Gene Mutations
DNA replication is extraordinarily accurate, but not perfect, and DNA can also be damaged by radiation and chemicals. A change in the base sequence of a gene is a gene mutation, and because the base sequence codes for the amino acid sequence of a polypeptide, a mutation may change the polypeptide that is made. This note covers the three types of gene mutation, substitution, insertion and deletion, and explains, using the nature of the genetic code, why some mutations have no effect while others change a single amino acid or completely alter the polypeptide. Exam questions usually give you a DNA sequence, a mutated sequence and part of a codon table, and ask you to work out and explain the effect.
What a gene mutation is
A gene mutation is a change in the sequence of base pairs in a DNA molecule that may result in an altered polypeptide.
Gene mutations happen:
- spontaneously, as random errors during DNA replication in S phase (a wrong nucleotide is inserted and not corrected, or a nucleotide is added or missed);
- more often when DNA is exposed to mutagens: ionising radiation (X-rays, gamma rays, alpha and beta particles), ultraviolet light, and certain chemicals (such as some in tobacco smoke). Mutagens that cause cancer are called carcinogens.
A mutation in a body cell affects only that cell and its descendants produced by mitosis (it may, for example, contribute to a tumour). A mutation in a cell that gives rise to gametes can be inherited by offspring.
A mutation in a gene produces a new allele of that gene. Alleles are different versions of the same gene, with different base sequences.
Three types of gene mutation
Because the genetic code is read in non-overlapping triplets from a fixed start point, the effect of a mutation depends on what it does to the triplets.
Substitution
One base pair is replaced by a different base pair. For example, on the non-transcribed strand, a triplet GAG becomes GTG.
- Only one triplet (and so one codon) is changed. All other codons are read normally.
- Because the code is degenerate, the new codon may still code for the same amino acid: a silent mutation, with no change to the polypeptide. (Changes in the third base of a codon are often silent: for example, GCA, GCC, GCG and GCU all code for alanine.)
- The new codon may code for a different amino acid: one amino acid in the polypeptide is changed (a missense mutation). The effect depends on how different the new R group is and where it is in the protein.
- The new codon may be a stop codon: translation ends early, producing a shortened (truncated) polypeptide, which is usually non-functional (a nonsense mutation).
Insertion
One or more extra nucleotides (base pairs) are added to the sequence.
Deletion
One or more nucleotides (base pairs) are removed from the sequence.
Frameshift: why insertions and deletions are usually serious
If one nucleotide (or any number not a multiple of three) is inserted or deleted, every triplet after the mutation is changed, because the code is read in threes from a fixed point. This is a frameshift.
- Many amino acids after the mutation are changed, so the primary structure is very different.
- A stop codon may appear early in the new reading frame (truncated polypeptide), or the original stop codon may be lost (an abnormally long polypeptide).
- The protein is almost always non-functional.
- The closer to the start of the gene the frameshift is, the more amino acids are affected.
If three nucleotides (one whole triplet) are inserted or deleted, there is no frameshift: one amino acid is added or lost (and perhaps one is changed if the mutation falls across two triplets), and the rest of the polypeptide is normal. This often has a smaller effect.
| Mutation | Change to DNA | Effect on codons | Possible effect on polypeptide |
|---|---|---|---|
| Substitution | one base pair replaced by another | one codon changed; no frameshift | none (silent, due to degenerate code); one amino acid changed; or premature stop codon (shortened polypeptide) |
| Insertion | one or more base pairs added | frameshift: all codons after the mutation changed (unless 3 or a multiple of 3 are inserted) | many amino acids changed from that point; early stop codon or loss of stop codon; usually non-functional |
| Deletion | one or more base pairs removed | frameshift: all codons after the mutation changed (unless 3 or a multiple of 3 are deleted) | many amino acids changed from that point; early stop codon or loss of stop codon; usually non-functional |
From base sequence to protein function
When a mutation changes the amino acid sequence, the chain of consequences is the one from Proteins:
- The primary structure (sequence of amino acids) changes.
