Difficulty: Intermediate to Advanced | Prerequisites: Part 1 (Central Dogma, Gene Structure), Part 2 (Bacterial Transcription), basic chromatin biology.
Eukaryotic transcription is where the complexity ramps up. Instead of one RNA polymerase, eukaryotes use three. Instead of a single sigma factor, RNA Pol II requires six general transcription factors, a mediator complex, and often chromatin remodelling to get started. On top of that, RNA processing (capping, splicing, polyadenylation) happens co-transcriptionally and is coordinated through the C-terminal domain (CTD) of Pol II. This section also covers the experimental techniques you need to know for interpreting and designing transcription experiments. If you can explain how the pre-initiation complex assembles and how CTD phosphorylation orchestrates elongation and processing, you are well prepared for the exam.
Eukaryotes use three RNA polymerases (Pol I, II, III), each transcribing different gene classes. Pol II transcribes mRNA and requires six general transcription factors (GTFs) plus mediator to initiate. The CTD of Pol II is progressively phosphorylated during transcription, serving as a landing pad for RNA processing enzymes. Co-transcriptional processing includes 5' capping, splicing, and polyadenylation. Termination involves the torpedo or allosteric model.
RNA Polymerase I (Pol I)
The eukaryotic RNA polymerase that transcribes the large ribosomal RNA (rRNA) precursor gene (28S, 18S, 5.8S) in the nucleolus. Think of it as the rRNA specialist.
RNA Polymerase II (Pol II)
The eukaryotic RNA polymerase that transcribes all protein-coding genes (mRNAs) as well as snRNAs, miRNAs, and some other non-coding RNAs. This is the polymerase at the centre of gene regulation.
RNA Polymerase III (Pol III)
The eukaryotic RNA polymerase that transcribes small structural RNAs: tRNAs, 5S rRNA, and some snRNAs. Uses internal (downstream) promoter elements.
General transcription factors (GTFs)
A set of six protein complexes (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH) required for basal transcription by RNA Pol II at all class II promoters. In simple terms, they are the minimum crew needed to get Pol II to the start site.
TFIID
A GTF complex composed of TBP (TATA-binding protein) and TAFs (TBP-associated factors). It initiates pre-initiation complex assembly by recognising the TATA box or other core promoter elements.
TBP (TATA-binding protein)
A subunit of TFIID that binds the minor groove of the TATA box and sharply bends the DNA (~80°), nucleating the assembly of the pre-initiation complex.
TAFs (TBP-associated factors)
Subunits of TFIID that interact with activators, other GTFs, and core promoter elements. They are especially important at TATA-less promoters where TBP alone is insufficient.
TATA box
A conserved promoter element (consensus TATAAA) located ~25–30 bp upstream of the transcription start site, bound by TBP. Common in tightly regulated genes.
Inr (initiator element)
A core promoter element that spans the transcription start site. Some promoters use Inr instead of (or in addition to) the TATA box.
DPE (downstream promoter element)
A core promoter element located downstream of the start site, important in TATA-less promoters. Recognised by TAFs within TFIID.
BRE (TFIIB recognition element)
A promoter element adjacent to the TATA box that is bound directly by TFIIB, helping position Pol II.
Core promoter
The minimal set of DNA sequences sufficient to direct accurate transcription initiation by Pol II and the GTFs. In simple terms, the bare-minimum start signal.
Pre-initiation complex (PIC)
The large assembly of GTFs, mediator, and Pol II on the core promoter, formed before transcription begins.
Mediator complex
A large multi-subunit complex that acts as a bridge between gene-specific transcriptional activators/repressors and the general transcription machinery (Pol II + GTFs). Think of it as the switchboard that integrates regulatory signals.
C-terminal domain (CTD)
A long, repetitive tail on the largest subunit of Pol II, composed of heptad repeats (consensus: Tyr-Ser-Pro-Thr-Ser-Pro-Ser). Its phosphorylation state changes during the transcription cycle and recruits RNA processing factors.
