RNA Polymerases

Core Concept

RNA polymerases transcribe DNA into RNA. Prokaryotes use a single RNA polymerase; eukaryotes use three specialized enzymes: RNA polymerase I synthesizes the large ribosomal RNAs (28S, 18S, 5.8S), RNA polymerase II synthesizes messenger RNAs and many noncoding RNAs (snRNAs, miRNAs), and RNA polymerase III synthesizes transfer RNAs, 5S rRNA, and other small RNAs. All RNA polymerases synthesize RNA in the 5′→3′ direction, require a DNA template but no primer, and use ribonucleoside triphosphates as substrates — the central step converting genetic information into functional products.

Key Components

• RNA polymerase I — transcribes pre-rRNA (45S → 28S/18S/5.8S) in the nucleolus.
• RNA polymerase II — transcribes mRNA precursors (plus snRNA, miRNA); requires general transcription factors.
• RNA polymerase III — transcribes tRNA, 5S rRNA, and small structural RNAs (e.g., U6).
• General transcription factors (TFIIA–TFIIH) — assemble with Pol II at promoters; TFIIH unwinds DNA and phosphorylates the Pol II C-terminal domain.
• C-terminal domain (CTD) — repetitive tail of Pol II's largest subunit whose phosphorylation regulates the transcription cycle.
• Promoter — DNA sequence directing polymerase binding and start-site selection.

Mechanism

Transcription proceeds through initiation, elongation, and termination. The polymerase binds a promoter, unwinds a short DNA "transcription bubble," and selects a start site. It then adds ribonucleotides complementary to the template strand (read 3′→5′), forming phosphodiester bonds as it moves, using the energy of NTP hydrolysis. Elongation is processive; termination releases the RNA and the polymerase. Because only one DNA strand is copied, transcription is asymmetric and templated, and the RNA produced is a copy of the coding (sense) sequence.

How It Works

1. Pol II and general transcription factors assemble at a core promoter (TATA box recognized by TBP).
2. TFIIH unwinds the DNA to form an open complex; Pol II is positioned at the transcription start site.
3. TFIIH phosphorylates serine residues in the Pol II CTD, triggering promoter escape and elongation.
4. Pol II moves along the template, matching incoming rNTPs to the DNA and synthesizing RNA 5′→3′.
5. The RNA exits through a channel while the DNA duplex re-forms behind the polymerase.
6. Termination signals cause the polymerase to release the transcript and dissociate (e.g., polyadenylation-linked cleavage for Pol II).
7. Pol I and Pol III use analogous cycles at their own promoters to produce rRNA and tRNA/5S RNA.

Energy and Directionality

RNA synthesis is powered by ribonucleoside triphosphates: each nucleotide addition hydrolyzes one high-energy phosphoanhydride bond (NTP → NMP + pyrophosphate), and pyrophosphate hydrolysis makes the reaction effectively irreversible. Synthesis is strictly 5′→3′ (nucleotides add to the growing 3′-OH), and the template is read 3′→5′. Promoter opening by TFIIH requires ATP, and CTD phosphorylation (ATP) licenses elongation. The directionality of the promoter fixes which strand is the template, so transcription is directional.

Experimental Evidence

• α-Amanitin sensitivity — the mushroom toxin inhibits Pol II at low, Pol III at higher, and Pol I only at very high concentrations, proving the three enzymes are distinct.
• Nuclear fractionation — rRNA, mRNA, and tRNA synthesis localize to different nuclear compartments (nucleolus vs nucleoplasm), matching Pol I/II/III activities.
• In vitro transcription — purified Pol II plus general transcription factors transcribe a template from the correct start site, defining the minimal machinery.
• CTD truncation/phosphorylation mutants — show that the Pol II CTD is required for promoter escape and for coupling to RNA processing.

Technique

Techniques include nuclear run-on assays (measure active transcription), in vitro transcription with purified polymerases, chromatin immunoprecipitation (ChIP) to map polymerase occupancy, RNA-seq to profile transcripts, and inhibitor studies (α-amanitin, actinomycin D) to assign function to specific polymerases.

How it works

1. Pol II and general transcription factors assemble at a core promoter (TATA box recognized by TBP).
2. TFIIH unwinds the DNA to form an open complex; Pol II is positioned at the transcription start site.
3. TFIIH phosphorylates serine residues in the Pol II CTD, triggering promoter escape and elongation.
4. Pol II moves along the template, matching incoming rNTPs to the DNA and synthesizing RNA 5′→3′.
5. The RNA exits through a channel while the DNA duplex re-forms behind the polymerase.
6. Termination signals cause the polymerase to release the transcript and dissociate (e.g., polyadenylation-linked cleavage for Pol II).
7. Pol I and Pol III use analogous cycles at their own promoters to produce rRNA and tRNA/5S RNA.

Common confusions

• "One enzyme makes all RNA" — eukaryotes use three specialized polymerases; only prokaryotes use one.
• "RNA polymerase needs a primer like DNA polymerase" — it does not; it can begin synthesis de novo.
• "Pol II makes only mRNA" — it also transcribes many noncoding RNAs (snRNAs, miRNAs).
• "Transcription copies both strands" — it copies only the template strand; the other strand is the coding (sense) strand.
• "Termination is identical in all cases" — Pol II termination is coupled to 3′-end processing/cleavage, distinct from Pol I/III mechanisms.

Quick review

• Pol I → rRNA; Pol II → mRNA/snRNA/miRNA; Pol III → tRNA/5S rRNA.
• 5′→3′ synthesis, NTP-driven, template read 3′→5′, no primer.
• Pol II cycle: TBP/TFIID → TFIIH unwinds + CTD phosphorylation → elongation → termination.
• α-Amanitin distinguishes the three polymerases.
• Transcription is the key regulated step of gene expression.