- A different R group is present at that position (or many are, after a frameshift).
- Different bonds form between R groups (hydrogen bonds, ionic bonds, disulfide bonds, hydrophobic interactions), or expected bonds are lost.
- The polypeptide folds differently, so its tertiary structure (and quaternary structure) changes.
- The function is altered: for example, an enzyme's active site may no longer be complementary to its substrate, a receptor may not bind its signal, or a transport protein may not work.
A change of one amino acid may have no noticeable effect if the new R group has similar properties (e.g. one non-polar R group for another), or if the amino acid is in a region that does not affect the shape of the functional part of the protein.
Mutations can also occur in introns, which are removed from the primary transcript; these usually have no effect on the polypeptide.
Example: sickle cell anaemia
Sickle cell anaemia is caused by a substitution in the gene for the β-globin polypeptide of haemoglobin (see Haemoglobin and collagen).
- In the normal allele, the sixth triplet on the non-transcribed strand is GAG (template CTC); the mRNA codon is GAG, coding for glutamic acid.
- In the sickle cell allele, the A is substituted by T: non-transcribed strand GTG (template CAC); mRNA codon GUG, coding for valine.
- Glutamic acid has a charged, hydrophilic R group; valine has a non-polar, hydrophobic R group.
- The hydrophobic valine on the surface of the haemoglobin molecule makes molecules of the abnormal haemoglobin (HbS) stick together when oxygen concentration is low, forming long fibres.
- These distort red blood cells into a sickle shape. Sickle cells carry less oxygen, are destroyed more quickly (anaemia), and can block capillaries.
One base substitution, changing one amino acid out of 146, has severe consequences, because of where that amino acid is and how different its R group is.
The non-transcribed strand of part of a gene reads:
ATG GCA TTC AAA GGT TGG CAT TAA
The mRNA codons and amino acids are:
AUG GCA UUC AAA GGU UGG CAU UAA → Met–Ala–Phe–Lys–Gly–Trp–His–stop
Use this information and the codon table in Protein synthesis to describe the effect of each mutation on the polypeptide.
(a) The 6th base, A, is replaced by G. (b) The 10th base, A, is replaced by G. (c) The 18th base, G, is replaced by A.
Solution
(a) Second triplet GCA → GCG; mRNA GCG = alanine. Same amino acid: silent substitution; polypeptide unchanged (because the code is degenerate).
(b) Fourth triplet AAA → GAA; mRNA GAA = glutamic acid instead of lysine. One amino acid changed (Lys → Glu). Lysine has a positively charged R group and glutamic acid a negatively charged one, so ionic bonds may change, altering the tertiary structure.
(c) Sixth triplet TGG → TGA; mRNA UGA = stop. Translation stops after Gly: polypeptide Met–Ala–Phe–Lys–Gly, shortened (truncated), lacking Trp and His and anything after; likely non-functional.
Using the same original sequence, ATG GCA TTC AAA GGT TGG CAT TAA:
(a) Give the amino acid sequence if the 7th base (T) is deleted. (b) Give the amino acid sequence if a G is inserted after the 3rd base. (c) Give the amino acid sequence if bases 7, 8 and 9 (TTC) are deleted. (d) Explain why (c) is likely to have less effect than (a).
Solution
(a) New sequence: ATG GCA TCA AAG GTT GGC ATT AA…
mRNA: AUG GCA UCA AAG GUU GGC AUU → Met–Ala–Ser–Lys–Val–Gly–Ile… Every amino acid after the second is changed, and the original stop codon is lost, so translation continues into the following sequence.
(b) New sequence: ATG GGC ATT CAA AGG TTG GCA TTA A…
mRNA: AUG GGC AUU CAA AGG UUG GCA UUA → Met–Gly–Ile–Gln–Arg–Leu–Ala–Leu… All amino acids after Met are changed.
