Gene Regulation in Prokaryotes

• All of our cells have the same DNA

  • What allows them to have unique functions and properties is regulating (controlling) which genes get expressed
• Gene regulation can occur at just about any step in the Central Dogma
• Transcriptional level

  • Epigenetic regulation* (make DNA sequences more/less accessible for transcription)
  • Transcription factor binding to non-coding, “regulatory” sequences on DNA
• Post-transcriptional level*

  • How mRNA is processed (e.g., splicing)
  • mRNA stability (remember 5’ cap and 3’ polyA tail?)
• Translational level

  • Where, whether, and how much mRNA is translated
• Post-translational level

  • How proteins are processed (which affects their activity/function)
• Prokaryotes can’t regulate gene expression as intricately as eukaryote
• Prokaryotic cells regulate

  • whether and how much a gene is transcribed.
  • whether and how much an mRNA is translated
• Prokaryotic cells regulate

  • mRNA stability (how long it lasts)
  • how proteins are processed (which affects their activity/ function)
• A promoter is a sequence of DNA that is not transcribed into RNA

  • Both eukaryotic and prokaryotic genes have promoters
• Transcriptional Regulation

  • Genes can be positively regulated
  • Transcription is stimulated (“turned on”) by the binding of a protein to the DNA in response to an environmental signal
• Transcriptional Regulation

  • Genes can be negatively regulated
  • Transcription is repressed (“turned off”) by the binding of a protein to the DNA in response to an environmental signal.
• Prokaryotic gene transcription is regulated by proteins that bind to DNA sequences
• Positive regulation of prokaryotic transcription

  • The default of some genes is “don’t transcribe me.” For this kind of gene, RNA polymerase can’t bind well to its promoter and thus can’t start transcription on its own
• Positive regulation of prokaryotic transcription

  • When a situation arises in which the cell wants to express (“activate”) one of these genes, an activator protein specific for the gene will bind to a site near the gene’s promoter to help RNA polymerase initiate transcription
• Negative regulation of prokaryotic transcription

  • The default for some other genes is always “on.” For this kind of gene, RNA polymerase binds easily to the gene’s promoter and can initiate transcription just fine on its own
• Negative regulation of prokaryotic transcription

  • When the cell wants to inhibit the expression of this gene, a repressor protein specific to the gene binds to a DNA sequence near the beginning of the gene (usually in or after the promoter)
• Both activator proteins and repressor proteins can be regulated by molecules that bind to them (allosteric regulation)
• • In some cases, a molecule can bind to the protein to allow the protein to bind to the DNA.
  • In other cases, a molecule can bind to the protein to prevent it from binding to the DNA
• Bacterial gene regulation: Responding to nutrients in the environment... like your gut

  • E. coli’s (and all cells’) “favorite meal” is glucose
  • Metabolizing glucose provides greatest net energy “pay off”
  • If glucose isn’t available, E. coli will utilize other energy/carbon sources
• If glucose isn’t available, E. coli will utilize other energy/carbon sources

  • If lactose is present, E. coli can import it into the cell and break it down into glucose and galactose
• To avoid wasting the energy and materials for making lactose metabolism proteins if they aren’t needed, E. coli regulates the expression of the genes encoding these proteins, which are located in the lac operon
• What’s an operon?

  • A set of multiple bacterial genes that are regulated together and are transcribed into a single mRNA (they share a promoter)
• What’s an operon?

  • The part of the mRNA corresponding with each gene is translated into the protein encoded by that gene
• What’s an operon?

  • Operons usually contain multiple genes that functionally related proteins (they all contribute to the same cellular process)
• Anatomy of the lac operon

  • The lac operon contains 3 genes that code for 3 proteins involved in lactose metabolism
• Anatomy of the lac operon

  • lacZ
  • codes for B-gal, the enzyme that breaks down lactose into glucose and galactose
• Anatomy of the lac operon

  • lacY
  • codes for lactose permease, a transmembrane protein that transports lactose into the cell
• Anatomy of the lac operon

  • lacA
  • codes for another protein you don’t have to worry about
• Anatomy of the lac operon

  • One promotor (lacP) controls the transcription lacZ, lacY, and lacA
• Anatomy of the lac operon

  • A repressor binding site called an “operator” (lacO), which binds a repressor encoded by lacl, located outside the operon
• E. Coli can regulate lac operon expression in response to the amounts of glucose and lactose in their environment
• When lactose isn’t present, cells don’t need lactosemetabolism proteins

  • During these times, lacl is constitutively (continually, constantly) expressed, producing repressor proteins that bind to lacO and repress transcription of lac operon
  • negative regulation
• When lactose is present, the repressor gets repressed,inducing expression of lac operon

  • Lactose will bind to the repressor protein, which changes its shape
  • This makes repressor unable to bind to lacO sequence.
• When lactose is present, the repressor gets repressed, inducing expression of lac operon

  • RNA polymerase can bind to promoter (lacP), lac operon is transcribed, lacY, lacZ and lacA get expressed, lactose gets metabolized
  • Thus, lactose is an inducer of gene expression
• Anatomy of the lac operon: CRP binding site cAMP

  • CRP is a protein that binds to a signaling protein called cAMP
  • Binding of cAMP to CRP causes shape change that allows CRP/cAMP complex to bind to DNA
• Anatomy of the lac operon: CRP binding site cAMP

  • CRP on its own can’t bind to DNA
  • CRP/cAMP complex binds to DNA, this increases transcription of lac operon
  • CRP is thus a positive regulator of the lac operon
• What happens when lactose is available, butnot glucose?

  • If lactose levels are high, repressor can’t bind to DNA, so transcription of lac operon increases
• What happens when lactose is available, butnot glucose?

  • since glucose is low, there’s high cAMP – this means lots of CRP/cAMP complexes to bind to DNA and increase transcription of lac operon
  • CRP and lactose thus work together to ramp up lac operon expression
• What happens when neither lactose or glucoseis present?

  • CRP/cAMP complex binds to DNA, which would increase transcription of lac operon.
  • However, since lactose levels are low (or absent) repressor will remain bound to lacO, so overall transcription of lac operon will be very low (whether or not glucose is present)