REGULATING AND SIGNALING

Regulation: Signaling -> Metabolic Coordination

• How do cells know that they are low on phosphorus?
• Protein Activity: Ways to Control protein activity include:
  • Allosteric Change:
    • Can affect the velocity of a protein.
    • Involves the binding of a ligand to a site on a protein that is not the active site.
  • Reversible Modification (Covalent Modification):
    • Binding a phosphate onto a protein can control its activity.

Controlling Amount of Protein

• Promoter Recognition:
  • RNA polymerase specificity for the promoter is conferred by Sigma factors.
    • Sigma factors:
      • Changing the concentration of Sigma factors can change protein production.
• Transcriptional Regulators:
  • Binding of regulatory protein to DNA.
    • Active Repressor: decreases expression.
      • Repressor
    • Active Activator: increases expression.
      • Activator
      • Enhancers
        • Enhancers are not near the promoter but can influence transcription by binding elsewhere.

Lac Operon

• Transcriptional Regulators: Enhancers, Activators, Repressors.
• Why study the lac operon:
  • Lactose metabolism in E. coli demonstrates how regulation happens using activators and repressors in the cell.
    • Structural genes:
      • lacZ, lacY, lacA
    • Beta-galactosidase (encoded by lacZ).
    • Operator: where the repressor can bind.
      • Repressor (LacI):
        • Made regularly and binds the operator.
        • Serves as a barrier preventing RNA polymerase from transcribing genes.
        • When lactose is present, LacI binds to allolactose, becoming inactive.
        • Inactive LacI will not bind to the operator, allowing RNA polymerase to transcribe.

Lac Operon Behavior Relative to Lactose and Glucose

• Lactose present, no glucose: lots of beta-galactosidase.
• Lactose present, lots of glucose: very little beta-galactosidase.
• Allolactose inhibits LacI (LacI) binding, leading to increased beta-galactosidase production.

Glucose and Catabolite Repression

• Glucose is preferred in E. coli.
• When glucose is present, the metabolism of other sugars is repressed; this is catabolite repression.
  • Catabolite Repression: One substrate represses the catabolism of another substrate.

CAP (Catabolite Activator Protein)

• Binds to the promoter.
  • CAP = Activator
• Increases the efficiency of RNA polymerase binding to the promoter.
  • If the efficiency of RNA polymerase binding is increased, more expression occurs.
• CAP causes the DNA helix to bend slightly, enabling CAP to bind to RNA polymerase.
• CAP binds to cAMP (cyclic AMP), a small molecule regulator.

cAMP

• Signaling molecule in bacteria used to regulate gene expression in response to the environment (glucose levels).
• cAMP is formed from ATP by adenylate cyclase.
  • ATP → cAMP.
  • Adenylate cyclase is inhibited by glucose.
  • When glucose is present, less cAMP is formed because the enzyme is inhibited.
• Glucose controls cAMP levels.
• cAMP levels control the activity of CAP.

Allosteric Regulation

• Positive regulator: CAP activated by cAMP.
• Negative regulator: LacI inhibited by allolactose.

sRNA (Small RNA) Signaling

• Can affect transcription and translation.
  • Can work both transcriptionally and translationally.
  • Can also affect mRNA stability.
    • If mRNA is stable, there are many opportunities to make lots of protein.
    • If mRNA very unstable, decreases opportunities for making protein.
• Mechanism:
  • Bind DNA near promoter affecting RNA polymerase recognition of the promoter, blocking RNA polymerase from binding, or blocking regulators.
  • Terminate transcription by inducing a termination loop.
  • sRNA can bind at the 5' end of mRNA preventing translation.

DNA Topology

• Accessibility to transcription apparatus.
  • Making DNA accessible to RNA polymerase to get transcription.
• Transcription only happens at the interface of the nucleoid and cytoplasm.
  • DNA must be accessible to RNA polymerase.
  • If DNA is not accessible, transcription decreases.

Translational Repression / Activation

• Regulatory proteins bind to mRNA influencing translation.

Attenuation

• The structure of mRNA regulates transcription.
• Mechanism for transcription termination, identified in the trp operon.
  • Attenuator has parts: leader sequence and attenuator.
  • Leader sequence has two Trp codons.
    • The speed of translation and abundance of Trp tRNA control the speed of translation.
  • Attenuator exists in two configurations based on the speed of translation.
• In bacteria, attenuation controls transcription.

Attenuation Details

• If translation is fast there is a termination loop.
• If translation is slow (not enough tryptophan), the ribosome pauses, leading to no terminator hairpin.

mRNA Stability

• More stable mRNA leads to more protein synthesis because it can be used as a template to make more protein.
  • Small RNAs (sRNA) can decrease mRNA stability by degrading it.

Protein Proteolysis

• Commonly applied to regulatory proteins, not metabolic enzymes.