Gene Regulation and Bacterial Genetics

lac operon induction

The lac operon of Escherichia coli is an inducible system that is normally switched off. In the absence of lactose, the LacI repressor binds the operator and blocks transcription of lacZ, lacY and lacA. Allolactose, an isomer of lactose formed from lactose by a basal level of β-galactosidase, binds LacI, changes its shape, and releases it from the operator, so accumulation of allolactose induces transcription. Full expression also requires the catabolite activator protein. cAMP binds CAP, not the promoter DNA. The cAMP–CRP complex binds a site upstream of the lac promoter and stimulates RNA polymerase, so removing cAMP lowers induction rather than promoting it. The on-off response is also characteristic. After lactose is added, lac mRNA rises quickly, and permease and β-galactosidase appear after a short lag because the transcript must first be translated. When lactose is removed, the mRNA decays within minutes, whereas the enzymes are stable and persist. β-galactosidase accumulates to a higher level than permease because lacZ is translated more often than lacY. Key terms: inducer — a small molecule that inactivates a repressor and switches on transcription, e.g., IPTG, a non-metabolised analogue, induces the operon. CAP-cAMP complex — a positive regulator that must bind the promoter region before RNA polymerase can act efficiently.

Hfr conjugation gene mapping

In an Hfr strain, the F factor is integrated into the bacterial chromosome. Transfer begins at oriT and proceeds linearly, so the time at which a gene enters the F- recipient is proportional to its distance from oriT. Genes nearer the origin are transferred first and appear earlier as recombinants when the mating is interrupted, so the order of appearance reveals the gene order. Because the donor DNA is a linear fragment while the F- chromosome is circular, a single crossover cannot produce a stable recombinant. A double crossover is required, which exchanges a homologous segment and replaces the recipient allele with the donor allele. The minimum number of crossovers is therefore two. Key terms: oriT — the origin of transfer from which the Hfr chromosome enters the recipient. interrupted mating — a technique that stops conjugation at timed intervals to determine when each marker enters the recipient.

Genetic code combinatorics

The genetic code is read as codons, and the number of distinct codons of length n that can be built from k different nucleotides is k^n. The capacity of a code therefore depends on both the alphabet size and the codon length. To specify at least 20 amino acids, the codon length must satisfy k^n \ge 20. With k = 4 and n = 3, the standard code gives 64 codons, which is more than enough. With k = 2, n = 4 gives only 16 codons, which is too few, while n = 5 gives 32 codons, which is sufficient. Key terms: degenerate code — a code in which several codons specify the same amino acid, which is why the number of codons can exceed the number of amino acids.

Restriction enzyme digestion

Restriction endonucleases cut DNA at specific recognition sequences, which are usually palindromic. HindIII and EcoRI are examples of enzymes that leave sticky ends. The number of fragments produced depends on the topology of the DNA molecule. A linear DNA with n cut sites gives n + 1 fragments. A circular DNA with n cut sites gives n fragments, because the cut sites divide the circle into n arcs. For example, a circular plasmid with three sites that are all cut yields three fragments. Complete digestion means that every recognition site is cut.