Questions: Eukaryotic Promoters and the TFIID Complex

Question 1

A researcher mutates the TATA box of a eukaryotic gene's promoter to a random non-binding sequence. What is the most likely consequence for transcription of that gene?

• A. No significant effect — the TATA box is not required for all eukaryotic genes, so transcription continues via alternative elements
• B. Transcription increases because the mutant sequence no longer bends DNA, allowing more open access for RNA Pol II
• C. Transcription is severely reduced because TBP cannot bind, blocking assembly of the pre-initiation complex and preventing RNA Pol II from being correctly positioned
• D. Only the transition from initiation to elongation is impaired — RNA Pol II still assembles at the promoter but cannot begin moving along the template

Answer: (C)

For genes with TATA box-containing promoters, the TATA box is the nucleation point for pre-initiation complex (PIC) assembly. TBP within TFIID binds the TATA box directly, bending DNA ~80° and creating the structural platform for sequential recruitment of TFIIA, TFIIB, RNA Pol II/TFIIF, TFIIE, and TFIIH. If TBP cannot bind, this entire cascade fails and transcription is essentially abolished. Note that TATA-less promoters exist (many housekeeping genes use Inr, DPE, or CpG islands instead), so the answer is specifically about genes that do have a functional TATA box.

Question 2

Which step in eukaryotic pre-initiation complex assembly directly triggers the transition from initiation to elongation?

• A. TFIID binding to the TATA box and bending the DNA ~80°, which signals RNA Pol II to begin synthesis
• B. TFIIB bridging TFIID to RNA Polymerase II and positioning the enzyme at the transcription start site
• C. TFIIH phosphorylating the C-terminal domain (CTD) of RNA Pol II, releasing the polymerase from the promoter so it can begin elongation
• D. TFIIA stabilizing the TFIID-DNA interaction against competitive inhibitors

Answer: (C)

TFIIH is the 'launch' signal for elongation. It contains two critical enzymatic activities: helicase activity (to unwind the DNA double helix and create the transcription bubble) and kinase activity (to phosphorylate the RNA Pol II CTD). Phosphorylation of the CTD is the molecular trigger that releases RNA Pol II from the promoter-bound PIC and converts it from an initiation-competent to an elongation-competent form. TFIID binding (option A) nucleates the complex; TFIIB (option B) positions Pol II; TFIIA (option D) stabilizes — but none of these trigger the transition to elongation.

Question 3

TFIIH contributes both helicase activity (to unwind DNA at the transcription start site) and kinase activity (to phosphorylate the RNA Pol II CTD), making it essential for both transcription bubble formation and the initiation-to-elongation transition.

• T. True
• F. False

Answer: True

TFIIH is the most enzymatically active component of the pre-initiation complex and serves as the 'activation switch' for transcription. Its XPB and XPD subunits have helicase activity that uses ATP to unwind ~10 bp of DNA at the transcription start site, forming the open complex. Its CDK7 subunit phosphorylates serine residues in the heptapeptide repeats of RNA Pol II's C-terminal domain, which releases the polymerase from the promoter and recruits elongation factors and RNA processing machinery. TFIIH is also part of the nucleotide excision repair pathway, connecting transcription and DNA repair.

Question 4

The TATA box in eukaryotic promoters plays the same role as the −10 and −35 elements in prokaryotic promoters: both serve as direct recognition sequences where RNA polymerase itself binds to initiate transcription.

• T. True
• F. False

Answer: False

This is a key conceptual difference between prokaryotic and eukaryotic transcription initiation. In prokaryotes, the sigma factor — which is a direct subunit of the holoenzyme — recognizes the −10 and −35 elements. RNA polymerase itself makes the initial promoter contact. In eukaryotes, RNA Pol II never directly contacts the TATA box. Instead, TBP (within TFIID) binds the TATA box first, and RNA Pol II is recruited downstream through protein-protein interactions with TFIIB and TFIIF. The eukaryotic system requires a full pre-initiation complex assembled before Pol II arrives; the prokaryotic system uses sigma as a direct bridge between polymerase and DNA.

Question 5

Why does eukaryotic transcription initiation require a multi-protein pre-initiation complex rather than a single sigma-factor equivalent, and what feature of eukaryotic DNA contributes to this requirement?

Model answer: Eukaryotic DNA is wrapped around histones and compacted into chromatin, making promoter sequences physically inaccessible. A single factor analogous to sigma could not reliably access and open chromatin-embedded promoters. The multi-protein PIC provides a modular, tunable system: different combinations of general transcription factors, chromatin remodelers, and co-activators can be recruited under different conditions, allowing precise regulation of which genes are transcribed at what level in which cell type. The assembly sequence also provides multiple regulatory checkpoints — each step is a potential control point for activators or repressors.

The complexity of eukaryotic transcription initiation is a consequence of the chromatin packaging problem and the need for fine-grained gene regulation in multicellular organisms. Prokaryotic sigma factors work because bacterial DNA is largely accessible and the cell needs only modest transcriptional diversity. Eukaryotic cells must regulate thousands of genes independently across hundreds of cell types, requiring a combinatorial assembly system. The PIC architecture — where TFIID, GTFs, Pol II, Mediator, and chromatin remodelers all contribute — creates the regulatory flexibility that enables cell differentiation and tissue-specific gene expression.