as a:
a) Reversible process
b) Stochastic process
c) Processive process ✅
d) Discontinuous process

Section VI: Termination

RNA pol I termination is:
a) Factor-independent
b) Factor-dependent ✅
c) By poly-A signal
d) Involves snRNP
RNA pol II termination involves recognition of:
a) TATA box
b) GC box
c) Poly-A signal ✅
d) CCA sequence
CPSF recognizes:
a) Cap site
b) TATA box
c) Poly-A signal ✅
d) Transcription factors
Poly-A tail addition is carried out by:
a) CPSF
b) RNA polymerase
c) Poly A polymerase ✅
d) P-TEFb
RNA pol III termination requires:
a) Helicase
b) CPSF
c) Hairpin and poly-T sequence ✅
d) CTD phosphorylation

Miscellaneous / PYQs

CTD is located on which subunit of RNA polymerase II?
a) Smallest
b) Middle
c) Largest ✅
d) None
Which RNA polymerase is responsible for pre-mRNA transcription?
a) Pol I
b) Pol II ✅
c) Pol III
d) Pol IV
The role of TFIIE is to:
a) Bind TATA box
b) Recruit TFIIH ✅
c) Elongate mRNA
d) Cap RNA
Which of the following is NOT a general transcription factor?
a) TFIIH
b) TFIIF
c) P-TEFb ✅
d) TFIIB
Which polymerase terminates at poly-T signal?
a) RNA pol I
b) RNA pol II
c) RNA pol III ✅
d) DNA pol
The ‘closed complex’ in transcription refers to:
a) Melted DNA
b) DNA not yet separated ✅
c) Elongating RNA
d) Terminated transcript
The ‘open complex’ differs from the closed complex by:
a) Promoter presence
b) RNA synthesis
c) Strand separation ✅
d) Enhancer interaction
Which step involves ‘scrunching’ motion?
a) Elongation
b) Promoter escape
c) Abortive initiation ✅
d) Termination
RNA-DNA hybrid is formed during:
a) Melting
b) Initiation
c) Elongation ✅
d) Termination
RNA polymerase moves along the template strand in:
a) 3’ to 5’ direction ✅
b) 5’ to 3’
c) Random
d) Both directions
Which process is essential for transcript capping?
a) P-TEFb recruitment ✅
b) CPSF activation
c) TFIIE binding
d) DNA replication
Transcription bubble is typically:
a) 5 nt
b) 12–14 nt ✅
c) 50 nt
d) 100 nt
Role of CSTF in transcription is:
a) Splicing
b) Transcription activation
c) RNA cleavage and polyadenylation ✅
d) Translation
What ensures high fidelity in transcription elongation?
a) TFIID
b) Elongation factors ✅
c) TFIIH
d) Enhancers
Which enzyme adds adenine residues post-cleavage?
a) RNA polymerase
b) DNA ligase
c) Poly-A polymerase ✅
d) SPT5