Source: University of Florida Cell Biology lecture notes
Tags: transcription, RNA polymerase, promoter, terminator, sigma factor, TATA box, TFIID, TFIIB, TFIIH, TBP, prokaryotic transcription, eukaryotic transcription, transcription initiation complex, RNA pol I, RNA pol II, RNA pol III
Difficulty: Intermediate Prerequisites: Part 1 notes (Central Dogma, Gene Expression, DNA vs RNA). You should already know that transcription copies DNA into RNA.
Now you are inside the first half of the central dogma: how the cell actually copies a gene's information into RNA. Transcription is heavily regulated, and that regulation differs substantially between prokaryotes and eukaryotes. If the central dogma is the "what," this unit is the "how" of the DNA-to-RNA step. Understanding initiation, elongation, and termination here will make RNA processing and gene regulation (later units) far easier to follow.
RNA polymerase reads a DNA template strand and builds a complementary RNA in the 5′ to 3′ direction. Transcription has three phases: initiation (binding the promoter), elongation (adding nucleotides), and termination (releasing the transcript). Prokaryotes use one RNA polymerase with a sigma factor; eukaryotes use three RNA polymerases and a suite of general transcription factors.
RNA polymerase
The enzyme that synthesises RNA from a DNA template by adding ribonucleotides complementary to the template strand. Think of it as the molecular photocopier that reads one strand of DNA and produces an RNA copy.
Promoter
A specific DNA sequence upstream of a gene that signals where transcription should begin. It is the landing pad for RNA polymerase and its associated factors.
Template strand
The DNA strand that RNA polymerase reads (3′ to 5′) to build a complementary RNA strand (5′ to 3′). Also called the antisense strand.
Terminator
A DNA sequence that signals the end of transcription, causing RNA polymerase to release the completed RNA transcript.
Sigma factor (σ factor)
A prokaryotic protein that associates with the core RNA polymerase to form the holoenzyme, enabling it to recognise and bind specific promoter sequences. Once transcription begins, the sigma factor dissociates.
TATA box
A conserved AT-rich DNA sequence (consensus TATAAA) found in many eukaryotic promoters, typically about 25–30 base pairs upstream of the transcription start site. It is the initial anchor point for the transcription initiation complex.
TATA-binding protein (TBP)
A subunit of the general transcription factor TFIID that binds the TATA box and distorts the DNA, marking the spot where the rest of the initiation machinery assembles. In simple terms, TBP is the first factor to "sit down" on the promoter.
General transcription factors (GTFs)
Proteins (TFIID, TFIIB, TFIIH, and others) required for RNA polymerase II to initiate transcription at a eukaryotic promoter. They are called "general" because they are needed at virtually every Pol II promoter, unlike gene-specific regulatory factors.
TFIIH
A general transcription factor with helicase and kinase activity. It unwinds the DNA at the promoter and phosphorylates the C-terminal domain (CTD) of RNA polymerase II, which signals the transition from initiation to elongation.
Transcription initiation complex
The full assembly of RNA polymerase II and general transcription factors at a eukaryotic promoter, poised to begin RNA synthesis.
Initiation
RNA polymerase (with accessory factors) binds the promoter.
The DNA double helix unwinds locally, exposing the template strand.
The first ribonucleotides are laid down.
Elongation
Ribonucleotides (rNTPs) are added one by one, complementary to the template strand: A pairs with U, G pairs with C.
RNA is synthesised in the 5′ → 3′ direction (the polymerase reads the template 3′ → 5′).
As the polymerase moves forward, the newly made RNA peels away and the DNA helix reforms behind it.
Termination
The polymerase reaches a terminator sequence.
The RNA transcript is released.
The DNA helix fully reforms.
Unlike DNA replication, transcription copies only a specific gene segment, not the entire chromosome. The RNA transcript does not stay hydrogen-bonded to the DNA after synthesis.
Promoters sit upstream (5′ side) of the gene on the non-template strand.
The orientation of the promoter determines which DNA strand is the template and in which direction transcription proceeds.
In prokaryotes, key promoter elements include the −35 region and the −10 region (also called the Pribnow box).
In eukaryotes, the TATA box (around −25 to −30) is a common core promoter element, though not all promoters contain one.
Terminators vary: prokaryotes may use intrinsic (hairpin-forming) terminators or Rho-dependent mechanisms. Eukaryotic termination is coupled to RNA processing signals.
A single type of RNA polymerase handles all transcription.
The sigma factor binds the core enzyme, forming the holoenzyme, which can recognise promoter sequences.
Transcription occurs on naked DNA (no chromatin packaging).
Because prokaryotes lack a nucleus, transcription and translation are coupled: ribosomes can begin translating the mRNA before transcription is finished.
Three distinct RNA polymerases divide the work:
RNA Pol I: transcribes most ribosomal RNA genes.
RNA Pol II: transcribes protein-coding genes (produces mRNA) and some small regulatory RNAs. This is the polymerase you will deal with most.
RNA Pol III: transcribes tRNA genes, 5S rRNA, and other small RNAs.
Transcription occurs in the nucleus; translation occurs in the cytoplasm. The two processes are separated in both space and time.
Eukaryotic DNA is packaged into chromatin, so access to promoters depends on chromatin remodelling and histone modifications.
Genes are often spread out, with large non-coding regions and distant regulatory elements (enhancers, silencers).
Assembly follows a sequential, ordered process:
TBP (part of TFIID) binds the TATA box, bending/distorting the DNA.
