RNA Polymerase 1, 2, and 3: Functions and Differences

By Dr. Zubair Khalid, DVM, MS, PhD · 2026-08-11

Introduction to RNA Polymerases

RNA polymerase is the enzyme responsible for catalyzing the template-directed synthesis of RNA from a DNA template. This process, termed transcription, is the first step in gene expression and is fundamental to all life. In prokaryotes, a single RNA polymerase core enzyme (with a sigma factor for promoter recognition) synthesizes all cellular RNAs. In eukaryotes, however, the task is divided among three distinct nuclear RNA polymerases—RNA Polymerase I (Pol I), RNA Polymerase II (Pol II), and RNA Polymerase III (Pol III)—each dedicated to transcribing specific classes of genes. A fourth polymerase, Pol IV and Pol V, exists in plants for siRNA-mediated silencing, but the three nuclear polymerases are the focus here.

The division of labor is not arbitrary. Each polymerase recognizes different promoter architectures, transcribes different gene sets, and produces RNAs with distinct cellular fates. Pol I is devoted almost exclusively to the large ribosomal RNA (rRNA) precursor. Pol II synthesizes all messenger RNAs (mRNAs) and most small nuclear RNAs (snRNAs) and microRNAs (miRNAs). Pol III produces transfer RNAs (tRNAs), the 5S rRNA, and other small structural RNAs. Understanding the functions and differences among these enzymes is essential for grasping how eukaryotic gene expression is organized, regulated, and integrated with cell growth and stress responses.

RNA Polymerase I: Ribosomal RNA Synthesis

RNA Polymerase I is the most specialized of the three eukaryotic polymerases. Its sole function is to transcribe the ribosomal DNA (rDNA) repeat unit, which encodes three of the four ribosomal RNA components. In humans, the rDNA repeat is approximately 43 kb long and is present in 300–400 tandem copies on the short arms of chromosomes 13, 14, 15, 21, and 22 (the nucleolar organizer regions). Each repeat contains a single transcription unit that produces a 47S precursor rRNA, which is subsequently processed into the 18S, 5.8S, and 28S rRNAs.

Promoter Recognition and Transcription Factors

Pol I does not recognize its promoter directly. Instead, it relies on two core transcription factors: upstream binding factor (UBF) and selectivity factor 1 (SL1, also known as TIF-IB in mice). The Pol I promoter consists of two elements: the core promoter (spanning approximately −45 to +20 relative to the transcription start site) and the upstream control element (UCE, spanning approximately −180 to −107). UBF binds as a dimer to both the UCE and the core promoter, inducing DNA bending and facilitating the recruitment of SL1. SL1 is a complex of TATA-binding protein (TBP) and three TBP-associated factors (TAFs) specific to Pol I. Once SL1 is bound, it recruits Pol I itself, along with the initiation factors Rrn3 (a Pol I-associated factor essential for initiation) and the core factor complex (in yeast).

The transcription cycle for Pol I is tightly coupled to ribosome biogenesis. The 47S precursor is co-transcriptionally assembled with ribosomal proteins and small nucleolar ribonucleoproteins (snoRNPs) that mediate the cleavage and modification steps required to produce mature rRNAs. The mature rRNAs then combine with ribosomal proteins to form the 40S and 60S ribosomal subunits.

Regulation and Nucleolar Localization

Pol I transcription occurs within the nucleolus, a membrane-less nuclear organelle that forms around the rDNA repeats. The nucleolus is a dynamic structure; its size and number reflect the transcriptional activity of Pol I. Actively growing cells have large, prominent nucleoli, whereas quiescent cells have small, inconspicuous ones.

Pol I activity is regulated primarily by growth signaling pathways, including mTOR (mechanistic target of rapamycin) and MYC. mTOR signaling promotes Pol I transcription by enhancing the association of Rrn3 with Pol I and by promoting UBF phosphorylation. Conversely, cellular stress—such as DNA damage, nutrient deprivation, or inhibition of protein synthesis—leads to rapid repression of Pol I transcription. This repression is mediated in part by the tumor suppressor p53, which sequesters SL1, and by the dephosphorylation of Rrn3, which inactivates it. The rate of Pol I transcription is a major determinant of ribosome production and, consequently, of cell growth and proliferation.

