Lecture 3: Core Concepts of Transcription 📜✒

Key Questions Addressed:

  • What is the core process of transcription, and how does it work?

  • Compare and contrast the structure and function of Eukaryote and Prokaryote RNA Polymerase.

  • What is the difference between RNAP I, II, and III in eukaryotes?

  • How is RNAP assembled?

  • What is the role of Mg²⁺ in RNAP activity?

The Core Process of Transcription

Transcription is the synthesis of RNA from a DNA template. The general steps are:

  1. Binding: RNA Polymerase (RNAP) binds at the promoter region of a gene.

  2. Melting: The DNA is unwound (melted) to become single-stranded at the transcription start site. RNA ribonucleoside triphosphates (NTPs) are recruited.

  3. Initiation & Elongation: An RNA copy is synthesized from the antisense (template) strand of the DNA as the RNAP moves along the DNA. The mRNA transcript grows in the 5' to 3' direction.

  4. Termination: Transcription stops at a specific termination signal.

More detailed stages include:

  • Recruitment of the Pre-initiation complex (PIC).

  • Melting of the initiator/promoter region.

  • Promoter clearance (RNAP moves away from the promoter).

  • Processive elongation.

  • Termination.

Comparison of Transcription in Prokaryotes and Eukaryotes

There are four major differences in transcription between prokaryotes and eukaryotes:

  1. Location: Transcription occurs in the nucleus in eukaryotes. Consequently, co-transcriptional translation (translation of mRNA while it is still being transcribed), which occurs in prokaryotes, is not possible in eukaryotes.

  2. Chromatin Structure: Chromatin is more compacted in eukaryotes. DNA must be unwound from histones (nucleosomes) before it can be transcribed.

  3. Gene Regulation Complexity: Gene regulation is more complex in eukaryotes, involving numerous interactions between cis-acting DNA sequences (like enhancers and promoters) and trans-acting protein factors (Transcription Factors).

  4. mRNA Modification: In eukaryotes, mRNA undergoes extensive modification after transcription, including 5' capping, intron splicing, and 3' polyadenylation. The initial transcripts are known as heterogeneous nuclear RNAs (hnRNAs).

Bacterial RNAP is a common target for antibiotics and antimicrobials, which specifically inhibit bacterial protein expression.

Bacterial RNA Polymerase

Structure:

  • Bacterial RNAP is a multi-subunit enzyme. The core enzyme consists of 5 subunits:

    • 2 x α-Subunits (RpoA): Structural role, involved in assembly and interaction with regulatory proteins.

    • β Subunit (RpoB): Catalytic activity, involved in RNA synthesis.

    • β' Subunit (RpoC): Catalytic activity, binds DNA template.

    • ω-Subunit (RpoZ): Promoter specificity, involved in assembly and stability.

  • The σ Subunit (sigma factor) associates with the core enzyme to form the holoenzyme. The sigma factor is crucial for promoter recognition and binding, and for initiating transcription.

  • The β and β' subunits are similar in structure. The two α subunits form a homodimer. The sigma factor often forms a link between the beta subunits. The omega subunit's role is variable and less clear.

  • The overall structure has several 'lobes' or protruding parts, forming jaws and flaps that create a clamp to hold the DNA and the nascent mRNA.

Function & Assembly:

  • The holoenzyme scans the DNA for promoter sequences.

  • The sigma factor specifically recognizes and binds to promoter elements (e.g., -10 and -35 boxes). This forms a closed complex.

  • The polymerase then unwinds the DNA at the promoter to form an open complex.

  • After transcription initiation and the synthesis of a short RNA chain, the sigma factor typically dissociates from the core enzyme. The core enzyme continues elongation. The released sigma factor can then bind to another core enzyme.

  • The holoenzyme forms away from the DNA. The core enzyme moves from closed to open to initiation complexes.

  • Different Sigma Factors: Bacteria have different sigma factors (e.g., σ⁷⁰ and σ⁵⁴) that recognize different sets of promoters, allowing for coordinated gene expression.

  • RNAP Conformations: Bacterial RNAP can switch between a "tight form" (which is ratchetable and allows for 1-nt backtracking and RNA 3'-end fraying) and a "ratcheted form" (Gre-bound state, which allows for RNA long backtracking and hairpin-dependent termination). These forms are involved in processes like nucleotide addition, intrinsic RNA cleavage, pausing, and termination.

Eukaryotic RNA Polymerases

Eukaryotes have multiple RNA polymerases, each responsible for transcribing different sets of genes:

  • RNA Polymerase I (Pol I): Transcribes most rRNA genes (except 5S rRNA), which are major structural components of ribosomes and essential for translation.

  • RNA Polymerase II (Pol II): Transcribes mRNA genes (protein-coding genes), as well as most snRNAs and microRNAs. The amount of mRNA produced is generally proportional to the amount of protein.

  • RNA Polymerase III (Pol III): Transcribes tRNA genes, the 5S rRNA gene, some snRNAs, and other small RNAs.

Structure and Comparison to Prokaryotic RNAP:

  • Eukaryotic RNAPs are larger and more complex than bacterial RNAP, typically consisting of 10-17 subunits.

  • However, they share structural and functional similarities with bacterial RNAP, indicating evolutionary conservation. For example, eukaryotic Rpb1 and Rpb2 subunits are homologous to bacterial β' and β subunits, respectively. Rpb3 and Rpb11 are related to bacterial α subunits.

