14.1 Transcription initiation, elongation, and termination

RNA transcription copies genetic information from DNA into RNA. This is a central step in gene expression: before a cell can make a protein, it first needs an RNA copy of the gene's instructions. The process unfolds in three stages: initiation, elongation, and termination, each with distinct molecular machinery.

Transcription Process

Role of RNA polymerase in transcription

RNA polymerase is the enzyme that synthesizes RNA from a DNA template. It's a multi-subunit complex. In prokaryotes, the core enzyme (composed of $\alpha$, $\beta$, $\beta'$, and $\omega$ subunits) handles catalysis, while the sigma factor ($\sigma$) is a dissociable subunit that directs the enzyme to the correct promoter. In eukaryotes, three different RNA polymerases exist (I, II, and III), each transcribing different classes of genes. RNA Pol II is the one responsible for mRNA.

RNA polymerase carries out a different job at each stage of transcription:

• Initiation: The enzyme binds the promoter region, separates the two DNA strands to expose the template, and positions itself at the transcription start site (+1).
• Elongation: It moves along the template strand in the 3' → 5' direction, reading the template and synthesizing a complementary RNA strand in the 5' → 3' direction. It catalyzes phosphodiester bonds between incoming ribonucleotides (NTPs).
• Termination: The enzyme encounters a termination signal, releases the newly made RNA transcript, and dissociates from the DNA.

Transcription initiation: prokaryotes vs eukaryotes

Initiation is the most heavily regulated stage, and it works quite differently in prokaryotes and eukaryotes.

Prokaryotic initiation is relatively straightforward. The sigma factor guides RNA polymerase to the promoter by recognizing two conserved sequence elements:

• The -10 element (Pribnow box, consensus $TATAAT$), located ~10 base pairs upstream of the start site
• The -35 element (consensus $TTGACA$), located ~35 base pairs upstream

Once $\sigma$ binds these sequences, the holoenzyme (core + $\sigma$) melts open the DNA to form an open complex, and transcription begins. After the first ~10 nucleotides are synthesized, $\sigma$ dissociates, and the core enzyme continues on its own. No large assembly of accessory factors is needed.

Eukaryotic initiation is far more elaborate. RNA Pol II cannot recognize the promoter on its own. Instead, general transcription factors (GTFs) must assemble at the promoter in a specific order to build the preinitiation complex (PIC):

1. TFIID binds first. Its TBP (TATA-binding protein) subunit recognizes the TATA box (typically ~25–30 bp upstream of the start site).
2. TFIIA and TFIIB join, stabilizing the TFIID-DNA interaction and helping position RNA Pol II.
3. RNA Pol II is recruited to the promoter, brought in as a complex with TFIIF.
4. TFIIE and TFIIH bind last. TFIIH is particularly important because it has helicase activity (to unwind DNA and form the open complex) and kinase activity (to phosphorylate the C-terminal domain, or CTD, of RNA Pol II, triggering the transition to elongation).

Beyond the GTFs, regulatory transcription factors (activators and repressors) fine-tune whether and how efficiently the PIC assembles, allowing the cell to control gene expression in response to signals.

Promoters and transcription factors

Promoters are DNA sequences located upstream of the transcription start site. They serve as the landing pad for the transcriptional machinery. In prokaryotes, the key elements are the -10 and -35 boxes. In eukaryotes, promoters can include the TATA box, the Inr (initiator) element right at the start site, and downstream promoter elements (DPE). Not all eukaryotic promoters contain a TATA box; many housekeeping genes use TATA-less promoters with other recognition elements instead.

Transcription factors are proteins that bind specific DNA sequences to regulate transcription:

• Activators bind to promoter-proximal elements or distant enhancers and help recruit or stabilize the transcriptional machinery, increasing transcription rates. They often work through coactivator complexes like Mediator.
• Repressors bind to silencer sequences and block activator binding, recruit corepressors, or directly interfere with the assembly of the PIC, reducing or shutting off transcription.

These factors allow cells to respond to signals like hormones, growth factors, or developmental cues by turning specific genes on or off.

Steps of transcription elongation and termination

Elongation:

1. RNA polymerase clears the promoter (called promoter escape) and enters the elongation phase. In eukaryotes, this requires CTD phosphorylation by TFIIH.
2. The enzyme maintains a transcription bubble of about 12–14 base pairs of unwound DNA. Within this bubble, the nascent RNA forms a short DNA-RNA hybrid (~8–9 bp) with the template strand.
3. Incoming nucleoside triphosphates (NTPs) are selected by complementary base pairing with the template (A pairs with U in RNA). RNA polymerase catalyzes the addition of each NTP to the 3' end of the growing RNA, releasing pyrophosphate ($PP_i$).
4. The enzyme moves along the template at roughly 40–80 nucleotides per second in prokaryotes (somewhat slower in eukaryotes), re-annealing the DNA behind it and extruding the single-stranded RNA transcript.
5. RNA polymerase has a basic proofreading ability: it can backtrack and cleave misincorporated nucleotides, though its error rate (~$10^{-4}$ to $10^{-5}$) is higher than DNA polymerase's.

Termination:

Termination happens when RNA polymerase receives a signal to stop and release the transcript. The mechanism differs between prokaryotes and eukaryotes.

Prokaryotic termination uses two mechanisms:

• Rho-independent (intrinsic) termination: The RNA transcript contains a self-complementary GC-rich sequence followed by a run of U's. The GC-rich region folds into a stem-loop (hairpin) structure that stalls RNA polymerase. The weak rU-dA base pairs in the DNA-RNA hybrid then destabilize, and the transcript dissociates.
• Rho-dependent termination: The Rho ($\rho$) protein, an ATP-dependent helicase, binds to a C-rich, unstructured region on the nascent RNA called the rut site (rho utilization site). Rho translocates along the RNA toward the polymerase and, when the polymerase pauses, unwinds the DNA-RNA hybrid to release the transcript.

Eukaryotic termination (for RNA Pol II) is coupled to 3' end processing of the mRNA:

1. RNA Pol II transcribes past the polyadenylation signal (consensus $AAUAAA$) in the pre-mRNA.
2. CPSF (cleavage and polyadenylation specificity factor) recognizes the $AAUAAA$ signal, and CstF (cleavage stimulation factor) binds a GU-rich element downstream.
3. The pre-mRNA is cleaved 10–30 nucleotides downstream of the $AAUAAA$ signal.
4. Poly(A) polymerase (PAP) adds a poly(A) tail (~200 adenines) to the 3' end of the cleaved RNA, which protects the mRNA and aids in export and translation.
5. RNA Pol II continues transcribing briefly after cleavage, but the unprotected 5' end of the remaining transcript is degraded by a 5' → 3' exonuclease (Rat1/Xrn2), which catches up to the polymerase and triggers its dissociation from the DNA. This is known as the torpedo model of termination.