RNA Polymerase II: Messenger RNA and More

RNA Polymerase II is the workhorse of eukaryotic gene expression. It transcribes all protein-coding genes into messenger RNA (mRNA), as well as many non-coding RNAs, including most small nuclear RNAs (snRNAs), microRNAs (miRNAs), and long non-coding RNAs (lncRNAs). Pol II is also responsible for synthesizing the RNA component of the spliceosome (U1, U2, U4, U5, and U6 snRNAs, though U6 is transcribed by Pol III in some organisms) and the telomerase RNA component (TERC).

Pol II is a 12-subunit enzyme (in yeast and mammals) with a total molecular mass of approximately 550 kDa. Its largest subunit, RPB1, contains a unique C-terminal domain (CTD) consisting of tandem heptapeptide repeats with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. In humans, this repeat is present 52 times; in yeast, 26 times. The CTD is a platform for the recruitment of RNA processing factors and is extensively modified by phosphorylation during the transcription cycle.

Core Promoter Elements

Unlike Pol I and Pol III, Pol II promoters are highly diverse. The core promoter—the minimal DNA region required for accurate transcription initiation—typically spans from approximately −40 to +40 relative to the transcription start site (TSS). Several sequence elements are found within core promoters, though no single element is present in all genes:

• TATA box: A conserved AT-rich sequence (consensus TATAAA) located approximately 25–30 bp upstream of the TSS. It is bound by the TATA-binding protein (TBP) subunit of TFIID.
• Initiator (Inr): A pyrimidine-rich sequence (consensus YYANWYY in humans) that overlaps the TSS and is recognized by TAF1 and TAF2 of TFIID.
• Downstream promoter element (DPE): Located approximately +28 to +32, it is recognized by TAF6 and TAF9 in TATA-less promoters.
• TFIIB recognition element (BRE): Located immediately upstream (BREu) or downstream (BREd) of the TATA box, recognized by TFIIB.

The general transcription factors (GTFs)—TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH—assemble with Pol II at the core promoter to form the preinitiation complex (PIC). TFIID, which contains TBP and 13–14 TAFs, is the primary promoter recognition factor. TFIIH possesses both helicase (XPB and XPD subunits) and kinase (CDK7) activities; its helicase activity melts the promoter DNA to form the open complex, and its kinase activity phosphorylates the CTD at Ser5 to release Pol II from the promoter.

Enhancers, which can be located thousands of base pairs away from the promoter, regulate Pol II transcription by recruiting sequence-specific transcription factors that interact with the PIC via coactivators such as Mediator. Mediator is a large multi-subunit complex that bridges enhancer-bound activators and the PIC, stimulating both PIC assembly and the transition to productive elongation.

The CTD and Processing Coupling

The CTD is not merely a structural appendage; it orchestrates the coupling of transcription to RNA processing. During the transcription cycle, the CTD undergoes a dynamic pattern of phosphorylation:

1. Ser5 phosphorylation (by CDK7 in TFIIH) occurs during initiation and early elongation. It recruits the capping enzyme, which adds a 7-methylguanosine cap to the 5' end of the nascent RNA.
2. Ser2 phosphorylation (by CDK9 in P-TEFb) increases during productive elongation. It recruits splicing factors and the 3' end processing machinery (CPSF and CstF).
3. Ser7 phosphorylation is important for the expression of snRNAs and certain histone genes.

The transition from initiation to elongation is also marked by the exchange of the negative elongation factors NELF and DSIF for the positive elongation factor P-TEFb. P-TEFb phosphorylates both the CTD and the negative factors, converting them into positive regulators. This regulatory checkpoint is a major point of control for genes that are poised for rapid induction, such as heat shock genes and immediate-early response genes.

Termination of Pol II transcription is coupled to 3' end processing. For most mRNAs, cleavage and polyadenylation at a poly(A) site triggers termination. The "torpedo" model proposes that the 5'→3' exonuclease XRN2 degrades the RNA downstream of the cleavage site, catching up to Pol II and promoting its release. For replication-dependent histone genes, which lack poly(A) tails, a stem-loop structure and the histone-specific factor SLBP mediate a distinct termination pathway.

RNA Polymerase III: Small RNAs and tRNA

RNA Polymerase III transcribes a diverse set of small, essential RNAs. Its primary products are transfer RNAs (tRNAs), the 5S ribosomal RNA, the U6 snRNA, the signal recognition particle (SRP) RNA, RNase P RNA, and several other small RNAs involved in splicing and translation. In total, Pol III transcribes approximately 500–600 genes in the human genome, but these genes are highly transcribed, and Pol III products can constitute up to 15% of total cellular RNA.

