Prokaryote Gene Expression

• Regulation of prokaryote gene expression is vital for understanding bacterial genetics and molecular biology.
• Key information to learn include the organization and function of the E.coli lactose and arabinose operons, names of their genes, and their protein products.
• Knowledge of transcription and translation is required before proceeding.

Transcriptional Control

• Some bacterial genes are expressed constitutively to perform routine tasks, known as 'housekeeping genes'.
• Other bacterial genes require regulated transcription in response to environmental changes such as nutrients and temperature.
• Transcriptional regulation is the most common method of gene expression regulation in bacteria to control mRNA synthesis.
• Negative control involves repressor proteins preventing transcription while positive control uses activator proteins to initiate it.

E. coli Lactose Operon

• Operons are bacterial gene clusters under coordinated transcriptional regulation.
• The lactose (lac) operon in E. coli produces three polypeptides for lactose usage, which bacteria can uptake from the environment as a carbon source.
• The lac operon consists of a regulatory region and three protein-coding genes (lacZ, lacY, lacA).
• The regulatory region contains a promoter for RNA polymerase binding and an operator (lacO) for the lac repressor protein.
• In the absence of lactose, the lac repressor protein binds to the operator, blocking transcription.
• Lactose is an alternative carbon source, inducible by the lac operon with an inducible promoter, and the preferred energy source is glucose.
• Allolactose, derived from lactose, acts as the inducer compound.
• The protein-coding genes are transcribed as a single, polycistronic mRNA, then translated into three distinct polypeptides.
• lacZ encodes β-galactosidase which breaks down lactose.
• lacY encodes lactose permease which uptakes lactose.
• lacA encodes transacetylase.
• The lacl gene is next to, but not part of the lac operon.
• lacl encodes the lac repressor protein and is constitutively expressed from its promoter with no regulation.
• The lac repressor protein is a homotetramer with a DNA-binding domain that binds to the operator.
• Allolactose then binds to the lac repressor protein, causing it to dissociate from the operator.

Lactose Operon Regulation

• The lac operon has very low levels of expression without lactose meaning there is some β-galactosidase present.
• When lactose is present, β-galactosidase produces allolactose which binds to the lac repressor, preventing it from binding to the operator, therefore transcription is induced.
• The allolactose-lac repressor complex prevents the repressor from binding the operator.
• The allolactose-lac repressor complex alone isn't sufficient to generate enough copies of the lac operon mRNA for lactose metabolism.

CAP-cAMP Regulation

• Glycolysis can occur with glucose and no cAMP is produced by the cell.
• In the absence of glucose, cAMP is produced.
• CAP-cAMP then binds to the CAP-cAMP binding site in the lac promoter region and stimulates a higher level of transcription.

Lac Operon Mutations

• Mutations to the lac operon can affect its function which can be studied in E. coli.
• A lacl mutation affects the repressor proteins binding to the operator, producing constitutive synthesis of the lac operon.
• Mutant lac repressor protein cannot bind to the operator and the Lac operon is therefore constitutively expressed, so galactosidase is always present in E. coli.
• An operator site mutation prevents repressor protein binding, therefore leading to constitutive synthesis of the lac operon.
• The Lac operon is constitutively expressed, so -galactosidase is constitutively present. A super lac repressor also exists but results in the Lac operan never being expressed.
• Super lac repressor binds to the operator site, but cannot bind allolactose and the repressor always bounds to the operator (lacO).
• "Super” lac repressor is a mutant repressor protein that is unable to bind allactose.

Summary of the Lac Operon Structure

• CAP-cAMP binds here and promotes transcription.
• RNA polymerase binds here, essential for transcription.
• The lac repressor binds here, blocking transcription.

Plasmids

• Coding sequences for human growth hormone can be placed next to the lac operon promoter region.
• The coding sequence includes a (CAP-cAMP binding site, RNA polymerase binding site, lac repressor binding site).
• Expression of growth hormone can be induced using these plasmids.

E. coli Arabinose Operon

• The arabinose operon is expressed in the presence of arabinose in E. coli.
• Expression of the arabinose operon is induced by presence of arabinose in the gut.
• Arabinose is a pentose sugar released from plant cell walls after eating plant material, and is used as a nutrient source.
• The arabinose operon is induced with the presence of arabinose if glucose is absent.
• Expression of the arabinose operon is induced and the arabinose operon produces three enzymes (araB, araA and araD) that break down arabinose. AraC carries out transcriptional regulation.
• In the arabinose (ara) operon, a single regulatory protein (araC) carries out both positive and negative transcriptional regulation.
• Two araC protein monomers bind to aral and araO₂, which then induce a DNA loop formation.
• Because of the loop, RNA polymerase (and cAMP-CAP) are prevented from accessing the Para promoter which prevents epxression of the gene.
• Arabinose breaks the connection between araC proteins at aral and araO₂ and therefore opens the loop.
• A second araC-arabinose complex can bind to the promoter also increases its affinity for RNA polymerase,
• CAP-cAMP is produced when glucose is absent.

The Arabinose Promoter Plasmids

• The ara operon is not expressed in the absence of arabinose, but has a large increase in expression with arabinose.
• arac promoter and araC gene are present in plasmids.
• There are no arab, araA or araD genes in plasmids such as pGLO.
• There is no arabinose in the growth medium when arabinose is absent, meaning that the GFP is not expressed.
• E. coli containing pGLO results in a medium when arabinose is present in the growth medium, the GFP gene is expressed.
• araBAD and Para are both used to name the promoter in the arabinose operon.