Transcription & RNA Processing
Three polymerases, one death-cap toxin, and the cap-splice-tail assembly line
Transcription basics
DNA → RNA. RNA polymerase builds RNA 5'→3' while reading the template (antisense) strand 3'→5'. The transcript is therefore complementary to the template and matches the coding (sense) strand letter for letter, except that uracil replaces thymine. Unlike DNA polymerase, RNA polymerase needs no primer and does no proofreading.
To read an mRNA off a gene, copy the coding/sense strand and swap every T for U.
Exons and introns. The primary transcript (pre-mRNA, also called heterogeneous nuclear RNA) contains both. Exons are the expressed segments — kept in mature mRNA and usually coding. Introns are intervening sequences that are spliced out in the nucleus. Mature mRNA therefore holds only exons plus a 5' cap and a 3' poly-A tail.
RNA polymerases and their inhibitors
Three eukaryotic polymerases. Pol I makes rRNA, the most abundant RNA, in the nucleolus. Pol II makes mRNA, along with snRNA and miRNA. Pol III makes tRNA and 5S rRNA. Mnemonic: I, II, III → rRNA, mRNA, tRNA ("rmt").
One prokaryotic polymerase. Bacteria use a single multisubunit RNA polymerase to make every type of RNA; a sigma factor lets it recognize promoters.
| Polymerase | Products | Where / clinical hook |
|---|---|---|
| Eukaryotic pol I | rRNA (most abundant RNA) | Nucleolus |
| Eukaryotic pol II | mRNA, snRNA, miRNA | Inhibited by α-amanitin → hepatotoxicity |
| Eukaryotic pol III | tRNA, 5S rRNA | Smallest products (transfer RNA) |
| Prokaryotic RNA pol | All RNA | Sigma factor for promoters; inhibited by rifampin |
α-Amanitin. This toxin of Amanita phalloides, the death cap mushroom, inhibits RNA polymerase II and so halts mRNA synthesis, producing severe hepatotoxicity. The typical story is a forager with vomiting first and fulminant liver failure a couple of days later — the classic cause of mushroom-poisoning liver failure.
Rifampin. Inhibits the bacterial DNA-dependent RNA polymerase and blocks initiation of transcription, sparing the eukaryotic enzymes. Associations: red-orange body fluids, strong cytochrome P450 induction, and rapid resistance if used alone.
Promoters, enhancers, and silencers
Promoter. The site where RNA pol II and the general transcription factors assemble, just upstream of the gene: the TATA box about 25 bases upstream (recognized by the TATA-binding protein of TFIID), with a CAAT box farther up. A promoter mutation typically causes a sharp drop in transcription of that gene.
Enhancers and silencers. DNA regions that bind activator or repressor proteins to raise or lower transcription. They can lie far upstream, downstream, or within an intron; the DNA bends so the bound proteins reach the promoter. Enhancers work in either orientation.
Cis vs trans. Promoters, enhancers, and silencers are cis-acting — on the same DNA molecule as the gene they control. The proteins that bind them (transcription factors, activators, repressors) are trans-acting, encoded elsewhere in the genome.
| Feature | Promoter | Enhancer | Silencer |
|---|---|---|---|
| Position | Fixed, just upstream (TATA ~25 bases up; CAAT farther) | Anywhere: far upstream, downstream, intron | Anywhere, like enhancers |
| Binds | RNA pol II + general transcription factors | Activators | Repressors |
| Effect | Starts transcription; mutation sharply reduces it | Increases transcription | Decreases transcription |
Processing: cap, splice, tail
- Pre-mRNATranscribed by pol II in the nucleus
- 5' cap7-methylguanosine added to the 5' end
- SplicingsnRNPs remove introns and join exons
- 3' poly-A tailAdded after the AAUAAA signal
- Mature mRNAExported to the cytoplasm
5' cap. The nascent transcript receives a 7-methylguanosine cap. It is required for ribosome recognition at translation initiation and protects the mRNA from 5' exonucleases.
Splicing. The spliceosome — built from snRNPs (small nuclear ribonucleoproteins) and other proteins — recognizes intron boundaries (typically GU at the 5' end, AG at the 3' end) and a branch-point adenine. The intron is excised as a lariat anchored at that adenine, and the flanking exons are joined, all in the nucleus before export. Memory aid: exons are EXpressed; INtrons stay IN the nucleus.
3' poly-A tail. The transcript is cleaved downstream of the AAUAAA polyadenylation signal, and poly-A polymerase then adds about 200 adenines without a template. The tail stabilizes the mRNA and aids nuclear export and translation.
Splicing in disease and autoimmunity
Alternative splicing. Joining different exon combinations yields several protein isoforms from one gene. Classic examples: membrane-bound vs secreted antibody from the same B-cell gene, tissue-specific tropomyosin isoforms, and calcitonin (thyroid) vs CGRP (neurons).
Splice-site mutations. Errors at splice junctions produce abnormally spliced, often nonfunctional mRNA; some β-thalassemias arise this way. In spinal muscular atrophy, SMN1 mutation impairs snRNP assembly.
Anti-snRNP antibodies. Anti-Smith (anti-Sm) antibodies target snRNP core proteins and are highly specific for systemic lupus erythematosus, as is anti-dsDNA. Anti-U1 RNP antibodies, also directed at snRNPs, mark mixed connective tissue disease. By contrast, anti-histone antibodies suggest drug-induced lupus.
Spliceosome proteins as autoantigens: anti-Sm → SLE (specific); anti-U1 RNP → mixed connective tissue disease.