(c) New sequence: ATG GCA AAA GGT TGG CAT TAA.
mRNA: AUG GCA AAA GGU UGG CAU UAA → Met–Ala–Lys–Gly–Trp–His. Phenylalanine is missing; every other amino acid is unchanged.
(d) Deleting three bases removes one whole triplet, so there is no frameshift: only one amino acid is lost and the rest of the polypeptide is normal. Deleting one base causes a frameshift, changing every codon after the mutation, so many amino acids are changed and the stop codon is lost. The polypeptide in (c) is much more likely to fold into the correct shape and function.
Explain how a single base substitution in the β-globin gene results in the production of an abnormal haemoglobin in sickle cell anaemia.
Solution
- In the DNA, the triplet CTC on the template strand changes to CAC (non-transcribed strand GAG → GTG): a substitution of one base pair;
- the mRNA codon changes from GAG to GUG;
- during translation, a tRNA carrying valine instead of glutamic acid binds to this codon, so the sixth amino acid of the β-globin polypeptide is valine instead of glutamic acid;
- the primary structure changes: valine has a non-polar / hydrophobic R group, whereas glutamic acid has a charged / polar / hydrophilic one;
- the haemoglobin molecules (HbS) are less soluble and stick together at low oxygen concentration, forming fibres that distort red blood cells into a sickle shape.
A scientist sequenced the gene for an enzyme in 50 people. She found 12 different single-base substitutions. Only two of them caused a change in enzyme activity. Suggest explanations for these observations.
Solution
Why most had no effect (any four):
- The genetic code is degenerate: the new triplet may code for the same amino acid (silent mutation), so the primary structure is unchanged.
- The substitution may be in an intron, which is removed by splicing, so it does not appear in the mRNA.
- The new amino acid may have an R group with similar properties (e.g. both non-polar), so the same bonds form and the tertiary structure is unchanged.
- The changed amino acid may be away from the active site and not involved in bonds that maintain its shape, so the active site is still complementary to the substrate.
- The mutation may be in a non-coding region outside the gene's coding sequence.
Why two had an effect (any two):
- The new amino acid has a different R group that forms different bonds (or no longer forms an ionic/hydrogen/disulfide bond), so the tertiary structure and active site shape change; the substrate no longer fits, so fewer ES complexes form.
- A substitution may produce a stop codon, giving a shortened polypeptide that cannot fold correctly.
- The change may be in the active site itself, altering binding of the substrate.
- A gene mutation changes the base sequence of DNA. Do not define it as "a change in an amino acid" or "a change in a protein"; those are possible effects.
- Do not say a mutation "changes the gene into a different gene". It produces a different allele of the same gene.
- Substitutions do not cause frameshifts. Only insertions and deletions (not in multiples of three) do.
- "Silent" mutations are possible because the code is degenerate, not because it is "universal".
- Mutations are random: they are not caused by the organism needing a change.
- When working out mutated sequences, recount the triplets from the start codon after an insertion or deletion; do not keep the old triplet boundaries.
- Learn the syllabus definition word for word: "a change in the sequence of base pairs in a DNA molecule that may result in an altered polypeptide".
- In data questions, set out your answer in stages: identify the change in the DNA triplet → write the new mRNA codon → look up the amino acid → state the effect on the polypeptide. Each stage can be a mark.
- For "explain why a deletion has a greater effect than a substitution", the key words are frameshift, all subsequent codons/triplets changed, many amino acids changed, versus one codon / one amino acid for substitution.
- Link the effect of a changed amino acid to R groups → bonds → tertiary structure → function. Examiners reward this chain of reasoning.
- For sickle cell anaemia: GAG → GUG (mRNA), glutamic acid → valine, hydrophilic → hydrophobic.
- A gene mutation is a change in the sequence of base pairs in DNA that may result in an altered polypeptide.
- Mutations arise from errors in DNA replication or from mutagens (ionising radiation, UV, chemicals); they produce new alleles.
- Substitution: one base pair replaced; one codon changed; may be silent (degenerate code), change one amino acid, or create a stop codon.