P-TEFb
A kinase (CDK9/Cyclin T) that phosphorylates Ser2 of the CTD heptad repeat during elongation, promoting processive transcription and recruiting RNA processing enzymes.
NELF (negative elongation factor)
A factor that, together with DSIF, induces promoter-proximal pausing of Pol II shortly after initiation.
DSIF (DRB sensitivity-inducing factor)
A factor that cooperates with NELF to pause Pol II near the promoter. After phosphorylation by P-TEFb, DSIF switches to a positive elongation factor.
Promoter-proximal pausing
A regulatory checkpoint where Pol II pauses 20–60 bp downstream of the transcription start site. Release from this pause (by P-TEFb) is a key control point for many genes.
5' capping
The co-transcriptional addition of a methylated guanine cap (m7G) to the 5' end of the nascent mRNA, protecting it from degradation, promoting translation, and facilitating nuclear export.
Polyadenylation
The cleavage of pre-mRNA at a poly-A signal sequence followed by addition of ~200 adenine residues by poly-A polymerase (PAP). Enhances mRNA stability and translation.
CPSF (cleavage and polyadenylation specificity factor)
Recognises the AAUAAA poly-A signal on the pre-mRNA and directs cleavage.
CstF (cleavage stimulation factor)
Binds downstream of the poly-A site and works with CPSF to ensure accurate cleavage.
Spliceosome
A large ribonucleoprotein complex that removes introns from pre-mRNA and ligates exons together. Composed of snRNPs (small nuclear ribonucleoproteins) and associated proteins.
Torpedo model (of termination)
A model for Pol II termination in which, after cleavage at the poly-A site, a 5' to 3' exonuclease (Rat1 in yeast, Xrn2 in mammals) degrades the residual RNA still attached to Pol II, eventually catching up and dislodging the polymerase.
Allosteric model (of termination)
A model proposing that passage through the poly-A signal triggers a conformational change in Pol II that reduces its processivity, leading to spontaneous dissociation.
UBF and SL1
Transcription factors specific to RNA Pol I, required for rRNA gene transcription. UBF binds upstream control elements; SL1 positions Pol I.
TFIIIB and TFIIIC
Transcription factors for RNA Pol III. TFIIIC recognises internal promoter elements (e.g., A and B boxes in tRNA genes); TFIIIB positions Pol III at the start site.
TRF1 (TBP-related factor)
An alternative to TBP that some promoters use, particularly in certain developmental contexts.
NC2
A factor that can act as either an activator or repressor of transcription depending on the promoter context, highlighting the flexibility of the initiation machinery.
RNA Pol I: transcribes the large rRNA precursor (which is processed into 28S, 18S, and 5.8S rRNAs) in the nucleolus. Uses upstream control elements and the factors UBF and SL1.
RNA Pol II: transcribes all protein-coding genes (mRNAs), plus snRNAs and some regulatory RNAs. The most heavily regulated polymerase and the focus of most exam questions.
RNA Pol III: transcribes tRNAs, 5S rRNA, and some small RNAs. Unique in that it often uses internal (downstream) promoter elements recognised by TFIIIC, with TFIIIB positioning the polymerase.
Pol II cannot find promoters on its own. It requires six GTFs:
TFIID (TBP + TAFs) binds first, recognising the TATA box (via TBP) or other core promoter elements (via TAFs). TBP inserts into the minor groove and bends DNA sharply.
TFIIA stabilises the TFIID-DNA complex.
TFIIB binds next, positioning Pol II at the correct start site and contacting the BRE.
TFIIF escorts Pol II to the promoter.
TFIIE recruits TFIIH.
TFIIH has two critical enzymatic activities: a helicase that melts the promoter DNA (opens the bubble) and a kinase that phosphorylates the CTD of Pol II.
The mediator complex integrates signals from gene-specific activators and repressors, bridging them to the PIC.
Activator proteins enhance transcription by recruiting GTFs, mediator, and/or chromatin-modifying enzymes (e.g., histone acetyltransferases, HATs).
TATA box (~25–30 bp upstream): bound by TBP; common in tightly regulated genes but absent from many housekeeping genes.