  • Pol I and Pol III are structurally more similar to each other than to Pol II.

  • Eukaryotic RNAP II Structure:

    • Has two major domains: a CORE and a STALK.

    • Subunits assemble into subcomplexes (e.g., Rpb1 subassembly, Rpb2 subassembly, Rpb3 subassembly, Rpb4/7 subcomplex) before the entire enzyme is formed.

    • Features a CLEFT where DNA binds.

  • Assembly of RNAP II Holoenzyme: The holoenzyme, comprising many independent subunits, forms before it binds to DNA.

    • PIC Formation: Transcription initiation by Pol II requires the assembly of a Pre-Initiation Complex (PIC) at the promoter. This involves Pol II and a set of General Transcription Factors (GTFs) including TFIIA, TFIIB, TFIID (containing TATA-binding protein, TBP), TFIIE, TFIIF, and TFIIH, as well as the Mediator complex.

    • The PIC forms, the DNA is opened (by TFIIH helicase activity), and Pol II initiates transcription. After promoter clearance, elongation proceeds, and the complex can be reinitiated.

  • RNAP II Biogenesis: This is a complex process involving the synthesis of subunits in the cytoplasm, their assembly into subcomplexes (some mediated by assembly factors like R2TP and HSP90), import into the nucleus via the Nuclear Pore Complex (NPC) (involving importins like Crm1), and final assembly of the holoenzyme in the nucleoplasm. Some subunits may be degraded if assembly is incorrect.

Role of Magnesium (Mg²⁺) in RNAP Activity

  • Two Mg²⁺ ions are crucial components of the RNAP active site and are essential for catalysis.

  • Their functions include:

    • Performing the deprotonation of the 3'-OH group of the terminal nucleotide on the growing RNA chain. This makes the 3'-OH a stronger nucleophile to attack the α-phosphate of the incoming NTP.

    • Facilitating phosphodiester bond formation between the incoming NTP and the growing RNA chain.

    • Aiding in the release of pyrophosphate (PPi) after nucleotide addition.

    • Stabilizing the transition state during the nucleotidyl transfer reaction.

    • Neutralizing the negative charge on the leaving pyrophosphate group.

Structural Features of Eukaryotic RNAP II During Transcription

  • DNA Interaction: DNA is held within a clamp-like structure of RNAP II.

  • Accessory Proteins (TFs): Numerous GTFs and other accessory proteins assist in the association of RNAP II at the promoter. Most of these are released after transcription has initiated (around 25bp synthesized).

  • Bridge Helix: A key structural element within RNAP II that is important for melting the DNA helix downstream of the active site, allowing the template strand to enter the active site for base pairing with incoming NTPs. The bridge helix undergoes conformational changes (bending) during the catalytic cycle.

  • Rudder, Lid, and Fork Loop: These are other structural elements within RNAP II.

    • The rudder helps to separate the newly synthesized RNA from the DNA template strand.

    • The lid and fork loop also contribute to DNA melting and maintaining the transcription bubble.

  • Trigger Loop: This flexible loop is located near the active site and plays a critical role in nucleotide selection and addition. It changes conformation upon binding of the correct NTP, facilitating catalysis.

  • Translocation Mechanism: RNAP II moves along the DNA template. This process involves oscillations between pre-translocation (after NTP addition, before RNAP moves) and post-translocation (after RNAP has moved one nucleotide forward) states. The enzyme recognizes the correct incoming base largely via the geometry of the complex formed in the active site. Backtracking can also occur if an incorrect nucleotide is incorporated or due to other factors.

  • RNAP II C-Terminal Domain (CTD):

    • The largest subunit of RNAP II (Rpb1) has a long, unstructured C-Terminal Domain (CTD) consisting of multiple repeats of a heptapeptide consensus sequence (Tyr-Ser-Pro-Thr-Ser-Pro-Ser in mammals).

    • The CTD undergoes dynamic phosphorylation and dephosphorylation at specific serine residues (Ser2, Ser5, Ser7) during the transcription cycle.

      • Pre-initiating RNAPII: CTD is largely unphosphorylated.

      • Initiating RNAPII: TFIIH (a GTF with kinase activity) phosphorylates Ser5 (Ser5P).

      • Elongating RNAPII: Positive Transcription Elongation Factor b (P-TEFb, also known as CTDK-I) phosphorylates Ser2 (Ser2P), leading to a Ser2,5P pattern during early elongation, and predominantly Ser2P during later elongation.

      • Terminating RNAPII: Ser5P is removed by a phosphatase (PPase), leaving a Ser2P pattern that is also eventually dephosphorylated.

    • These phosphorylation patterns create binding sites for various protein complexes involved in co-transcriptional RNA processing (5' capping, splicing, 3' polyadenylation), as well as transcription elongation and termination.

Summary

  • Transcription involves several distinct stages from initiation to termination.

  • Prokaryotic and Eukaryotic RNAPs share structural similarities but have fundamental differences in subunit composition and regulatory mechanisms.

  • Eukaryotes utilize three distinct RNA Polymerases (I, II, and III) for transcribing different classes of RNA.

  • RNAP is assembled sequentially into a functional holoenzyme.

  • Mg²⁺ ions are essential cofactors for the catalytic activity of RNA Polymerases.