14.2 Pre-mRNA Splicing & Eukaryotic Transcription

Eukaryotic Transcription by RNA Polymerase II

In eukaryotes, three nuclear RNA polymerases divide transcription duty: RNA polymerase I transcribes the large rRNA precursor (28S, 18S, 5.8S), RNA polymerase II transcribes all protein-coding genes to produce pre-mRNA, and RNA polymerase III transcribes tRNAs, 5S rRNA, and other small RNAs. PA-CAT items typically emphasize Pol II because it produces the mRNA that the translation machinery reads, and because its product is the substrate for all the processing steps that follow.

Unlike bacterial RNA polymerase, which needs only a sigma factor to recognize promoters, RNA polymerase II requires a set of general transcription factors (GTFs) — TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH — to assemble at the promoter into the pre-initiation complex. TFIID (via its TATA-binding protein subunit, TBP) recognizes the TATA box located roughly 25–30 nucleotides upstream of the transcription start site. TFIIH supplies helicase activity that melts the DNA and kinase activity that phosphorylates the Pol II C-terminal domain (CTD), releasing the polymerase from the promoter to begin elongation.

Promoters, Enhancers, and Silencers

A promoter is the proximal regulatory region immediately upstream of a gene where the transcription machinery assembles. Core promoter elements include the TATA box, the initiator (Inr) at the start site, and the downstream promoter element (DPE). Enhancers are distal, orientation-independent elements that can lie tens of kilobases upstream, downstream, or within an intron; they bind activator proteins and, through DNA looping, recruit the Mediator complex that bridges them to Pol II. Silencers bind repressors and dampen transcription. A common PA-CAT framing asks how enhancers can act at a distance — the answer is DNA looping mediated by Mediator and cohesin, which brings enhancer-bound activators into physical proximity with the promoter.

Co-transcriptional Processing of Pre-mRNA

As Pol II transcribes, the nascent pre-mRNA is co-transcriptionally processed in three reactions that convert it into mature, export-ready mRNA.

5' Capping

Shortly after transcription begins, the 5' end is modified with a 7-methylguanosine cap linked by an unusual 5'-5' triphosphate bond. The cap protects the transcript from 5' exonucleases, promotes ribosome binding during translation initiation, and is recognized by the cap-binding complex for nuclear export.

3' Cleavage and Polyadenylation

Near the 3' end, the pre-mRNA is cleaved at a polyadenylation signal (canonical sequence AAUAAA) ~10–30 nucleotides downstream, and poly(A) polymerase adds a tail of 150–250 adenylate residues without a template. The poly-A tail protects the 3' end, aids nuclear export, and synergizes with the cap to enhance translation through a closed-loop poly(A)-binding protein/eIF4E interaction. Note that the poly-A tail is added enzymatically, not encoded in the DNA.

Pre-mRNA Splicing

Eukaryotic genes are interrupted: exons (expressed sequences) are separated by introns (intervening sequences). Splicing removes introns and ligates exons to produce a continuous coding sequence. The reaction is carried out by the spliceosome, a megadalton machine built from small nuclear ribonucleoproteins (snRNPs) — U1, U2, U4, U5, U6 — and ~150 accessory proteins.

The spliceosome recognizes three key sequences: the 5' splice site (consensus GU), the 3' splice site (consensus AG), and the branch point (an adenine ~20–40 nt upstream of the 3' splice site). Splicing proceeds through two transesterification reactions:

1. The 2'-OH of the branch-point adenine attacks the 5' splice-site GU, forming a lariat (looped) intron and freeing the 5' exon.
2. The 3'-OH of the freed 5' exon attacks the 3' splice-site AG, joining the two exons and releasing the intron lariat, which is debranched and degraded.

snRNP Roles at a Glance

Alternative Splicing

A single pre-mRNA can be spliced in different combinations — exon skipping, intron retention, mutually exclusive exons, or alternative 5'/3' splice sites — to produce multiple protein isoforms from one gene. This is the primary mechanism by which the ~20,000 human protein-coding genes yield an estimated >100,000 distinct proteins. Mutations that disrupt splice sites cause a substantial fraction of genetic disease (~15% of disease-causing point mutations), a clinically relevant point for PA-CAT items linking molecular biology to pathology.

Splicing vs. Other Processing — Quick Comparison

Why This Matters on the PA-CAT

The Bulletin groups transcription with RNA structure because the two are inseparable: Pol II makes the very substrate that tRNA and ribosomes will read. Expect items that test: (1) which polymerase makes mRNA (Pol II), (2) which factor recognizes the TATA box (TFIID/TBP), (3) the chemistry of splicing (two transesterifications, lariat intermediate), (4) the role of the 5' cap and poly-A tail, and (5) how alternative splicing multiplies protein diversity. Be ready to distinguish prokaryotic (no introns, coupled transcription-translation, polycistronic) from eukaryotic (introns, nuclear processing, monocistronic) gene expression — the PA-CAT loves this comparison.