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.
- 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.
- 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.
- 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.