Inr (initiator): overlaps the +1 start site; can function with or without TATA.
DPE (downstream promoter element): located ~+30 bp; particularly important in TATA-less promoters; recognised by TAFs.
BRE: contacted by TFIIB; flanks the TATA box.
Core promoter: the minimal combination of these elements needed for accurate initiation.
Not all promoters require TBP or the full set of TAFs. Some use TRF1 instead of TBP, and NC2 can function as either an activator or repressor depending on promoter architecture.
GTFs assemble on the promoter in a stepwise manner, recruiting Pol II.
Promoter melting is catalysed by the helicase activity of TFIIH (using ATP), creating the open complex.
Abortive initiation occurs (as in bacteria): short transcripts are made and released repeatedly.
Promoter escape requires phosphorylation of Ser5 on the CTD by the kinase subunit of TFIIH. This modification destabilises interactions with the PIC and allows the polymerase to move into productive elongation.
After initiation, Pol II enters a promoter-proximal pause regulated by NELF and DSIF. The polymerase sits 20–60 bp downstream, waiting for a signal to proceed.
P-TEFb (CDK9/Cyclin T) phosphorylates Ser2 of the CTD, NELF, and DSIF. This releases the pause and converts DSIF into a positive elongation factor.
The CTD phosphorylation code acts as a recruitment platform:
Ser5-P (from TFIIH): recruits the capping enzyme.
Ser2-P (from P-TEFb): recruits splicing and polyadenylation factors.
RNA processing is therefore coupled to transcription through the CTD.
5' capping:
Added almost immediately after the first ~20–30 nucleotides are synthesised.
An enzyme complex adds a methylated GTP cap (m7G) to the 5' end via a 5'-5' triphosphate linkage.
Functions: protects RNA from 5' exonucleases, facilitates nuclear export through the nuclear pore, and promotes translation by helping the ribosome bind.
Splicing:
Introns are removed and exons are joined by the spliceosome.
Splicing occurs co-transcriptionally, guided by consensus sequences at the 5' splice site (GU), 3' splice site (AG), and the branch point.
Splicing enhancers and silencers in the pre-mRNA fine-tune which exons are included (alternative splicing).
Polyadenylation:
The pre-mRNA is cleaved at a poly-A signal (AAUAAA), typically 10–30 nt upstream of the cleavage site.
CPSF recognises the AAUAAA signal; CstF binds a downstream G/U-rich element.
After cleavage, poly-A polymerase (PAP) adds ~200 adenine residues.
The poly-A tail enhances stability and translation efficiency.
PolyA-dependent termination (Pol II):
After the polymerase transcribes past the poly-A site and the pre-mRNA is cleaved, termination occurs by one of two proposed mechanisms:
Torpedo model: a 5' → 3' exonuclease (Xrn2/Rat1) degrades the uncapped RNA still associated with Pol II, eventually catching and dislodging the polymerase.
Allosteric model: transcription through the poly-A signal induces a conformational change in Pol II that makes it less processive, leading to spontaneous release.
In practice, both mechanisms may contribute.
In yeast and some genes: additional termination signals and factors such as Nrd1 are involved, particularly for non-coding RNA genes.
Reporter assays: a reporter gene (e.g., luciferase, GFP) is placed under the control of a test promoter. The amount of reporter protein produced indicates promoter strength.
DNA footprinting: identifies the exact DNA sequences bound by a protein. Protected regions appear as "footprints" on a sequencing gel.
Electrophoretic mobility shift assay (EMSA / gel shift): detects protein-DNA binding. A protein-DNA complex migrates more slowly than free DNA on a native gel.
In vitro transcription assays: purified components are used in a test tube to measure transcription from a defined template, allowing dissection of factor requirements.
ChIP-seq (chromatin immunoprecipitation + sequencing): maps where a protein of interest binds across the entire genome. Essential for studying transcription factor binding and histone modifications.
Northern blot: detects specific RNA species by size and abundance using gel electrophoresis and hybridisation with a labelled probe.
