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Question 1: Explain the molecular mechanisms underlying the regulation of the lac operon in E. coli and discuss how it adapts to changes in glucose and lactose availability.

Expert Solution:

The lac operon in Escherichia coli is a classic example of gene regulation in prokaryotes. It consists of structural genes (lacZ, lacY, and lacA), a promoter, an operator, and a regulatory gene (lacI). The operon functions through an inducible system controlled by lactose availability and catabolite repression via glucose.

1. Regulation by Lactose:

   • In the absence of lactose, the lacI gene encodes the lac repressor protein, which binds to the operator region, preventing RNA polymerase from transcribing the operon’s genes.

   • When lactose is available, it is converted into allolactose, which acts as an inducer by binding to the repressor. This binding alters the repressor’s conformation, preventing it from binding to the operator, thereby allowing transcription of the operon.

2. Effect of Glucose via Catabolite Repression:

   • When glucose levels are high, E. coli prefers using glucose over lactose due to catabolite repression.

   • The cyclic AMP (cAMP) level is inversely proportional to glucose concentration. In low glucose conditions, cAMP binds to the catabolite activator protein (CAP), which then attaches to the promoter region of the lac operon, enhancing RNA polymerase binding and promoting transcription.

   • When glucose levels are high, cAMP levels decrease, leading to reduced CAP binding and decreased transcription of the lac operon, even if lactose is available.

Thus, the lac operon efficiently regulates gene expression based on nutrient availability, ensuring metabolic efficiency for the bacterium.

Question 2: Describe the key steps involved in eukaryotic DNA replication and explain how cells ensure accuracy during the process.

Expert Solution:

Eukaryotic DNA replication is a complex, highly regulated process that ensures faithful transmission of genetic information. The process occurs in the S-phase of the cell cycle and involves the following key steps:

1. Initiation:

   • DNA replication begins at multiple origins of replication along the chromosomes.

   • Origin Recognition Complex (ORC) binds to replication origins, recruiting additional factors like MCM helicase, which unwinds the DNA.

   • The pre-replication complex forms, and once the cell transitions to S-phase, replication is initiated.

2. Elongation:

   • DNA polymerase α synthesizes short RNA-DNA primers to initiate synthesis.

   • Leading strand synthesis is carried out by DNA polymerase ε, which continuously extends in the 5′ to 3′ direction.

   • Lagging strand synthesis occurs discontinuously, forming Okazaki fragments, which are later joined by DNA ligase.

   • DNA polymerase δ extends the Okazaki fragments and has exonuclease activity for error correction.

3. Termination:

   • Replication forks converge, and synthesis is completed.

   • Telomerase extends the telomeres in certain cell types, preventing loss of genetic material.

Mechanisms Ensuring Accuracy:

• Proofreading Activity: DNA polymerases have a 3′ to 5′ exonuclease activity that removes misincorporated nucleotides.

• Mismatch Repair (MMR): Post-replication, the MMR system corrects base-pair mismatches.

• Cell Cycle Checkpoints: Surveillance mechanisms halt progression if errors are detected, allowing repair before continuation.

These stringent control mechanisms ensure minimal errors, maintaining genetic integrity across generations.

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