Transcription

Transcription is the first stage of gene expression. It converts the digital, double-stranded, base-coded information stored in DNA into a single-stranded, transportable, translatable messenger RNA (mRNA) intermediate. In eukaryotic cells, transcription is spatially separated from translation: the mRNA is synthesised in the nucleus, processed within the nucleus, and then exported through nuclear pores to ribosomes in the cytoplasm or on the rough endoplasmic reticulum. This separation creates an opportunity for extensive post-transcriptional regulation — capping, polyadenylation, splicing, and alternative splicing — that is absent from the simpler prokaryotic system.

Spec mapping: This lesson sits in AQA 7402 Section 3.4.2 — DNA and protein synthesis, with extension into Section 3.8 (control of gene expression) through the role of transcription factors. The relevant content covers the mechanism of transcription, the role of RNA polymerase, and the production of mature mRNA from pre-mRNA in eukaryotes. (Refer to the official AQA specification document for exact wording.)

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Overview — Why Transcription Is Necessary

DNA is the long-term, faithful, double-stranded store of genetic information. It is too valuable to send to the ribosomes — the working copies must therefore be derived from it. Transcription generates those working copies as mRNA, a single-stranded molecule that can:

• be produced rapidly in many copies (amplification);
• be processed and edited (capping, polyadenylation, splicing);
• be exported from the nucleus to the cytoplasm without taking the entire genome with it;
• be degraded relatively quickly, providing a kinetic mechanism for switching genes off.

The relationship between DNA and mRNA is asymmetric in another important way: only one strand of the DNA double helix is used as a template for any given gene. The other strand carries the complementary sequence and is bypassed by the polymerase.

Strand	Role	Alternative names
Template strand	Read by RNA polymerase 3′→5′; complementary to mRNA	Antisense strand, non-coding strand
Coding strand	Same sequence as mRNA (except T replaces U); not used as template	Sense strand, non-template strand

Key Definition: Transcription is the process by which the base sequence of one strand of a gene's DNA (the template strand) is used to synthesise a complementary single-stranded messenger RNA molecule. The mRNA is built 5′→3′ from free ribonucleoside triphosphates by the enzyme RNA polymerase.

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The Mechanism of Transcription

Initiation

1. Transcription factors assemble at the promoter region — a specific DNA sequence located upstream (5′ side) of the gene. In eukaryotes a common promoter element is the TATA box (a T-A-rich consensus sequence ~25–30 bp upstream of the transcription start site).
2. The transcription factors recruit RNA polymerase II (the enzyme that synthesises mRNA in eukaryotes) to the promoter. Unlike DNA polymerase, RNA polymerase does not require a primer.
3. RNA polymerase positions itself on the DNA and begins to unwind a short region of the double helix (~10–14 bp), breaking the hydrogen bonds between complementary base pairs and creating a transcription bubble.

Elongation

4. RNA polymerase reads the template strand in the 3′→5′ direction.
5. Free ribonucleoside triphosphates (ATP, GTP, CTP, UTP — note U replaces T in RNA) are tested for complementarity with the template:
   • Template A → mRNA U
   • Template T → mRNA A
   • Template C → mRNA G
   • Template G → mRNA C
6. RNA polymerase catalyses the formation of phosphodiester bonds between adjacent ribonucleotides, releasing pyrophosphate and extending the mRNA chain in the 5′→3′ direction.
7. As RNA polymerase moves forward, the DNA double helix re-anneals behind it. The transcription bubble travels with the polymerase like a moving zip.

