Questions: Eukaryotic Transcription Initiation: TFIID, Mediator, and Chromatin

Question 1

A gene has a perfect TATA box and all necessary general transcription factors are present in abundant quantities. However, the promoter region is embedded in tightly packed heterochromatin. What will happen?

• A. Transcription will proceed normally — TFIID can recognize the TATA box regardless of chromatin packaging
• B. TFIIH will use its helicase activity to melt the chromatin and expose the promoter before preinitiation complex assembly
• C. Transcription will not initiate — chromatin accessibility is a prerequisite; nucleosomes occluding the promoter must be remodeled or displaced before any transcription factor can reach the DNA
• D. RNA Pol II will transcribe the heterochromatin directly as a non-specific template

Answer: (C)

Chromatin accessibility is the first gate in eukaryotic transcription. The presence of general transcription factors and a perfect promoter sequence is irrelevant if the DNA is physically inaccessible under nucleosomes packed into heterochromatin. ATP-dependent chromatin remodeling complexes (like SWI/SNF) must slide or eject nucleosomes to expose the promoter, and histone-modifying enzymes must create the right chemical environment, before TFIID can bind and PIC assembly can begin. This is a fundamental difference from prokaryotic transcription, where RNA polymerase and sigma factor can access naked DNA directly.

Question 2

A transcriptional activator protein is bound to an enhancer 80 kb upstream of a gene's promoter. RNA Pol II is located at the core promoter. How does the activator's signal reach the polymerase?

• A. The transcription factor diffuses along the DNA in three-dimensional space until it contacts RNA Pol II directly at the promoter
• B. The transcription factor synthesizes a signaling molecule that diffuses through the nucleus to reach the preinitiation complex
• C. DNA looping brings the enhancer into physical proximity with the promoter; the Mediator complex bridges the enhancer-bound activator and the preinitiation complex, transmitting the activation signal
• D. TFIIB scans the chromatin for bound transcription factors and relays their activation signals to the core promoter

Answer: (C)

Long-range transcriptional regulation works through DNA looping — the chromosomal region between the enhancer and promoter loops out, bringing the two regulatory elements into physical contact. The Mediator complex serves as the molecular bridge: its tail domain contacts the enhancer-bound transcription factor, and its head domain contacts the preinitiation complex at the promoter. Mediator integrates signals from multiple enhancer-bound factors and relays them to RNA Pol II, explaining how a single gene can respond to dozens of different regulatory inputs simultaneously.

Question 3

The Mediator complex directly recognizes the TATA box and serves as the primary DNA-binding component of the preinitiation complex.

• T. True
• F. False

Answer: False

TATA box recognition is the job of TBP (TATA-binding protein), a subunit of TFIID. TBP binds the minor groove of the TATA box consensus sequence approximately 25 bp upstream of the transcription start site, bending the DNA and creating the platform for preinitiation complex assembly. Mediator does not bind DNA at the core promoter; it bridges between enhancer-bound transcription factors and the already-assembled PIC. Confusing Mediator's role with TFIID's role is a common error because both are essential for initiation.

Question 4

TFIIH plays a dual role in transcription initiation: its helicase activity separates the DNA strands to form the transcription bubble, and its kinase activity phosphorylates the CTD of RNA Pol II to trigger the transition from initiation to elongation.

• T. True
• F. False

Answer: True

TFIIH is the most enzymatically active general transcription factor. Its XPB/XPD subunits provide ATP-dependent helicase activity to melt about 11-15 bp of DNA at the transcription start site, generating the open complex (transcription bubble) that allows RNA synthesis to begin. Its CDK7 kinase subunit phosphorylates Ser5 of the heptapeptide repeat in the CTD of RNA Pol II, converting the polymerase from its initiation conformation to an elongation-competent form and also recruiting RNA processing factors. Both activities are required for productive transcription.

Question 5

Why does eukaryotic transcription initiation require both chromatin remodeling and the Mediator complex, while prokaryotic transcription requires neither? What regulatory capability does this complexity provide?

Model answer: Prokaryotic DNA is not packaged into nucleosomes and lacks enhancer-based regulation; a sigma factor alone can recognize the promoter and recruit RNA polymerase. Eukaryotic DNA is compacted into chromatin, making the promoter physically inaccessible by default — chromatin remodeling is required to expose it. And eukaryotic genes must integrate regulatory signals from multiple enhancers located far from the promoter across the genome; Mediator provides the physical bridge for this long-range communication. Together, these two requirements create a system where a gene's expression integrates many inputs: developmental stage, cell type, environmental signals, all encoded in which transcription factors are bound to which enhancers. The complexity enables the precise, combinatorial gene regulation that underlies eukaryotic development and differentiation.

This architectural logic explains why a human genome with ~20,000 genes can generate hundreds of distinct cell types: the same genes are regulated differently in each cell type by different combinations of transcription factors, chromatin states, and active enhancers — all converging through Mediator onto the core machinery.