Control of Gene Expression in Prokaryotes

Control of Gene Expression in Prokaryotes

Learning Objectives

  • Understand why control of gene expression is important.

  • Understand how prokaryotes (bacteria) control gene expression using repressors, activators, and inducers.

  • Learn how the lac operon works as an example of controlling gene expression in a biochemical pathway.

Prokaryotic Cells

  • Prokaryotic cells (bacteria) lack organelles, making gene expression control simpler than in eukaryotes.

  • Bacterial DNA resides in the nucleoid without a nucleus.

  • DNA contains genes for replication, RNAs, and proteins needed for new bacteria.

Central Dogma and Control of Gene Expression

  • Control of gene expression can occur at any level of the central dogma (DNA to RNA to protein).

  • Organisms must control gene expression for proteins in biochemical pathways, structural and functional RNAs.

  • Gene expression must be controlled so that it occurs in the right place at the right time.

Importance of Control of Gene Expression

  • Bacteria:

    • Respond to environmental changes e.g. temperature by turning on expression of specific genes.

    • Availability of nutrients e.g. switch on new digestive enzymes to metabolise new nutrients.

    • Production of virulence factors e.g. for pathogenesis and competition, requiring new gene sets.

    • Change gene expression during different life cycle stages, including division.

    • Have constitutively active genes that are always expressed.

  • Eukaryotes:

    • Regulate gene expression during development as the organisms grows and changes and throughout life.

    • Express different genes in different tissues (e.g., brain, skin, liver).

Examples of why gene expression is so important

  • Biofilms: Bacteria produce biofilms when threatened, requiring a switch to make a mucopolysaccharide coat.

  • Bacterial Growth Curve: Different growth stages require different factors.

  • Invasion of Eukaryotic Cells: Bacteria express enzymes that help them to degrade the plasma membrane.

  • Immune Response: Bacteria express genes to evade immune detection.

Stages of Gene Expression Control

  • DNA Level:

    • More gene copies = more genes expressed = more protein (e.g., trypanosomes with over 200 tubulin gene copies).

  • DNA to RNA (Transcription):

    • Heavily regulated in bacteria and eukaryotes.

    • production of RNA via transcription

    • Controls the amount of RNA produced, influencing the amount of protein produced

  • RNA to Protein (Translation):

    • Post-transcriptional regulation of a protein

    • Differential RNA stability and export from the nucleus.

    • mRNA degradation if protein is unneeded.

  • Protein Level:

    • Proteins can be directed for degradation if misfolded or unneeded.

Control of gene expression at a transcriptional level in Prokaryotes

  • Simpler than in eukaryotes; regulation occurs at the DNA to RNA level. (production of RNA)

  • Involves specific DNA sequences binding to DNA binding proteins.

  • very important protein-protein interactions involved

  • RNA polymerase interacts with the promoter region of a gene to initiate transcription.

  • Repressors and activators are essential for this process

Negative regulation by a repressor

  • RNA polymerase binds to the promoter, transcription occurs and gene is switched on

  • Repressor bound, preventing RNA polymerase binding therefore transcription cannot occur and gene is switched off

  • the repressor blocks expression

Positive regulation by an activator

  • Activator is required for RNA polymerase to bind and initiate transcription.

  • Without the activator, RNA polymerase cannot bind and gene is switched off

  • the activator enables expression

Bacterial Gene Elements

  • Promoter: Where RNA polymerase binds.

  • Activator: Facilitates RNA polymerase binding.

  • Operator Sequence: Where repressors bind, blocking RNA polymerase.

Inducers of Transcription

  • Inducers bind to repressors, changing their conformation and preventing it from binding to the operator.

  • RNA polymerase can then bind, switching the gene on.

Bacterial Metabolism Examples

  • Bacteria switch off old biochemical pathways and switch on new ones when entering a new environment with a new nutrient.

Lactose Metabolism in E. Coli

  • E. coli prefers glucose as an energy source but can use lactose in the absence of glucose.

  • require beta galactosidase to hydrolyse lactose into galactose and glucose

    Experiment/graph

  • No beta galactosidase is produced until lactose is added.

  • Beta galactosidase is rapidly produced after lactose is added until lactose is removed.

Lactose metabolism by beta-galactosidase - Hydrolysis Reaction

  • \text{Lactose} + H_2O \rightarrow \text{Galactose} + \text{Glucose}

  • Water molecule is added in the cleavage

  • reaction is catalysed by beta galactosidase.

Three enzymes are expressed

  • When beta galactosidase increases, so do two other enzymes: galactoside permease, and thiogalactoside transacetylase.

  • Galactoside permease: Transports lactose across the cell membrane.

  • Thiogalactoside transacetylase: Detoxifies non-metabolizable pyranoside compounds by acetylation.

  • these three enzymes work together in response to specific change in environment and are expressed co-ordinately

  • genes encoding these enzymes are clustered together on the genome in an operon

  • operon = region where the genes are all coordinately expressed

Operons

  • Operon: A region where genes are coordinately expressed.

  • Most prokaryotic genes are organised into operons.

  • Operons allow coordinated regulation because the genes have related functions or are part of the same biochemical pathway.