Primer extension / S1 mapping: locates the precise transcription start site of an mRNA.
RNA-Seq: high-throughput sequencing of all RNA in a sample, providing a genome-wide snapshot of expression levels, splicing patterns, and transcript variants.
Yeast two-hybrid: detects protein-protein interactions in vivo by fusing a "bait" protein to a DNA-binding domain and a "prey" protein to an activation domain. Interaction reconstitutes a functional transcription factor and activates a reporter gene.
CTD heptad repeat consensus: Tyr₁-Ser₂-Pro₃-Thr₄-Ser₅-Pro₆-Ser₇
TATA box consensus: TATAAA, located ~25–30 bp upstream of +1
Poly-A signal: AAUAAA
Poly-A tail length: ~200 adenines
Number of GTFs for Pol II: six (TFIIA, B, D, E, F, H)
Promoter-proximal pausing is a widespread regulatory mechanism in metazoans. Many developmentally important genes (and immediate-early response genes) are regulated at the pause-release step rather than at PIC assembly. This allows rapid activation: the polymerase is already loaded and waiting, so the cell can produce mRNA within minutes of receiving a signal. Dysregulation of P-TEFb is implicated in cancer (e.g., through the BRD4 pathway, which is a target for the experimental drug class known as BET inhibitors).
Students often think the TATA box is present in all eukaryotic promoters. Many housekeeping genes lack a TATA box entirely and rely on Inr, DPE, or other elements instead.
The CTD is sometimes confused with an enzymatic domain. The CTD is a structural/regulatory tail, not a catalytic site. Its role is to serve as a phosphorylation-dependent docking platform for processing factors.
Splicing is sometimes described as happening "after transcription." In eukaryotes, splicing is largely co-transcriptional, occurring while the polymerase is still elongating.
Students sometimes assume that Pol I, II, and III are interchangeable. Each polymerase transcribes a distinct class of genes and uses its own set of transcription factors and promoter elements.
⚠️ Know which polymerase transcribes which gene class (Pol I = rRNA, Pol II = mRNA/snRNA, Pol III = tRNA/5S rRNA). This is a classic multiple-choice target.
⚠️ Be able to list the six GTFs for Pol II in order of assembly and state the key function of each (especially TFIID/TBP for TATA recognition and TFIIH for melting + CTD phosphorylation).
⚠️ Understand the CTD phosphorylation code: Ser5-P recruits capping enzymes; Ser2-P recruits splicing and polyadenylation factors.
⚠️ Expect a question on promoter-proximal pausing: what causes it (NELF + DSIF), what releases it (P-TEFb), and why it matters (rapid gene activation).
⚠️ Know the torpedo vs allosteric models of termination and be able to explain each in 2–3 sentences.
⚠️ Experimental techniques are commonly tested. Be able to match each technique to the question it answers: "Where does this protein bind?" → ChIP-seq or DNA footprinting. "Does this protein interact with another protein?" → yeast two-hybrid or co-IP.
True or false: RNA Pol III transcribes mRNA in eukaryotes.
Fill in the blank: The kinase activity of _______ phosphorylates Ser5 of the CTD during initiation.
True or false: The mediator complex directly synthesises RNA.
Fill in the blank: In the torpedo model, the exonuclease _______ degrades residual RNA after poly-A site cleavage.
True or false: NELF promotes elongation by releasing the promoter-proximal pause.
Answers: 1. False (Pol II transcribes mRNA). 2. TFIIH. 3. False (mediator is a regulatory bridge, not a polymerase). 4. Xrn2 (Rat1 in yeast). 5. False (NELF induces the pause; P-TEFb releases it).
Q: List the three eukaryotic RNA polymerases and state what each transcribes.
A: RNA Pol I transcribes the large rRNA precursor (28S, 18S, 5.8S rRNAs) in the nucleolus. RNA Pol II transcribes all protein-coding genes (mRNAs), snRNAs, and some regulatory RNAs. RNA Pol III transcribes tRNAs, 5S rRNA, and certain other small RNAs.
