Questions: Bacterial Transcription and Operon Regulation

Question 1

E. coli cells are growing in medium containing both glucose and lactose. What is the predicted expression level of the lac operon, and why?

• A. Fully expressed, because lactose is present and the lac repressor is inactivated by allolactose
• B. Largely repressed, because glucose keeps cAMP levels low, preventing CAP-cAMP from activating transcription
• C. Partially expressed, because one activating signal (lactose) and one inhibiting signal (glucose) cancel out
• D. Completely silent, because glucose directly binds the lac promoter and blocks RNA polymerase

Answer: (B)

The lac operon functions as a logical AND gate: full expression requires lactose present (repressor off) AND glucose absent (CAP-cAMP on). When glucose is present, adenylyl cyclase is inhibited, cAMP levels fall, and CAP cannot form the CAP-cAMP complex needed to recruit RNA polymerase. Even though lactose is present and deactivates the repressor, the operon is transcribed at very low levels without positive CAP-cAMP activation. This catabolite repression ensures bacteria consume the most energetically favorable carbon source (glucose) before investing in enzymes for alternative sugars.

Question 2

What is the functional significance of bacteria possessing multiple sigma factors?

• A. Each sigma factor proofreads a different class of transcripts for errors before they are translated
• B. Different sigma factors direct RNA polymerase to different sets of promoters, enabling global transcriptional reprogramming in response to environmental conditions
• C. Multiple sigma factors serve as backup subunits in case the primary sigma factor is degraded
• D. Multiple sigma factors allow several RNA polymerases to transcribe the same gene simultaneously, increasing output

Answer: (B)

Sigma factors are the address-recognition modules of RNA polymerase: they bind promoter sequences and determine which genes are transcribed. Bacteria maintain multiple sigma factors with different sequence specificities — σ⁷⁰ for housekeeping genes, σ³² for heat shock genes, σˢ for stationary-phase survival, σ⁵⁴ for nitrogen metabolism. By changing which sigma factor is loaded onto the core polymerase, the cell can globally redirect transcription to an entirely different set of genes. This is faster and more comprehensive than individually regulating hundreds of genes, making sigma factor switching a powerful regulatory strategy for responding to stress and environmental change.

Question 3

Removing glucose from E. coli growth medium causes intracellular cAMP levels to rise, which promotes CAP-cAMP binding and boosts lac operon transcription.

• T. True
• F. False

Answer: True

When glucose is present, it inhibits adenylyl cyclase (the enzyme that synthesizes cAMP from ATP), keeping cAMP low and CAP inactive. When glucose is removed, adenylyl cyclase activity increases, cAMP levels rise, and cAMP binds CAP. The CAP-cAMP complex then binds upstream of the lac promoter, bends the DNA, and directly contacts RNA polymerase, stimulating transcription roughly 50-fold. This explains catabolite repression: glucose's indirect effect on cAMP is the mechanism by which its presence suppresses lac operon expression.

Question 4

In the lac operon system, the absence of glucose alone is sufficient to drive full lac operon transcription, even if lactose is also absent from the medium.

• T. True
• F. False

Answer: False

Full lac operon expression requires two conditions simultaneously: lactose must be present (to generate allolactose, which inactivates the lac repressor) AND glucose must be absent (to raise cAMP levels and activate CAP). Without lactose, the lac repressor remains bound to the operator regardless of glucose levels, blocking transcription. CAP-cAMP activation increases transcriptional efficiency, but it cannot overcome a repressor that is physically blocking the polymerase. The AND logic prevents wasteful enzyme synthesis: there is no point in making lactose-metabolizing enzymes if there is no lactose to metabolize.

Question 5

Why does E. coli express the lac operon at high levels only when lactose is present AND glucose is absent, and what does this two-condition requirement reveal about how bacteria prioritize energy sources?

Model answer: The two conditions implement a nutrient hierarchy. Glucose is the preferred carbon source because it is metabolized more efficiently. By requiring glucose absence (high cAMP → active CAP) for full lac operon expression, the cell ensures it invests in lactose-metabolizing enzymes only when glucose is unavailable. Lactose presence (allolactose → repressor off) ensures the enzymes are made only when there is actually lactose to metabolize. The AND gate prevents two kinds of waste: making lactose enzymes when glucose is already available (unnecessary), and making them when lactose is absent even if glucose is gone (pointless). This regulatory logic is a general principle of bacterial metabolism — major catabolic operons are only fully activated when the preferred substrate is gone and the alternative substrate is present.

The broader principle is catabolite repression: glucose and its metabolites suppress the expression of operons for alternative carbon sources throughout the bacterial genome. This is not glucose directly repressing the lac operon — it is glucose indirectly keeping cAMP low and thus CAP inactive. The system is therefore responsive to the cell's overall metabolic state, not just to the presence of any one molecule.