Mutations, Gene Regulation and Biotechnology
General Biology · Section 2.3 · 18 study cards
How DNA sequence changes affect proteins, how cells switch genes on and off, and the lab tools built on both.
Practice this set → Spaced repetition, card by card. No account needed.
Method
Mutations: work from the protein backwards
Never classify a mutation from the DNA alone. Transcribe, translate, and compare the polypeptides; the label follows from what happened to the protein. The decision is short: if the amino acid is unchanged it is silent, if it is a different amino acid it is missense, if it became a stop it is nonsense, and if every residue after the site changed it is a frameshift.
Regulation: ask what is normal and what the small molecule does
- Decide whether the operon is normally on or normally off. Catabolic pathways such as lactose breakdown are normally off; anabolic pathways such as tryptophan synthesis are normally on.
- Find the small molecule. An inducer inactivates a repressor and turns the operon on; a corepressor activates a repressor and turns it off.
- Check whether anything gives positive control. In the lac operon that is CAP with cyclic AMP, which reports low glucose and is entirely separate from the repressor.
- State the outcome as a level of transcription, not merely on or off, since both controls act together.
For eukaryotes there is no operon. Instead, walk down the chain of opportunities in order: chromatin state, transcription initiation, RNA processing, mRNA lifespan, translation, protein activity and protein destruction. Any exam question about eukaryotic control is asking you to name the right rung of that ladder.
Biotechnology: each tool solves one problem
Learn the tools by the problem each one answers. Not enough DNA, so amplify it: PCR. Need to know the sizes present, so separate them: gel electrophoresis. Need to cut and paste, so use restriction enzymes and ligase. Need many copies inside a living cell, so use a plasmid and transformation. Need the actual bases, so sequence it. Need to change a specific base in a living genome, so use CRISPR-Cas9. Exam questions usually describe a goal and expect you to pick the tool, so this mapping is more useful than the protocols themselves.
Definitions and theorems
Worked example
A strain of E. coli carries a mutation in the operator of its lac operon that stops the lac repressor binding there. Predict whether beta-galactosidase is produced when the cells are grown in a medium with lactose and no glucose, with neither sugar, and with both sugars present. Explain each answer in terms of the two control systems acting on this operon.
State the normal control. In a wild-type cell the repressor sits on the operator and physically blocks RNA polymerase, so the operon is off. Allolactose, made from lactose, binds the repressor and releases it, which switches the operon on.
Work out what the mutation removes. If the repressor cannot bind the mutant operator, the block is gone permanently. The operon is transcribed whether or not lactose is present, so the strain is constitutive for the lac genes.
Lactose, no glucose. Transcription proceeds, as it would in a wild-type cell under the same conditions. Beta-galactosidase is produced at a high level.
Neither sugar. The wild type would make none, but this strain still transcribes the operon because nothing blocks the promoter. Beta-galactosidase is produced, wastefully, with no substrate for it to act on. This is the result that identifies the mutation as an operator defect.
Both sugars. The repressor is irrelevant here, but the second control system is untouched. Plenty of glucose means low cyclic AMP, so CAP stays inactive and does not help RNA polymerase bind the promoter. Transcription therefore falls to a low basal level, and only a little enzyme is made.
Draw the conclusion. The operator mutation removes negative control but leaves positive control intact, so expression becomes independent of lactose but remains sensitive to glucose. Stating both halves is what the question is testing; an answer that says only always on misses the glucose effect.
Common mistakes
- Assuming any base change alters the protein. Redundancy means many substitutions, especially at the third position of a codon, are silent. Always check the codon table before declaring an effect, and remember that a changed DNA sequence and a changed protein are different claims.
- Saying lactose binds the lac repressor. The inducer is allolactose, an isomer of lactose made from it. Marks are routinely allocated to that word, and the same care applies to naming tryptophan itself as the trp corepressor.
- Treating the glucose effect as another repressor. Catabolite repression is positive control through CAP and cyclic AMP acting at the promoter. Glucose does not switch on a repressor; it lowers cyclic AMP, so the activator stops helping. Questions that vary both sugars are testing precisely this distinction.
- Applying operons to eukaryotes. Eukaryotic genes are transcribed individually and regulated by chromatin state, transcription factors and enhancers. Writing about an operator or an inducer in a eukaryotic answer signals the wrong model outright.
- Confusing gel electrophoresis with a sequencing method. A gel separates fragments by size and tells you nothing about which bases are present. Sequencing reads the bases; PCR only makes more copies of a region you already knew how to target with primers.
Practice it
Reading the method is not the same as being able to recall it under pressure. This set drills 18 cards one at a time and schedules each card separately, so the ones you keep missing come back sooner.