Pol III is a 17-subunit enzyme (in humans) with a total mass of approximately 700 kDa. It shares five core subunits with Pol I and Pol II (RPC5, RPC6, RPC8, RPC9, and RPB10), and it has three Pol III-specific subunits (RPC1, RPC2, and RPC3) that are homologous to Pol II subunits. Pol III also has three additional subunits (RPC4, RPC5, and RPC7) that are unique to it.

Type 1, 2, and 3 Promoters

Pol III promoters are unusual in that they are often located within the transcribed region (internal promoters) rather than upstream of the transcription start site. Three promoter types are recognized:

Type 1 promoters are found in the 5S rRNA genes. They consist of three internal elements: the A box (positions +50 to +64), the intermediate element (IE, +67 to +72), and the C box (+80 to +97). The 5S rRNA gene is transcribed by Pol III only after the assembly of a complex containing TFIIIA, TFIIIC, and TFIIIB. TFIIIA is a zinc-finger protein that binds specifically to the internal control region; it is the only transcription factor known to be gene-specific for a Pol III transcript.

Type 2 promoters are found in tRNA genes. They contain two internal elements: the A box (positions +8 to +19) and the B box (+52 to +62). The A and B boxes correspond to the D- and T-arm sequences of the mature tRNA, respectively. TFIIIC binds directly to these internal elements and recruits TFIIIB, which then recruits Pol III.

Type 3 promoters are found in the U6 snRNA gene and other Pol III-transcribed genes that resemble Pol II promoters. They contain an upstream TATA box (recognized by TBP), a proximal sequence element (PSE), and a distal sequence element (DSE). These promoters are recognized by the snRNA-activating protein complex (SNAPc) and the Oct-1 transcription factor. Notably, the U6 promoter has a TATA box, which is a hallmark of Pol II promoters, but the presence of the PSE and the absence of a downstream promoter element direct the gene to Pol III.

In all three promoter types, the central event is the recruitment of TFIIIB, which contains TBP and the Pol III-specific factors Brf1 (or Brf2 for type 3 promoters) and Bdp1. TFIIIB is the principal Pol III initiation factor; once bound, it positions Pol III at the start site and remains bound during multiple rounds of initiation.

Termination Signals

Pol III termination is remarkably simple and does not require auxiliary factors. A run of four or more thymidine residues (T-tract) on the non-template strand is sufficient to signal termination. The Pol III-specific subunit RPC11 (C11 in yeast) contains a zinc-ribbon domain that is required for this intrinsic termination. When Pol III encounters the T-tract, it pauses, and the RNA:DNA hybrid in the active site becomes unstable, leading to transcript release. The efficiency of termination increases with the length of the T-tract; five or six T residues are typically more efficient than four. This simple termination signal is in stark contrast to the complex, factor-dependent termination mechanisms of Pol I and Pol II.

Structural and Functional Differences

The three RNA polymerases share a conserved core architecture, reflecting their common evolutionary origin. The active site, formed by the two largest subunits (RPB1/RPA1/RPC1 and RPB2/RPA2/RPC2), is nearly identical among the three enzymes. However, the polymerases differ substantially in their total subunit count, their sensitivity to inhibitors, and their subnuclear localization.

Subunit Similarities and Specifics

The largest subunits of Pol I and Pol III lack the CTD found in Pol II. Instead, Pol I and Pol III have specific subunits (RPA43 and RPC11, respectively) that serve analogous functions in recruiting processing factors. The additional subunits in Pol III (RPC4, RPC5, RPC7) are involved in promoter recognition and in the intrinsic termination mechanism.

Differential Inhibition by Toxins

The fungal toxin alpha-amanitin, produced by the death cap mushroom Amanita phalloides, is a powerful tool for distinguishing the three polymerases. Alpha-amanitin binds to the bridge helix of the largest subunit, trapping the polymerase in a state that prevents translocation after nucleotide addition. The three polymerases differ dramatically in their sensitivity:

• Pol II is exquisitely sensitive, with a half-maximal inhibitory concentration (IC50) of approximately 1–10 ng/mL (1–10 nM).
• Pol III is moderately sensitive, with an IC50 of approximately 1–10 µg/mL (1–10 µM).
• Pol I is essentially resistant, even at concentrations exceeding 1 mg/mL.

This differential sensitivity arises from a single amino acid difference in the bridge helix. In Pol II, the residue at the critical position is a proline; in Pol I, it is a leucine; in Pol III, it is a valine. The bulky side chain of leucine in Pol I sterically prevents alpha-amanitin from binding, while the smaller valine in Pol III allows weak binding.