  • Transcription produces a polycistronic mRNA, which contains multiple coding sequences for individual proteins.

  • (5 prime end)promoter — operator — gene 1 — gene 2 — gene 3 ect = this gene is currently switched OFF because there is no RNA polymerase bound to the promoter region

  • in this example with 3 genes there are 3 open reading frames (ORFs)

Lac Operon

  • LacI gene: Codes for the lacI repressor protein, located upstream of the operon.

  • The promoter and operator control expression of the lac operon genes: lacZ, lacY, lacA.

  • lacZ: Beta galactosidase gene.

Lac Operon in Absence of Lactose

  • The LacI gene is constitutively expressed, producing the LacI repressor.

  • The repressor binds to the operator region of the lac operon, preventing transcription of the operon

  • The lac operon is switched off.

Lac Operon in Presence of Lactose

  • Lactose binds to the lacI repressor, changing its conformation, and preventing it from binding to the operator.

  • RNA polymerase can bind to the promoter and produce a polycistronic transcript.

  • The three open reading frames are transcribed to produce beta galactosidase, permease, and transacetylase enzymes.

  • This conformational change is an example of allosteric regulation

  • as a result of the lactose binding to LacI repressor = lac operon is fully switched on.

  • This system is an example where the presence of a nutrient removes the repression and switches on the expression of the genes required to metabolize that nutrient.

How was this mechanism worked out?

  • Jacob, Monneau, and Lvoff used classical genetic techniques:

    consequences of mutations

    genetic complementation of different mutants

  • They isolated constitutive mutants that transcribe the lac operon in the absence of lactose.

E. Coli Growth and Diauxy

  • In a nutrient medium containing both glucose and lactose, E. coli uses glucose first.

  • After glucose is depleted, there is a lag phase while the lac operon is activated, before lactose can be used.

  • This sequential use of two substrates is called diauxy.

Inducer Exclusion

  • The lac operon is slightly ‘leaky’, (so not completely repressed by the LacI inhibitor) - there is normally some lactose permease present, allowing a small amount of lactose to enter the cell → therefore some low level transcription of lac operon

  • Glucose is transported into the cell via a phosphotransferase system, which inactivates lactose permease.

  • so lactose is excluded from the cell in the presence of glucose.

  • therfore with no inducer(lactose), there is no relief of repression of the lac operon → therefore lac operon switched OFF in the presence of glucose

  • Once glucose is lowered, lactose permease is no longer inhibited and can transport lactose into the cell.

  • Lactose binds with the lacI repressor to prevent its binding to the operator of the lac operon, which then activates the lac operon fully.

Carbon Catabolite Repression (CCR)

  • Operates to suppress other nutrient utilisation operons in the presence of glucose.

  • Glucose lowers the concentration of cyclic AMP (cAMP).

  • At low glucose levels, cAMP is high.

  • cAMP acts on the catabolite activator protein (CAP), a transcriptional regulatory protein.

  • The cAMP-CAP complex binds to a region upstream of the transcriptional start site.

  • Stimulates binding of RNA polymerase

  • complex activates the expression of many catabolic enzymes (NOT the lac operon)

  • It can increase transcription by as much as 50-fold.

High and Low Glucose Levels

  • At high glucose, there is no cAMP, and these genes are repressed → low levels of trancription

  • At low glucose levels, cAMP is high, forming a cAMP-CAP complex → stimulates high levels of transcription.

Tryptophan (TRP) Operon

  • The operon codes for five enzymes that convert chorismate into tryptophan.

  • The operon is expressed only when tryptophan levels are low.

  • The tryptophan repressor switches off expression when tryptophan levels are high.

  • The tryptophan repressor cannot bind DNA until it is bound to tryptophan that is produced by the trp operon

  • Binding of tryptophan leads to a conformational change in the repressor.

  • This causes the genes in the operon to be switched off.

    → product repression

  • complete opposite of the LacI repressor which cannot bind to the operator when bound to loctose (i.e. lactose induces expression)

    → substrate induction

Regulation of Trp Operon Expression at the Promoter of the Tryptophan Operon

  • When there is a lot of tryptophan present, it binds the repressor and makes it active.

  • There's a conformational change that then allows it to bind DNA, and this then prevents the RNA polymerase binding.

  • This completely switches off transcription of the TRP operon.

Summary

  • Control of gene expression is crucial in all species.

  • Prokaryotes regulate gene expression via repressors, activators, and inducers.

  • Prokaryotic operons are clusters of genes with related function.

  • The lac operon expresses three enzymes in the absence of glucose and presence of lactose.

  • The lac repressor (lacI protein) binds to the operator, preventing RNA polymerase binding and turning genes off.

  • When LacI repressor binds to lactose, it cannot bind to the operator, so RNA polymerase can bind and genes are turned on.

  • Mutation of either the operator region or the LacI gene itself can lead to constitutive expression of the lac operon.

  • A process called nutrient exclusion causes the initial glucose utilisation and then lactose, in diauxy.

  • Carbon catabolite repression (CCR) causes initial glucose utilisation for other nutrients and operons.

  • The lac operon is induced by substrate binding to its repressor, whereas the TRP operon is inhibited by product binding to its repressor.