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

Worked example

A patient has a mutation in the beta-globin gene at codon 39 that changes CAG to TAG.

Classification. Substitution (point mutation).

Effect. CAG codes for glutamine; TAG is a stop codon. This is a nonsense mutation producing a truncated beta-globin chain.

Phenotype. Beta-thalassaemia, with severe anaemia because functional beta-globin is not produced.

Common traps

Assuming all substitutions change the protein. Silent substitutions exist precisely because the genetic code is degenerate. Always check the codon table before claiming an amino acid change.

Ignoring non-coding mutations. They can be just as important; splice-site mutations are a major cause of inherited disease.

Forgetting the property of the substituted amino acid. Markers reward the chemistry argument (charged vs hydrophobic, large vs small) because it explains why the protein function changes.

Calling a 3-base deletion a frameshift. A multiple of three preserves the reading frame; only indels not divisible by three frameshift.

In one sentence

A mutation in coding DNA can be silent (no amino acid change), missense (one amino acid changed), nonsense (premature stop) or frameshift (reading frame shifted), while a mutation in non-coding DNA can change the amount, timing or splicing of the protein; the severity of the phenotype depends on which protein is affected and how much function is retained.