Cell Biology | DNA Transcription 🧬

DNA Transcription Overview
📌 Transcription is the process of converting double-stranded DNA into RNA.
🧬 This process requires specific proteins or enzymes, namely RNA polymerase and transcription factors.
🦠 Differences exist between prokaryotic (e.g., bacteria) and eukaryotic (human) cells regarding transcription machinery.

Prokaryotic Transcription Machinery
🔬 Prokaryotic cells use a single enzyme complex called the RNA polymerase holoenzyme to make all RNA types (rRNA, mRNA, tRNA).
⚙️ The holoenzyme consists of the core enzyme (made of $\alpha_2, \beta, \beta', \omega$ subunits) and the sigma ($\sigma$) subunit.
🔑 The sigma subunit is responsible for binding the polymerase to the promoter region of the DNA, while the core enzyme performs the RNA synthesis.

Eukaryotic Transcription Machinery and Products
🧬 Eukaryotic transcription utilizes three distinct RNA polymerases, each requiring general transcription factors (GTFs) to bind the promoter:
* RNA Polymerase I ($\text{Pol I}$): Synthesizes rRNA (incorporated into ribosomes).
* RNA Polymerase II ($\text{Pol II}$): Synthesizes mRNA and small nuclear RNAs ($\text{snRNA}$). GTF TFII$\text{D}$ (containing the TATA-binding protein) is crucial here.
* RNA Polymerase III ($\text{Pol III}$): Primarily synthesizes tRNA, with minor production of $\text{snRNA}$ and some rRNA.

Gene Regulation via Transcription Modulation
⬆️ Transcription rate can be increased by enhancers, which, when bound by specific transcription factors, cause DNA looping to bring them near the promoter, stimulating RNA polymerase activity.
⬇️ Transcription can be decreased by silencers, which fold the DNA to inhibit promoter access.
🔗 Signaling molecules (like hormones such as TSH or testosterone) activate specific transcription factors that modulate gene expression.

Stages of Transcription: Initiation and Elongation
🛑 Initiation involves the binding of polymerases to specific promoter sequences:
* Prokaryotes: Bind near the $-35$ region and the Pribnow box ($-10$ region).
* Eukaryotes ($\text{Pol II}$): Bind near the TATA box, CAAT box, or GC box with the help of GTFs, notably $\text{TFII}\text{D}$.
🔗 Elongation is consistent across both cell types: RNA polymerase reads the template strand (antisense) from $3' \to 5'$ direction and synthesizes the new RNA strand from $5' \to 3'$.
🧪 RNA polymerase possesses intrinsic helicase activity to unwind DNA but lacks a confirmed proofreading function like DNA polymerase.

Inhibitors of Transcription
🍄 $\alpha$-Amanitin (a mushroom toxin) inhibits eukaryotic RNA polymerase.
💊 Rifampicin (an antibiotic) inhibits RNA polymerase in prokaryotic cells (bacteria).

Termination of Transcription
🛑 Termination mechanisms differ between cell types:
* Prokaryotes (Rho-dependent): The Rho protein binds the nascent RNA and physically knocks the RNA polymerase off the DNA.
* Prokaryotes (Rho-independent): Formation of a hairpin loop in the synthesized RNA (due to inverted repeats in the template) signals cleavage enzymes to detach the RNA.
* Eukaryotes: Termination occurs after the RNA polymerase synthesizes the polyadenylation signal ($\text{AAUAAA}$), which triggers enzymes to cleave the RNA transcript.

Post-Transcriptional Modifications (Eukaryotes Only)
🔬 Immature RNA, termed heterogeneous nuclear RNA ($\text{hnRNA}$), undergoes three major modifications to become mature mRNA:
1. $5'$ Capping: A $5'$ cap (a 7-methylguanosine group) is added to the $5'$ end to facilitate translation initiation and prevent degradation by nucleases. This involves RNA triphosphatase and guanylyl transferase (using SAM as a methyl donor).
2. $3'$ Polyadenylation: Poly-A Polymerase adds a string of up to 200 adenine (A) nucleotides, forming the poly-A tail to aid transport out of the nucleus and reduce degradation.
3. Splicing: Non-coding sequences called introns are removed, and coding sequences called exons are stitched together by small nuclear ribonucleoproteins ($\text{snRNPs}$ or "snurps").

Splicing Mechanism Detail
✂️ $\text{snRNPs}$ bind to splice sites: the $3'$ splice site (marked by GU sequence) and the $5'$ splice site (marked by AG sequence), involving a branch point Adenine (A) residue.
🔄 The $\text{snRNP}$ action involves cleaving the $3'$ site, an attack by the branch point's $\text{OH}$ group on the $\text{GU}$ site, and finally, the $3'\text{OH}$ of Exon 1 attacks the $5'\text{AG}$ site of Exon 2, releasing the intron as a lariat structure and joining the exons.
⚠️ Splicing defects can cause severe diseases like spinal muscular atrophy or $\beta$-thalassemia.

Alternative RNA Splicing and RNA Editing
🎭 Alternative RNA splicing allows one $\text{hnRNA}$ transcript to yield multiple distinct mRNAs (and thus protein variants) by selectively including or excluding different exons (e.g., in antibodies or tropomyosin isoforms).
✏️ RNA Editing involves chemically altering nucleotides in the mRNA:
* In enterocytes, the enzyme cytidine deaminase converts a $\text{CAA}$ codon in the $\text{apoB}$ gene transcript to $\text{UAA}$ (a stop codon), resulting in the shorter protein ApoB-48 instead of the full ApoB-100 found in hepatocytes.

Key Points & Insights
➡️ The key functional component for recognizing the prokaryotic promoter is the sigma subunit of the holoenzyme.
➡️ RNA Polymerase II is uniquely responsible for synthesizing mRNA in eukaryotes and relies on numerous General Transcription Factors.
➡️ $5'$ Capping and the $3'$ Poly-A tail both protect the mRNA from degradation by nucleases and help initiate translation.
➡️ Alternative splicing is a major source of protein diversity, allowing one gene to code for multiple protein variants through differential exon inclusion.

📸 Video summarized with SummaryTube.com on Nov 16, 2025, 14:27 UTC