- Insertion and deletion: cause a frameshift (unless a multiple of three bases), changing every codon downstream; usually produce a non-functional polypeptide.
- A changed amino acid alters R groups and bonds, so tertiary structure and function (e.g. active site) may change.
- Sickle cell: substitution in β-globin gene, mRNA GAG → GUG, glutamic acid → valine, HbS forms fibres at low oxygen, red cells sickle.
Practice questions
- Define the term gene mutation.
- Name three types of gene mutation.
- State two factors that increase the rate of mutation.
- Explain why a substitution may have no effect on the polypeptide produced. (2 marks)
- Explain what is meant by a frameshift and which types of mutation cause one.
- The mRNA sequence AUG UUA GGC CAU UGA codes for Met–Leu–Gly–His. A substitution changes the second codon to UAA. State and explain the effect on the polypeptide.
- The non-transcribed strand of DNA reads ATG CCA GAT TGA. A mutation inserts a T after the fourth base. Using the codon table, give the original and the new amino acid sequences.
- Explain why a deletion near the beginning of a gene usually has a greater effect than a deletion near the end. (2 marks)
- Explain why the substitution that causes sickle cell anaemia has such a large effect on haemoglobin, even though only one of 146 amino acids in the β-globin chain is changed. (4 marks)
- A gene for an enzyme has the following mutations in different individuals: (P) substitution of the third base of a codon for leucine, CUU → CUC; (Q) deletion of a single nucleotide in the 5th codon; (R) deletion of three adjacent nucleotides forming the 200th codon of 400. Predict and explain the effect of each on the enzyme's activity. (6 marks)
Answers
- A change in the sequence of base pairs in a DNA molecule that may result in an altered polypeptide.
- Substitution; insertion; deletion.
- Any two: ionising radiation (X-rays, gamma rays); ultraviolet light; certain chemicals (mutagens, e.g. in tobacco smoke); errors during DNA replication.
- The genetic code is degenerate, so the new triplet may code for the same amino acid (silent); or the substitution may be in an intron (non-coding, removed by splicing); or the new amino acid may have similar properties / not affect the shape of the protein.
- A frameshift is a change in the reading frame of the genetic code: because codons are read in non-overlapping triplets from a fixed start point, adding or removing one (or two) nucleotides changes every codon after the mutation. It is caused by insertions and deletions (of numbers of nucleotides that are not multiples of three).
- UAA is a stop codon, so translation stops after methionine; the polypeptide is only Met (truncated), so it is non-functional.
- Original mRNA: AUG CCA GAU UGA → Met–Pro–Asp (stop). New DNA: ATG T CCA GAT TGA → ATG TCC AGA TTG A…; mRNA AUG UCC AGA UUG → Met–Ser–Arg–Leu… (frameshift; original stop codon lost, so translation continues).
- A frameshift changes every codon after the mutation; near the beginning, almost all codons and amino acids are changed, whereas near the end only a few are changed, so the polypeptide is more likely to keep its structure and function.
- Glutamic acid (hydrophilic, charged R group) is replaced by valine (hydrophobic, non-polar R group); this amino acid is on the surface of the haemoglobin molecule; the hydrophobic valine causes haemoglobin molecules to stick together / bind to each other at low oxygen concentration, forming long fibres; these distort red blood cells into a sickle shape, so they carry less oxygen, are destroyed rapidly and block capillaries.
- P: silent, no effect: CUC still codes for leucine (degenerate code), so the primary structure and enzyme are unchanged. Q: very likely loss of activity: frameshift near the start changes almost every subsequent codon, many amino acids changed and probably a premature stop codon; the polypeptide cannot fold into the correct tertiary structure, so there is no functional active site. R: smaller effect: one whole codon removed, so one amino acid is missing and there is no frameshift; the rest of the sequence is normal; activity may be unchanged or reduced, depending on whether the missing amino acid is in or near the active site or involved in bonds that maintain the tertiary structure.