Question

Distinguish between inducible operons and repressible operons and explain how they work. Describe the three types of prokaryotic genetic recombination (conjugation, transformation, and transduction). Explain how recombination might interfere with the metabolic functions of operons, such as the lac operon or trp operon of E. coli.

Answer 1

Distinguish between inducible operons and repressible operons and explain how they work.

            An operon is a cluster of coordinately regulated genes. Inducible operons are switched on in presence of a metabolite (a small molecule undergoing metabolism) that regulates the operon. E.g. the lac operon get induced in the presence of lactose through the action of allolactose (a metabolic by-product).

            However, repressible operons are turned off in response to a small regulatory molecule. E.g., the trp operon is repressed in the presence of tryptophan.

Type of Operon	Presence of	Effect	Metabolite	Operon
Inducible	Metabolite	ON	Lactose	Lac
Repressible	Metabolite	OFF	Trytophan	Trp

            Control of an operon is a type of gene regulation that enables organisms to regulate the expression of various genes depending on environmental conditions. Operon regulation can be either negative or positive by induction or repression.

In negative inducible operons, a regulatory repressor protein is generally bound to the operator that prevents the transcription of the genes on the operon. In presence of inducer molecule, it binds to the repressor and modify its conformation which makes it unable to bind to the operator. This allows for expression of the operon. The lac operon is a negatively controlled inducible operon when inducer molecule is allolactose.

In negative repressible operons, transcription of the operon generally occurs. Regulator gene produces repressor proteins, but unable to bind to the operator in their normal conformation. However, specific molecules called corepressors are bound by the repressor protein and produce changes in the conformation of active site and the activated repressor protein binds to the operator that prevents transcription. The trp operon, involved in the synthesis of tryptophan (which itself acts as the corepressor), is a negatively controlled repressible operon.

In positive inducible operons, activator proteins are generally not capable to bind to the relevant DNA. However, when an inducer is bound by the activator protein, it cause changes in conformation, that make it to bind to the DNA and activate transcription process.

In positive repressible operons, the activator proteins are generally bound to the relevant DNA segment. However, when an inhibitor is bound by the activator, it is prevented from binding the DNA and this stops activation and transcription of the system.

Description of the three types of prokaryotic genetic recombination (conjugation, transformation, and transduction)

Conjugation

            Conjugation is the process which is bacterial equivalent of sex which involves physical contact between two bacterial cells mainly via pilus structure on the surface of the bacterial cell. Donor cells must contain a small DNA segment called the F-plasmid, which is absent in the recipient bacterial cell. The donor cell provides a single strand of DNA from the F-plasmid and transfers it to the recipient. DNA polymerase enzyme starts synthesis of a complementary strand to two-stranded DNA structure. In some cases, the donor also contributes chromosomal DNA beyond that of the F-plasmid. The recipient combines the donor DNA with its own genome.

Transformation

            Some specific species of bacteria can ingest DNA segments, like plasmids, incorporate the DNA segment into their own chromosomes. The bacterium must first enter a special state, called competence, which allows transformation to occur. To achieve competence, the bacteria must activate a number of genes, which can express the required amount of proteins. Bacteria usually transform DNA of the same species. Scientists use transformation to introduce foreign DNA into prokaryotic cells by incorporating the DNA in the growth medium.

Transduction: it is the transfer of DNA from one bacterium to another through the action of viruses. Virus infects a bacterium and injects its genetic material into its host and using the bacterium’s machinery for synthesizing DNA, RNA and proteins. Virus causes the host to replicate many copies of the virus genome. The virus then causes the bacterial cell to rupture and release new virus particles, which continues the repetition of the cycle.

Explaination of how recombination might interfere with the metabolic functions of operons, such as the lac operon or trp operon of E. coli.

Escherichia coli is one of the most preferred organisms for the production of recombinant proteins. The lac operon of E. coli contains genes responsible for lactose metabolism. It has expressed only when lactose is present and glucose is absent.

The Lac operon is also under positive gene regulation. While the removal of the repressor protein in the presence of lactose is required for synthesis of the lacZ, lacY, and lacA genes, the gene expression will remain low. The level of gene expression is controlled by the amount of the preferred energy source, glucose, in the cell. This control is regulated by an allosteric regulatory protein, catabolite activator protein (CAP). When glucose levels in the cell are low, the organic molecule cyclic AMP is in high concentration. Cyclic AMP activates CAP by binding to the allosteric sites, causing CAP to attach to the Lac operon promoter. Unlike the repressor proteins, binding of CAP to the Lac operon stimulates gene expression. When the cell glucose levels increase, the cyclic AMP levels in the cell decrease, and the activator protein will disassociate from the promoter. Transcription will return to low levels, or will turn off if the repressor protein reattaches.

The Trp operon is responsible for synthesis of the amino acid trytophan when it is not available in the environment. The Trp operon is made up of a promoter with an operator, and five genes that encode enzymes for tryptophan synthesis. The Trp operon is regulated by the regulatory gene trpR, a gene that is located at a distance from the Trp operon.

The Trp operon is an example of a repressible operon; it is on unless turned off by a repressor protein. The repressor protein is synthesized by trpR. While the repressor protein is always present in the cell, it is synthesized in an inactive form. When a corepressor is present, in this case tryptophan, it binds to the repressor protein in an allosteric site. This changes the conformation of the protein such that it can bind to the operator and block transcription by preventing the binding of RNA polymerase to the promoter. In this way the cell saves energy by not producing tryptophan when it is already present.