Step-by-Step Guide: Transcription Initiation in Eukaryotes 🧬

1. Recognizing the Promoter

Transcription begins at promoter regions in the DNA, specific sequences that signal RNA polymerase II where to start transcription. The core promoter typically includes:

• TATA box: A sequence (~25-35 base pairs upstream of the transcription start site) that helps guide the assembly of the transcription machinery.
• Other sequences like BRE, Inr (Initiator element), and DPE (Downstream Promoter Element) may also assist.

2. Binding of TFIID (First Step of Assembly)

TFIID, a multi-protein complex, is the first to bind the promoter. It consists of:

• TBP (TATA-Binding Protein): Recognizes and binds to the TATA box.
  • When TBP binds, it bends the DNA, helping to open it slightly and prepare for transcription.
• TAFs (TBP-Associated Factors): Help stabilize TBP binding and recognize other promoter elements.

3. Recruitment of TFIIB

TFIIB binds next, interacting with both:

• The DNA (at the BRE element near the TATA box).
• TBP (from TFIID).

TFIIB serves as a bridge, guiding RNA polymerase II to the correct position at the promoter.

4. Recruitment of RNA Polymerase II and TFIIF

RNA Polymerase II is recruited to the promoter with the help of TFIIF.

• TFIIF:
  • Helps RNA polymerase II bind to the DNA.
  • Stabilizes RNA polymerase II's interaction with the transcription complex.

5. Recruitment of TFIIE and TFIIH

TFIIE binds next, creating a platform for the final factor, TFIIH.

TFIIH is a multi-functional protein complex with two critical roles:

• Helicase Activity: It unwinds the DNA at the transcription start site, creating a single-stranded "bubble" so RNA polymerase II can access the template strand.
• Kinase Activity: It phosphorylates the carboxy-terminal domain (CTD) of RNA polymerase II.

6. Formation of the Pre-Initiation Complex (PIC)

With all these factors assembled (TFIID, TFIIB, TFIIF, TFIIE, TFIIH, and RNA polymerase II), the pre-initiation complex (PIC) is complete.

The DNA is still mostly double-stranded at this point.

7. Melting of DNA and Open Complex Formation

TFIIH's helicase activity melts (unwinds) the DNA around the transcription start site (+1 position). This creates the open complex, exposing the template strand so RNA polymerase II can begin transcription.

8. Phosphorylation of RNA Polymerase II CTD

TFIIH's kinase activity phosphorylates the carboxy-terminal domain (CTD) of RNA polymerase II.

• The CTD has multiple repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser, and phosphorylation of serine residues in these repeats is a key step.
• Phosphorylation of the CTD:
  • Triggers RNA polymerase II to release from the promoter.
  • Allows RNA polymerase II to transition from initiation to elongation.

9. Escape of RNA Polymerase II and Promoter Clearance

RNA polymerase II starts synthesizing a short RNA strand (abortive transcription may occur briefly). Once the RNA reaches a certain length (around 10 nucleotides), RNA polymerase II clears the promoter and enters the elongation phase, leaving behind most of the general transcription factors.

Key Players Recap:

Factor	Function
TFIID (TBP + TAFs)	Recognizes and binds the promoter (TATA box).
TFIIB	Positions RNA polymerase II correctly and bridges it with TFIID.
TFIIF	Stabilizes RNA polymerase II binding to the complex.
TFIIE	Recruits TFIIH and helps regulate its activity.
TFIIH	Unwinds DNA (helicase activity) and phosphorylates RNA polymerase II CTD.
RNA Polymerase II	Synthesizes the RNA molecule from the DNA template.