Termination

8. RNA polymerase reaches a termination sequence on the DNA.
9. The polymerase dissociates from the template, releasing the newly synthesised RNA.
10. In eukaryotes the released RNA is the pre-mRNA (also called the primary transcript) and undergoes further processing before becoming functional mature mRNA.

flowchart TD
  A["Initiation: TFs + RNA Pol II bind to promoter (TATA box)"] --> B["Transcription bubble opens"]
  B --> C["Elongation: free NTPs added 5'→3'; template read 3'→5'"]
  C --> D["DNA re-anneals behind the moving polymerase"]
  D --> E["Termination at terminator sequence"]
  E --> F["Pre-mRNA released for processing"]

Worked example — transcribing a DNA template

A DNA template strand has the sequence 3′-TAC GCG ATT CCG GGA ATT-5′. The mRNA synthesised from this template by RNA polymerase will have a complementary sequence in the 5′→3′ direction. Write the sequence of the resulting mRNA.

Step 1. Note the template strand is presented 3′→5′ — the direction in which RNA polymerase reads.

Step 2. Build the complementary mRNA 5′→3′, replacing T with U:

Template (3′→5′)	T	A	C	G	C	G	A	T	T	C	C	G	G	G	A	A	T	T
mRNA (5′→3′)	A	U	G	C	G	C	U	A	A	G	G	C	C	C	U	U	A	A

Step 3. mRNA sequence: 5′-AUG CGC UAA GGC CCU UAA-3′.

Note this happens to contain an AUG start codon, a UAA stop codon (at position 4), and another UAA at the end. In a real gene the start and stop would be widely separated and the intervening codons would form the open reading frame.

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Prokaryotic vs Eukaryotic Transcription

The fundamental chemistry is identical, but the regulation, location and processing differ substantially:

Feature	Prokaryotes	Eukaryotes
Location	Cytoplasm (no nucleus)	Nucleus
RNA polymerase	Single type	Three types — Pol I (rRNA), Pol II (mRNA), Pol III (tRNA + small RNAs)
Transcription factors	Largely unnecessary — RNA polymerase binds directly via a σ factor	Required for RNA polymerase to bind the promoter
Promoter elements	Pribnow box (−10), −35 element	TATA box (−25 to −30), CpG islands, enhancers
mRNA processing	None — translation begins immediately	Extensive — 5′ cap, poly-A tail, splicing
Coupling with translation	Yes — ribosomes attach to nascent mRNA while transcription is ongoing	No — mRNA must be processed and exported first
Introns	Rare	Common — must be spliced out
Operons	Common — multiple genes co-transcribed (e.g. lac operon)	Rare — most genes have individual promoters

This regulatory complexity is one reason why eukaryotic gene expression is more amenable to fine-grained tissue-specific and developmental control: every step from chromatin remodelling onwards offers a potential regulatory checkpoint.

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Post-Transcriptional Modifications

In eukaryotes the pre-mRNA undergoes three crucial modifications before it can be exported and translated:

1. Addition of a 5′ cap

• A modified guanine nucleotide (7-methylguanosine, m⁷G) is added to the 5′ end of the pre-mRNA via an unusual 5′-to-5′ triphosphate linkage.
• Functions:
  • Protects the mRNA against degradation by 5′→3′ exonucleases.
  • Acts as a recognition element for the small ribosomal subunit during translation initiation (the cap-binding complex eIF4F binds m⁷G).
  • Facilitates export from the nucleus through nuclear pores.

2. Addition of a poly-A tail

• A chain of 100–250 adenine ribonucleotides is added to the 3′ end of the pre-mRNA. This is catalysed by poly-A polymerase, which does not require a template — it simply adds A residues one at a time.
• Functions:
  • Protects against 3′→5′ exonuclease degradation.
  • Provides binding sites for poly-A-binding proteins (PABPs), which loop the mRNA and bring its 3′ end into contact with the 5′ cap during translation, enhancing ribosome recruitment.
  • Determines mRNA stability — a longer tail correlates with longer cytoplasmic half-life; the tail is progressively shortened by deadenylases until critical destabilisation triggers degradation.

