Eukaryotic transcription

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.