the expression of a gene to form a functional protein in a eukaryotic cell, including transcription, RNA processing (5' capping, polyadenylation and splicing) and translation, and the role of mRNA, tRNA and ribosomes

What this dot point is asking

VCAA wants the eukaryotic pathway from a gene to a functional protein, named in order: transcription, RNA processing (5' cap, poly-A tail, splicing) and translation. You also need the role of mRNA, tRNA and ribosomes.

The answer

Gene expression is the process by which the information in a gene is used to make a functional product (usually a protein). In eukaryotes it occurs in two main locations: transcription and processing in the nucleus, translation in the cytosol.

Transcription (nucleus)

Initiation

Transcription factors and RNA polymerase II assemble at the promoter region of the gene (a TATA box sits upstream of the start site). The DNA double helix unwinds, exposing the template strand.

Elongation

RNA polymerase moves along the template 3' to 5', adding complementary RNA nucleotides to the growing pre-mRNA in the 5' to 3' direction. Pairing follows A with U, T with A, G with C. The newly built pre-mRNA carries the same sequence as the non-template (coding) strand, with U in place of T.

Termination

RNA polymerase reaches a terminator sequence, releases the pre-mRNA, and the DNA re-zips.

RNA processing (nucleus)

Pre-mRNA undergoes three modifications before it can leave the nucleus.

1. 5' capping. A 7-methylguanosine cap is added to the 5' end. It protects against exonucleases and is recognised by ribosomes for translation initiation.
2. 3' polyadenylation. A poly-A tail of roughly 100 to 250 adenine nucleotides is added to the 3' end. It increases mRNA stability and aids export through the nuclear pore.
3. Splicing. The spliceosome removes introns (non-coding regions) and joins the exons (coding regions). Alternative splicing can join different combinations of exons, so one gene can code for several related proteins.

The mature mRNA exits the nucleus through a nuclear pore to the cytosol.

Translation (cytosol or rough ER)

Initiation

The small ribosomal subunit binds the 5' cap and scans for the first AUG start codon. An initiator tRNA carrying methionine binds with anticodon UAC. The large subunit then joins to form a complete ribosome with three sites: A (aminoacyl), P (peptidyl) and E (exit).

Elongation

A charged tRNA enters the A site, and its anticodon pairs with the mRNA codon. The ribosome (rRNA acting as a ribozyme) catalyses a peptide bond between the amino acid in the A site and the polypeptide in the P site. The ribosome translocates one codon along the mRNA; the empty tRNA shifts to the E site and exits.

Termination

When a stop codon (UAA, UAG or UGA) reaches the A site, a release factor binds, the polypeptide is freed, and the ribosome dissociates.

Roles at a glance

Why processing matters

Without the 5' cap, the ribosome cannot recognise the start. Without the poly-A tail, mRNA is rapidly degraded. Without splicing, introns would be translated as nonsense codons or premature stops, producing a non-functional protein.

Examples in context

Example 1. Sickle-cell anaemia and a single base change at WEHI. WEHI molecular biologists explain sickle-cell anaemia as a transcription-translation problem. The HBB gene has a single A-to-T substitution in exon 1, changing codon 6 from GAG (glutamate) to GTG (valine). After transcription, 5' capping, polyadenylation and splicing, the mature mRNA carries the altered codon. tRNA carrying valine docks at the ribosome instead of the tRNA carrying glutamate. The resulting beta-globin chain has valine at position 6, which causes haemoglobin to polymerise into long fibres when deoxygenated, deforming red blood cells into the sickle shape. The whole pathway from gene to phenotype runs through transcription, RNA processing and translation.

Example 2. mRNA splicing variants in breast cancer at Peter MacCallum. Peter MacCallum researchers found that BRCA1 mRNA undergoes alternative splicing, producing multiple protein isoforms from one gene. Some isoforms retain DNA-repair function; others, lacking key exons, lose it. Splicing requires the spliceosome to cut introns precisely; mutations in splice sites cause exons to be skipped. In familial breast cancer, certain BRCA1 splice-site mutations remove a critical exon, producing a truncated protein that cannot repair DNA double-strand breaks. The Peter Mac genetic counselling clinic uses RNA sequencing alongside DNA sequencing to identify splicing defects that DNA tests alone would miss.

Try this

Q1. Outline the three steps of RNA processing in eukaryotes and state the role of each. [3 marks]

• Cue. 5' capping (protects mRNA, helps ribosome bind), polyadenylation (stabilises mRNA, aids export), splicing (removes introns, joins exons).

Q2. A DNA template strand reads 3'-TACGGCATT-5'. (a) Write the mRNA produced. (b) Using a codon table, translate the mRNA into amino acids. [2+2 marks]

• Cue. (a) 5'-AUGCCGUAA-3'. (b) AUG (Met, Start) - CCG (Pro) - UAA (Stop). Peptide is Met-Pro.

Q3. Refer to translation. (a) State the function of mRNA, tRNA and the ribosome. (b) Explain how a single base substitution can change a polypeptide. (c) Predict the effect of a mutation in the start codon. [2+2+2 marks]

• Cue. (a) mRNA carries codons; tRNA brings amino acids matching codons via anticodons; ribosome catalyses peptide bond formation. (b) New codon may code for a different amino acid, altering protein structure and function. (c) No translation initiates; no protein produced.