3. Splicing

• Introns are excised from the pre-mRNA and exons are joined to form the continuous coding sequence of the mature mRNA.
• Splicing is catalysed by the spliceosome, a ribonucleoprotein complex composed of five small nuclear RNAs (U1, U2, U4, U5, U6 — collectively the snRNAs) plus associated proteins (small nuclear ribonucleoproteins, snRNPs).
• The process:
  1. The spliceosome recognises specific consensus sequences at the intron-exon boundaries (5′ splice site: GU at the 5′ end of the intron; 3′ splice site: AG at the 3′ end; branch point: a conserved A within the intron).
  2. The intron is cut at the 5′ splice site and forms a lariat (loop) structure by linking the 5′ end of the intron to the branch-point A.
  3. The intron is then cleaved at the 3′ splice site and released as a lariat.
  4. The two flanking exons are joined by a phosphodiester bond.
  5. The excised intron is degraded and its nucleotides are recycled.

flowchart TD
  A["Pre-mRNA: Exon1 — Intron1 — Exon2 — Intron2 — Exon3"] --> B["snRNPs assemble at splice sites"]
  B --> C["Intron 1 cut at 5' splice site; forms lariat at branch point"]
  C --> D["Intron 1 released as lariat; Exon 1 joined to Exon 2"]
  D --> E["Process repeats for Intron 2"]
  E --> F["Mature mRNA: Exon1-Exon2-Exon3 + 5' cap + poly-A tail"]
  F --> G["Export through nuclear pore to cytoplasm"]

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Alternative Splicing — Expanding the Proteome

Key Definition: Alternative splicing is the process by which different combinations of exons from the same pre-mRNA are joined to produce different mature mRNA molecules and therefore different polypeptides from a single gene.

This is the dominant mechanism by which complex organisms expand their proteome beyond their modest gene count:

• A single gene with multiple exons can yield many different mRNAs depending on which exons are retained or skipped.
• The choice of splice pattern is regulated by tissue-specific splicing factors (SR proteins, hnRNPs) and by signalling pathways that modulate spliceosome assembly.
• The choice can vary between cell types, developmental stages, and environmental conditions.
• ~95% of human multi-exon genes undergo some form of alternative splicing.
• Common alternative splicing patterns include exon skipping (a cassette exon is included or excluded), mutually exclusive exons (one exon from a pair is included, the other excluded), alternative 5′ or 3′ splice sites (variants of the same splice junction), and intron retention (an intron is occasionally retained in the mature mRNA).

Example — calcitonin / CGRP

In humans, the calcitonin / CGRP gene illustrates tissue-specific alternative splicing:

• In thyroid C-cells, exons 1–4 are joined, producing the hormone calcitonin (which lowers blood Ca²⁺).
• In neurones, exons 1–3 and 5–6 are joined, producing calcitonin gene-related peptide (CGRP) — a vasodilator and neurotransmitter implicated in migraine pathophysiology.

The same gene, the same genome, the same pre-mRNA — but two functionally distinct proteins generated by tissue-specific splicing decisions.

Example — Drosophila Dscam

The fruit fly Dscam (Down syndrome cell adhesion molecule) gene contains four clusters of mutually exclusive exons (12, 48, 33, and 2 alternatives respectively). The combinatorial choice gives 12 × 48 × 33 × 2 = 38,016 possible mRNA variants from a single gene — more than twice the total number of genes in the Drosophila genome. This expansion underlies the specificity of neuronal wiring in the developing fly brain: different neurones express different Dscam isoforms, allowing them to recognise "self" axons (matching Dscam isoform → repulsion to prevent self-contact) while remaining unresponsive to "non-self" axons (different Dscam isoform).

The Dscam example is a striking illustration of how alternative splicing can expand a single gene's functional output to combinatorial levels that exceed what would be achievable by gene duplication alone.

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Worked Example — Calculating the Effect of Splicing

A gene transcribes a 12 kb pre-mRNA containing 8 exons (each ~500 bp) and 7 introns (each ~1 kb). After splicing, what is the size of the mature mRNA?