TFIIB binds next, positioning RNA Pol II correctly on the promoter.
RNA Pol II is recruited to the complex.
TFIIH joins. It has two critical enzymatic activities:
Helicase activity: uses ATP to unwind the DNA, creating the transcription bubble.
Kinase activity: phosphorylates the CTD tail of RNA Pol II, which is the signal to shift from initiation into productive elongation.
After initiation, most general transcription factors dissociate, and the polymerase moves along the template on its own (with elongation factors).
This elaborate, multi-step assembly provides multiple points at which the cell can regulate whether a gene is transcribed, making it a major control hub for gene expression.
Antibiotics such as rifampicin work by inhibiting bacterial RNA polymerase, blocking transcription and killing the bacterium. Because eukaryotic RNA polymerases are structurally different, rifampicin does not harm host cells. Alpha-amanitin, the toxin in death cap mushrooms, works the opposite way: it inhibits eukaryotic RNA Pol II, which is why it is lethal to humans.
Students often confuse the template strand with the coding (non-template) strand. The RNA sequence matches the coding strand (with U in place of T), but the polymerase reads the template strand.
"Prokaryotes have no transcription factors" is incorrect. Prokaryotes have the sigma factor and various regulatory proteins. They lack the elaborate general transcription factor apparatus that eukaryotes use.
Students sometimes think all eukaryotic genes have a TATA box. Many promoters are TATA-less and use other elements (e.g., Inr, DPE) for initiation.
Confusing the three eukaryotic RNA polymerases is common on exams. Remember: Pol I makes rRNA, Pol II makes mRNA, Pol III makes tRNA and 5S rRNA.
⚠️ The ordered assembly of the transcription initiation complex (TBP → TFIIB → Pol II → TFIIH) is a favourite exam topic. Know the sequence and what each factor contributes.
⚠️ Be ready to compare prokaryotic and eukaryotic transcription in a table format. Common axes: number of polymerases, role of sigma factor vs GTFs, coupling of transcription/translation, chromatin involvement.
⚠️ Understand why phosphorylation of the Pol II CTD by TFIIH matters: it marks the transition from initiation to elongation.
⚠️ Know the direction of RNA synthesis (5′ → 3′) and the base-pairing rules (A:U, G:C).
True or false: RNA polymerase synthesises RNA in the 3′ to 5′ direction. (False, it is 5′ to 3′.)
Fill in the blank: In prokaryotes, the ______ associates with core RNA polymerase to recognise promoters. (Sigma factor)
True or false: Eukaryotic transcription and translation can occur simultaneously on the same mRNA. (False, they are spatially separated by the nuclear envelope.)
Fill in the blank: The general transcription factor ______ phosphorylates the CTD of RNA Pol II. (TFIIH)
True or false: RNA Pol III transcribes protein-coding genes. (False, that is RNA Pol II.)
Q: Describe the three phases of transcription.
A: Initiation: RNA polymerase and associated factors bind the promoter and unwind the DNA locally. Elongation: the polymerase adds complementary ribonucleotides one by one in the 5′ to 3′ direction, building the RNA strand. Termination: the polymerase reaches a terminator signal, releases the RNA transcript, and the DNA helix reforms.
Q: Compare prokaryotic and eukaryotic transcription in terms of RNA polymerases used and coupling with translation.
A: Prokaryotes use a single RNA polymerase for all genes, and because they lack a nucleus, transcription and translation are coupled (ribosomes translate mRNA while it is still being transcribed). Eukaryotes use three RNA polymerases (Pol I, II, III) for different gene classes, and transcription (in the nucleus) is separated from translation (in the cytoplasm).
Q: What is the role of TBP in eukaryotic transcription initiation?
A: TBP (TATA-binding protein), a subunit of TFIID, binds the TATA box in the promoter and distorts the DNA. This marks the site where the transcription initiation complex assembles and is the first step in recruiting RNA Pol II to the promoter.
Q: A researcher adds alpha-amanitin to a eukaryotic cell culture. Which RNA polymerase is inhibited, and what consequence would you predict for the cell?
A: Alpha-amanitin inhibits RNA Pol II. This would block transcription of protein-coding genes, preventing new mRNA synthesis and eventually halting protein production, leading to cell death.
Q: Explain why the orientation of a promoter matters.
A: The promoter's orientation determines which DNA strand serves as the template and in which direction RNA polymerase travels along the gene. Reversing the promoter would cause the polymerase to read the opposite strand in the opposite direction, producing a completely different (and non-functional) RNA.
The initiation complex and promoter architecture connect directly to gene regulation topics, where you will learn how activators and repressors modulate transcription by interacting with enhancers, silencers, and the mediator complex. The distinction between prokaryotic and eukaryotic transcription is also essential for understanding the lac operon and other prokaryotic regulatory systems. RNA processing (Part 3 of these notes) happens during and immediately after transcription, so the events described here flow directly into capping, splicing, and polyadenylation.
transcription, RNA polymerase, RNA pol I, RNA pol II, RNA pol III, promoter, TATA box, TBP, TFIID, TFIIB, TFIIH, sigma factor, general transcription factors, transcription initiation complex, elongation, termination, terminator, template strand, coding strand, antisense strand, 5 prime to 3 prime, prokaryotic transcription, eukaryotic transcription, CTD phosphorylation, holoenzyme, Pribnow box, chromatin, coupled transcription translation