Advanced Microbiology Lecture 13: Gene Expression

Advanced Microbiology: Lecture 13 - Gene Expression: Genes & Operons

• Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product.
• Negative regulation: genes in an operon are expressed unless turned off by a repressor protein.
• Positive regulation: genes in an operon are not expressed unless turned on by an activator protein.
• Regulation can be inducible (increased gene expression), repressible (decreased gene expression), or both.
• Inducers: increase gene expression, activate an activator or inactivate a repressor.
• Co-repressors: decrease gene expression, activate a repressor or inactivate an activator - Apo vs. holo protein: the differences between the inactive (apo) and active (holo) forms of a protein, typically related to ligand binding.

Negative Regulation

• Negative inducible system (lac operon - lactose):
  • In the absence of lactose, the repressor protein (lacI) is bound to the operator, preventing transcription.
  • In the presence of lactose, allolactose (inducer) binds to the repressor, changing its shape; the repressor detaches from the operator, allowing RNA polymerase to bind and transcribe the lac genes.
• Negative repressible system (trp operon - tryptophan):
  • In the absence of tryptophan, the repressor protein (trpR) is inactive and cannot bind to the operator. Transcription occurs.
  • In the presence of tryptophan, tryptophan (co-repressor) binds to the repressor, activating it; the activated repressor binds to the operator, preventing transcription.

Positive Regulation

• Positive inducible system (ara operon - arabinose):
  • In the absence of arabinose, the activator protein (araC) is inactive and cannot bind to the promoter. Transcription is low.
  • In the presence of arabinose, arabinose binds to the activator, activating it; the activated protein binds to the promoter, increasing transcription.

Regulation of Gene Expression

• Transcriptional regulation: controls the rate at which genes are transcribed into mRNA.
• Posttranscriptional regulation: controls the processing of mRNA and its stability.
• Translational regulation: controls the rate at which mRNA is translated into protein.
• Posttranslational regulation: controls the activity of proteins after they've been translated.

Riboswitches

• RNA elements in mRNA leaders that sense metabolites.
• RNA folds into a 3D structure that allows a ligand to bind.
• Ligand binding leads to a conformational change, altering terminator/anti-terminator alterations or changing access to the ribosomal binding site.

T Box Mechanism

• First discovered for gene expression of aminoacyl-tRNA synthetases in B. subtilis.
• tRNA without an attached amino acid increases expression (anti-terminator forms).
• tRNA with an attached amino acid decreases expression (terminator forms).

Metabolite-binding Riboswitches

• Small molecules can bind directly to leader RNA.
• Typically involved in the same metabolic pathway.
• Found for many types of molecules (atoms): amino acids (lysine), vitamins (B12), nucleic acid bases (guanine), cofactors (SAM), and metal ions (Mg).
• Can regulate transcription through attenuation or blocking the transcription initiation region.

Regulation of mRNA Degradation

• Half-life: time taken for half of the template to be reduced.
• Increased half-life leads to increased protein production.
• RNase E targets its own RNA: Low RNase E increases mRNA message, and High RNase E degrades mRNA.

Regulation by sRNAs (small RNAs)

• Non-protein coding RNA involved in post-transcriptional regulation.
• Individual bacteria may encode hundreds of sRNAs.
• Can be encoded in intergenic regions, form secondary stem loop structures.
• Implicated in the regulation of physiology, metabolism, and virulence.

Mechanisms of sRNA-Interactions

• sRNAs can inhibit translation by blocking the ribosome binding site, destabilizing target RNA by recruiting RNase, or enhance translation by exposing an occluded ribosome binding site or stabilizing target RNA.

Regulation of Translation

• Initiation of translation is sensitive to mRNA structure.
• Proteins and sRNAs can bind mRNA and block translation initiation regions.
• Similar mechanisms can lead to activation of translation, where bound proteins or sRNAs can disrupt secondary structure, exposing the ribosome binding site.

Posttranslational Regulation

• Not all polypeptides are active proteins; some require modification (modification examples: phosphorylation, acetylation, methylation).
• Phosphorylation is a common signal in transduction systems.
• Two-component systems are common examples.

Posttranslational Regulation: Feedback Inhibition

• End products of a pathway bind to the first enzyme in the pathway to inhibit its activity.
• Common in biosynthetic pathways.
• Highly sensitive and fast for modulating the amount of end product.
• Trp operon is a good example.

Important Questions

• Why would an organism want so much regulation?
• Why do these mechanisms occur at different levels?
• Why make mRNA if not translating it?
• Why use sRNAs instead of repressors/activators?

Global Regulation: Regulons & Stimulons

• Bacteria must adapt to various conditions (nutrient availability, moisture/desiccation, temperature).
• Global regulatory mechanisms allow simultaneous regulation of multiple operons in response to environmental changes.
• Regulon: a set of several operons controlled by a single protein.
• Stimulon: a collection of regulons that respond to the same environmental condition. Examples: TrpR, LexA, and AdcR systems).

Catabolite-sensitive Operons

• Catabolites: smaller molecules resulting from the breakdown of larger molecules.
• Energy is expended for the production of enzymes in catabolism.
• Catabolite repression: cells preferentially utilize the most efficient energy source. Operons involved with using alternative carbon sources are frequently repressed when glucose is present (glucose effect).

Stress Responses in Bacteria

• Bacterial global regulons quickly respond to stress conditions (e.g., osmolarity, pH, temperature, metal availability, nutrient access).
• Responses are flexible and can address multiple stressors at once.
• E. coli heat shock response: unique heat shock proteins (Hsps) increase after temperature rise, supporting normal growth, protein folding, degradation of damaged proteins.
• Dnak is a cellular thermometer, generally binding to and helping new proteins fold or assisting denatured proteins to refold.

Signal Transduction Systems (STS) in Bacteria

• Some gene regulation requires bacteria to sense external environment.
• Two-component STS:
  • Histidine kinase: membrane-bound protein that senses environment and auto-phosphorylates.
  • Response regulator: intracellular protein that receives phosphate from histidine kinase and performs a regulatory function.

Explore the intricate mechanisms of gene expression in this quiz from Advanced Microbiology, Lecture 13. Learn about operons, negative and positive regulation, and the roles of inducers and co-repressors in gene activity. Test your understanding of the lac operon and other regulatory systems.