Transcription Mechanisms and Regulation

Despite their differences in promoter recognition and product identity, all three polymerases follow the same fundamental transcription cycle: initiation, elongation, and termination. The mechanistic details, however, differ substantially.

Initiation Complexes

Pol I: Initiation requires UBF, SL1, and Rrn3. UBF binds to the UCE and core promoter, SL1 provides sequence-specific recognition via TBP, and Rrn3 bridges Pol I to the SL1-UBF complex. Once initiation is complete, Rrn3 dissociates from Pol I and must be re-phosphorylated to participate in a new round of initiation.

Pol II: Initiation requires the ordered assembly of the PIC. The pathway is as follows:

1. TFIID binds to the TATA box (if present) and/or the Inr and DPE elements.
2. TFIIA stabilizes TFIID binding.
3. TFIIB binds to the BRE and to TBP, providing a bridge to Pol II.
4. Pol II, in complex with TFIIF, is recruited to the promoter.
5. TFIIE and TFIIH join the complex.
6. TFIIH melts the promoter DNA (using its XPB helicase) to form the open complex.
7. TFIIH phosphorylates the CTD at Ser5, triggering promoter escape.

Pol III: Initiation is the simplest. TFIIIC binds to the internal A and B boxes (for type 2 promoters), then recruits TFIIIB. TFIIIB, once bound, does not require TFIIIC for subsequent rounds of transcription; it remains stably associated with the promoter and recruits Pol III directly. This explains why Pol III genes can be transcribed at very high rates.

Elongation and Proofreading

All three polymerases are processive enzymes that can synthesize RNA at rates of 20–50 nucleotides per second in vivo. They all possess a conserved active site that catalyzes nucleotide addition and also performs proofreading. When a misincorporated nucleotide is detected, the polymerase backtracks by one or more nucleotides, and the intrinsic endonucleolytic activity (stimulated by TFIIS for Pol II, RPA12 for Pol I, and RPC11 for Pol III) cleaves the nascent RNA to remove the error. This proofreading mechanism reduces the error rate of transcription to approximately 1 in 10⁴ to 10⁵ nucleotides.

During elongation, Pol I and Pol II are associated with specific elongation factors. Pol I uses Spt4/Spt5 (DSIF) and the PAF complex, while Pol II uses DSIF, NELF (negative), P-TEFb (positive), and the elongation factors ELL and PAF. Pol III elongation is less regulated, consistent with the constitutive nature of most Pol III transcripts.

Termination Strategies

The three polymerases employ fundamentally different termination mechanisms:

• Pol I: Termination is mediated by the terminator element T1, which is bound by the Reb1 protein (in yeast) or TTF-I (in mammals). Transcription terminates approximately 200 bp downstream of the 28S rRNA coding region. The nascent RNA is then cleaved by the Rnt1 (yeast) or RNase III (mammals) endonuclease, and the 3' end is processed by the exosome.
• Pol II: Termination is coupled to 3' end processing. For polyadenylated mRNAs, the CPSF and CstF complexes recognize the poly(A) signal (AAUAAA) and the downstream GU-rich element, respectively. Cleavage at the poly(A) site generates a free 5' end that is degraded by XRN2, which "torpedoes" Pol II off the template. For histone genes, a stem-loop structure and the U7 snRNP mediate a similar process.
• Pol III: Termination is intrinsic, requiring only a run of four or more T residues on the non-template strand. The RPC11 subunit senses the T-tract and triggers transcript release.

Methods to Study RNA Polymerases

Several experimental approaches are used to investigate the functions and regulation of the three RNA polymerases.

In Vitro Assays

Run-on transcription: Nuclei are isolated from cells and incubated with radiolabeled or biotinylated nucleotides. The nascent RNA chains are extended by polymerases that were engaged at the time of nuclear isolation. This assay measures the density of engaged polymerases on a gene of interest and can distinguish Pol I, II, and III by using alpha-amanitin at the appropriate concentrations.

In vitro transcription with nuclear extracts: Whole-cell or nuclear extracts are incubated with a DNA template containing a promoter of interest. By adding alpha-amanitin at 1 µg/mL, Pol II is inhibited while Pol I and III remain active; at 100 µg/mL, both Pol II and III are inhibited. This allows the specific activity of each polymerase to be measured.

Promoter pulldown assays: Biotinylated promoter DNA is immobilized on streptavidin beads and incubated with nuclear extract. The bound proteins are then analyzed by Western blotting or mass spectrometry to identify the factors that assemble on the promoter.

