Assess the significance of 'coding' and 'non-coding' DNA segments in the process of mutation and investigate the effects of different mutations on a protein's amino acid sequence

What this dot point is asking

NESA wants you to trace the consequence of a mutation through the DNA, mRNA and protein levels, and explain why mutations in non-coding regions can also affect phenotype. The standard worked example is sickle cell anaemia.

The answer

A mutation's effect on protein depends on where it lands (coding vs non-coding region) and what kind of change it is (silent, missense, nonsense or frameshift).

Coding vs non-coding DNA

Coding DNA (exons). Translated into amino acids. A mutation here directly changes the protein sequence (or stops translation).

Non-coding DNA. Includes promoters, enhancers, introns, splice sites, untranslated regions (UTRs) and non-coding RNA genes. Not translated, but mutations here can still change the amount, timing or splicing of the protein.

In humans, less than 2% of the genome codes directly for protein. Most regulatory sequence is non-coding, so non-coding mutations are common and important.

Effects on amino acid sequence (coding region mutations)

Silent mutation. A substitution that does not change the amino acid because the genetic code is degenerate (e.g. GGA and GGC both code for glycine). No effect on protein sequence.

Missense mutation. A substitution that changes one amino acid for another. Effect depends on:

• Which amino acid changes. A conservative change (one hydrophobic for another) often preserves function. A non-conservative change (charged to non-polar, as in sickle cell) is more likely to disrupt folding or activity.
• Where in the protein. Changes at the active site of an enzyme or at a protein-protein interface are usually catastrophic; changes in loops or surface residues may be tolerated.

Nonsense mutation. A substitution that creates a premature stop codon (UAA, UAG, UGA). The protein is truncated and usually non-functional. Many Duchenne muscular dystrophy alleles are nonsense mutations in the dystrophin gene.

Frameshift mutation. An insertion or deletion of a number of bases not divisible by three shifts the reading frame from the mutation onward. The amino acid sequence past the mutation is essentially random, and a premature stop codon usually appears within a few codons, producing a truncated, non-functional protein.

Effects of non-coding region mutations

Promoter mutations

Alter transcription factor binding, increasing or decreasing transcription. A weaker promoter for a tumour suppressor reduces its expression and increases cancer risk.

Splice-site mutations

Disrupt the GT...AG signals at intron boundaries, causing exon skipping or intron retention. Many beta-thalassaemia and Marfan syndrome alleles are splice-site mutations.

Enhancer and silencer mutations

Change tissue-specific or developmental-stage expression.

Mutations in non-coding RNA genes

A mutation in a microRNA gene can dysregulate dozens of target mRNAs.

Worked example: sickle cell anaemia

DNA

Beta-globin gene, codon 6, sense strand changes from GAG to GTG (a single A to T substitution).

mRNA

Codon 6 changes from GAG to GUG.

Protein

Glutamic acid (charged, hydrophilic) is replaced by valine (uncharged, hydrophobic). This is a non-conservative missense mutation at a surface residue.

Cell level

The hydrophobic valine creates a sticky patch on the beta-globin surface. Under low oxygen, the deoxygenated haemoglobin (HbS) polymerises into long fibres, deforming red blood cells into rigid sickled shapes.

Organism level

Sickled cells block capillaries (vaso-occlusive pain crises), are destroyed by the spleen (chronic haemolytic anaemia) and have a shortened lifespan. Heterozygotes are carriers with partial resistance to malaria, which explains the high allele frequency in malarial regions.

Summary table

Examples in context

Example 1. Cystic fibrosis and the F508del mutation. Around 1 in 2500 babies in Australia is born with cystic fibrosis, and the most common cause is the F508del mutation in the CFTR gene. The mutation is an in-frame deletion of three nucleotides (CTT) in exon 11, which removes the codon for phenylalanine at position 508 of the CFTR chloride channel protein. Because exactly three bases are removed, the reading frame is preserved (this is not a frameshift), but the missing amino acid causes the protein to misfold and be degraded by the cell's quality control machinery before reaching the cell membrane. The lung epithelium cannot then move chloride ions, mucus thickens, and recurrent infection follows.

Example 2. A non-coding mutation in lactase persistence. Most adult mammals lose the ability to digest lactose, but roughly 35 percent of Australian adults of European ancestry can. The mutation is not in the coding region of the lactase gene (LCT) itself but in a regulatory region 14 kilobases upstream, within an intron of an adjacent gene. A single C to T substitution at position -13910 creates a stronger binding site for a transcription factor, keeping LCT transcription active in adulthood. Although the lactase protein itself is unchanged, the amount made is much higher. This is a textbook example of why non-coding DNA matters and why focussing only on coding regions misses important phenotypic variation.

Try this

Q1. A coding DNA sequence reads 5'-ATGCATCCATAA-3'. A point mutation changes the second codon from CAT to CAC. State the type of mutation and justify your answer using a codon table reference. [3 marks]

• Cue. CAT and CAC both code for histidine (His); this is a silent (synonymous) mutation that does not alter the amino acid sequence.

Q2. The mRNA sequence 5'-AUGGAAUUCUAA-3' is mutated by inserting a single G after the start codon. Predict the new amino acid sequence and identify the type of mutation. Use AUG = Met, GGA = Gly, AUU = Ile, CUA = Leu, A and other codons as needed. [3 marks]

• Cue. Insertion of one base shifts the reading frame: AUG-GGA-AUU-CUA-A... so the polypeptide becomes Met-Gly-Ile-Leu... This is a frameshift mutation.

Q3. Compare the phenotypic effects of a missense mutation, a nonsense mutation and a frameshift mutation occurring in the same coding region. (a) For each, describe the effect on the amino acid sequence. (b) Predict which is most likely to produce a non-functional protein and justify your answer. [3+3 marks]

• Cue. (a) Missense: one amino acid swapped; nonsense: premature stop truncates the protein; frameshift: changes every downstream codon. (b) Frameshift or nonsense typically more damaging because they disrupt the entire C-terminal portion of the protein.