Q: Describe the stepwise assembly of the pre-initiation complex on a TATA-containing promoter.
A: TFIID (via its TBP subunit) binds the TATA box and bends the DNA. TFIIA stabilises this complex. TFIIB binds next, contacting the BRE and positioning the start site. TFIIF escorts Pol II to the promoter. TFIIE then recruits TFIIH, whose helicase activity melts the promoter DNA and whose kinase activity phosphorylates the CTD at Ser5, triggering promoter escape.
Q: Explain how the CTD of RNA Pol II coordinates transcription with RNA processing.
A: The CTD contains heptad repeats that are phosphorylated at different stages. During initiation, TFIIH phosphorylates Ser5, which recruits the capping enzyme for 5' cap addition. During elongation, P-TEFb phosphorylates Ser2, which recruits splicing factors and the polyadenylation machinery (CPSF, CstF, PAP). This ensures that RNA processing occurs co-transcriptionally and in the correct order.
Q: What is promoter-proximal pausing, and why is it important for gene regulation?
A: Promoter-proximal pausing occurs when Pol II halts 20–60 bp downstream of the transcription start site, held in place by NELF and DSIF. The pause is released when P-TEFb phosphorylates Ser2 of the CTD (and NELF/DSIF). This mechanism allows cells to pre-load the polymerase at genes that need to be activated rapidly (e.g., in response to stress or developmental signals), bypassing the slow step of PIC assembly.
Q: Compare the torpedo and allosteric models of eukaryotic transcription termination.
A: In the torpedo model, after cleavage at the poly-A site, the downstream RNA (still attached to Pol II) is degraded by the 5' → 3' exonuclease Xrn2 (Rat1 in yeast). When the exonuclease catches up to the elongating polymerase, it destabilises the complex and causes dissociation. In the allosteric model, passage through the poly-A signal triggers a conformational change in Pol II that reduces processivity, leading to spontaneous release. Both models likely contribute in vivo.
Q: You want to determine where a transcription factor binds across the genome. Which technique would you use, and why?
A: ChIP-seq (chromatin immunoprecipitation followed by high-throughput sequencing). The protein of interest is crosslinked to DNA in living cells, the chromatin is fragmented, and the protein-DNA complexes are immunoprecipitated with a specific antibody. The bound DNA fragments are then sequenced to map binding sites genome-wide.
This material builds directly on Part 2 (bacterial transcription) by extending the same initiation-elongation-termination framework to a more complex system. The CTD phosphorylation cycle connects to RNA processing, which will be important when studying mRNA export, translation regulation, and mRNA decay. Chromatin modification (HATs, histone methylation) is introduced here but explored in depth in the epigenetics and chromatin regulation unit. The experimental techniques covered here (ChIP-seq, RNA-Seq, reporter assays) will recur throughout the course whenever new regulatory mechanisms are tested in the lab.
eukaryotic transcription, RNA Pol I, RNA Pol II, RNA Pol III, RNA polymerase II, general transcription factors, GTFs, TFIID, TBP, TATA-binding protein, TAFs, TBP-associated factors, TFIIA, TFIIB, TFIIF, TFIIE, TFIIH, TATA box, Inr, initiator, DPE, downstream promoter element, BRE, core promoter, pre-initiation complex, PIC, mediator complex, CTD, C-terminal domain, CTD phosphorylation, Ser5, Ser2, P-TEFb, CDK9, NELF, DSIF, promoter-proximal pausing, 5' capping, m7G cap, splicing, spliceosome, polyadenylation, poly-A tail, CPSF, CstF, PAP, torpedo model, allosteric model, Xrn2, Rat1, Nrd1, UBF, SL1, TFIIIB, TFIIIC, TRF1, NC2, ChIP-seq, EMSA, gel shift, DNA footprinting, reporter assay, luciferase, Northern blot, RNA-Seq, primer extension, S1 mapping, yeast two-hybrid, chromatin modification, histone acetylation, HATs, activator, repressor, Molecular Biology of the Gene Watson, UCF Molecular Biology II