Genomic Approaches

Chromatin immunoprecipitation (ChIP): Cells are cross-linked with formaldehyde, chromatin is sheared by sonication, and antibodies specific to each polymerase (e.g., anti-RPB1 for Pol II, anti-RPA194 for Pol I, anti-RPC155 for Pol III) are used to immunoprecipitate DNA fragments bound by the polymerase. The associated DNA is then identified by quantitative PCR (ChIP-qPCR) or by high-throughput sequencing (ChIP-seq). This reveals the genomic occupancy of each polymerase and its changes under different conditions.

RNA-seq: Total RNA is sequenced to quantify the steady-state levels of all transcripts. By comparing RNA-seq data with Pol II ChIP-seq data, one can distinguish between changes in transcription (reflected in polymerase occupancy) and changes in RNA stability (reflected only in RNA levels).

Nascent RNA sequencing: Techniques such as PRO-seq (precision run-on sequencing) and NET-seq (native elongating transcript sequencing) capture RNA that is being actively transcribed. These methods provide a snapshot of polymerase position at nucleotide resolution and can reveal promoter-proximal pausing, elongation dynamics, and termination sites.

CRISPR-based approaches: Catalytically dead Cas9 (dCas9) fused to transcriptional activators or repressors can be targeted to specific promoters to modulate polymerase recruitment. This approach has been used to study the consequences of activating or repressing Pol I transcription at individual rDNA repeats.

Common Misconceptions and Pitfalls

Prokaryotic vs. Eukaryotic Polymerases

A frequent source of confusion is the relationship between prokaryotic and eukaryotic RNA polymerases. Bacteria have a single RNA polymerase core enzyme (α₂ββ'ω) that associates with a sigma factor to recognize promoters. This enzyme transcribes all genes, including rRNA, tRNA, and mRNA. Eukaryotes, by contrast, have three distinct polymerases, each dedicated to a specific class of genes. The bacterial enzyme is most similar to Pol II in terms of its ability to transcribe protein-coding genes, but it is not homologous to any single eukaryotic polymerase. Instead, the eukaryotic polymerases evolved from a single ancestral enzyme through gene duplication and specialization.

Another common error is to confuse RNA polymerase with DNA polymerase. While both enzymes synthesize nucleic acids in the 5'→3' direction, they differ fundamentally in template usage (RNA vs. DNA), substrate (ribonucleotides vs. deoxyribonucleotides), and product (RNA vs. DNA). RNA polymerase does not require a primer, whereas DNA polymerase requires a pre-existing 3'-OH group. For a detailed comparison of the DNA polymerases, see DNA Polymerase 1 2 3 and Difference Between DNA Polymerase 1 and 3. The distinction between primase and polymerase is also commonly misunderstood; primase is an RNA polymerase that synthesizes short RNA primers for DNA replication, as discussed in Primase vs Polymerase.

Overlap in Functions

Students often assume that each polymerase transcribes a completely non-overlapping set of genes. While this is largely true, there are exceptions. For example, the U6 snRNA gene is transcribed by Pol III in most eukaryotes, but in some organisms (e.g., trypanosomes), it is transcribed by Pol II. Conversely, some snRNA genes that are typically transcribed by Pol II can be transcribed by Pol III under certain conditions. Additionally, the 5S rRNA gene is transcribed by Pol III in all eukaryotes, but the 5.8S rRNA is part of the Pol I transcript. This distinction is a common source of exam errors: Pol I makes the 47S precursor that contains 18S, 5.8S, and 28S rRNA; Pol III makes the 5S rRNA separately.

Another misconception is that Pol II transcribes only mRNA. In reality, Pol II also transcribes many non-coding RNAs, including most snRNAs, miRNAs, and lncRNAs. Conversely, Pol III transcribes some RNAs that are not tRNAs or 5S rRNA, such as the 7SL RNA (component of the signal recognition particle) and the RNase P RNA.

Finally, students sometimes assume that alpha-amanitin inhibits all eukaryotic transcription. In fact, Pol I is resistant, and Pol III is only moderately sensitive. At the concentrations typically used to inhibit Pol II (1–10 ng/mL), Pol I and Pol III are unaffected. This differential sensitivity is a valuable experimental tool, not a universal inhibitor.

Frequently Asked Questions

What is RNA polymerase 1, 2, and 3?

RNA polymerase 1 (Pol I), 2 (Pol II), and 3 (Pol III) are the three nuclear RNA polymerases in eukaryotic cells. Each is a multi-subunit enzyme that catalyzes DNA-dependent RNA synthesis but transcribes different classes of genes. Pol I synthesizes ribosomal RNA (rRNA), Pol II synthesizes messenger RNA (mRNA) and most small nuclear RNAs, and Pol III synthesizes transfer RNA (tRNA), 5S rRNA, and other small RNAs.

What is the function of RNA polymerase 1?

RNA polymerase 1 transcribes the ribosomal DNA repeat to produce a single 47S precursor RNA that is processed into the 18S, 5.8S, and 28S ribosomal RNAs. These rRNAs are essential components of ribosomes, and Pol I transcription is a major determinant of ribosome biogenesis and cell growth. Pol I is localized in the nucleolus and is regulated by growth signaling pathways such as mTOR and MYC.

What is the function of RNA polymerase 2?

RNA polymerase 2 transcribes all protein-coding genes into messenger RNA (mRNA), as well as many non-coding RNAs, including most small nuclear RNAs (snRNAs), microRNAs (miRNAs), and long non-coding RNAs (lncRNAs). Pol II is regulated by complex promoter and enhancer elements and is coupled to RNA processing events such as capping, splicing, and polyadenylation through its C-terminal domain (CTD).

What is the function of RNA polymerase 3?

RNA polymerase 3 transcribes transfer RNA (tRNA) genes, the 5S ribosomal RNA gene, the U6 snRNA gene, the 7SL RNA gene (signal recognition particle), and other small structural RNAs. Pol III promoters are often located within the transcribed region (internal promoters), and termination is signaled by a simple run of thymidine residues. Pol III products are essential for translation and RNA processing.

How do RNA polymerase 1, 2, and 3 differ?

The three polymerases differ in their subunit composition (14, 12, and 17 subunits for Pol I, II, and III, respectively), their promoter recognition mechanisms, their products, their sensitivity to alpha-amanitin, and their subnuclear localization. Pol I is resistant to alpha-amanitin, Pol II is highly sensitive (IC50 ~1–10 ng/mL), and Pol III is moderately sensitive (IC50 ~1–10 µg/mL). Pol I is localized in the nucleolus, while Pol II and III are in the nucleoplasm.

Which RNA polymerase is inhibited by alpha-amanitin?

RNA polymerase 2 is the most sensitive to alpha-amanitin, with a half-maximal inhibitory concentration of approximately 1–10 ng/mL. RNA polymerase 3 is moderately sensitive (IC50 ~1–10 µg/mL), and RNA polymerase 1 is resistant. This differential sensitivity is used experimentally to distinguish the activities of the three polymerases.

Where are RNA polymerase 1, 2, and 3 located?

RNA polymerase 1 is located in the nucleolus, where it transcribes ribosomal DNA. RNA polymerase 2 is distributed throughout the nucleoplasm, where it transcribes protein-coding genes. RNA polymerase 3 is also in the nucleoplasm, but it can be enriched in specific foci, and the 5S rRNA gene is transcribed near the nucleolus. The localization of each polymerase reflects the nuclear position of its target genes.

Key Takeaways

• Eukaryotes have three nuclear RNA polymerases—Pol I, Pol II, and Pol III—each dedicated to transcribing specific gene classes: rRNA (Pol I), mRNA and most snRNAs (Pol II), and tRNA and 5S rRNA (Pol III).
• Pol I transcribes a single 47S rDNA precursor that yields 18S, 5.8S, and 28S rRNAs; it is localized in the nucleolus and is regulated by growth signaling pathways.
• Pol II transcribes all protein-coding genes and is regulated by complex core promoter elements, enhancers, and the Mediator complex; its CTD couples transcription to RNA capping, splicing, and polyadenylation.
• Pol III transcribes tRNA, 5S rRNA, U6 snRNA, and other small RNAs; it uses internal promoters (types 1 and 2) or upstream promoters (type 3) and terminates at simple T-tracts.
• The three polymerases share a conserved catalytic core but differ in subunit number, alpha-amanitin sensitivity (Pol II > Pol III > Pol I), and nuclear localization.
• Alpha-amanitin is a useful experimental tool: at 1–10 ng/mL it inhibits Pol II, at 1–10 µg/mL it inhibits Pol III, and Pol I remains resistant even at milligram concentrations.
• Transcription by all three polymerases follows the same fundamental cycle—initiation, elongation, termination—but the regulatory mechanisms and accessory factors are polymerase-specific.

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