Bacterial Gene Expression & Regulation

• Bacteria adapt to their environment by regulating gene expression.
• This regulation allows them to synthesize products needed for DNA replication, recombination, repair, and cell division.

Inducible vs. Constitutive Enzymes

• Inducible enzymes are produced only when specific substrates are present in the environment.
• Constitutive enzymes are continuously produced, regardless of the environment's chemical makeup.

Repressible Systems

• A repressible system involves the presence of a specific molecule that inhibits gene expression.
• These systems often involve molecules that are end products of anabolic biosynthetic pathways.
• An abundance of the end product in the environment represses gene expression, conserving energy.
• Tryptophan production in bacteria is an example of a repressible system.

Positive vs. Negative Control

• Regulation of inducible or repressible systems can be under positive or negative control.
• Negative control: Genetic expression occurs unless shut off by a regulator molecule.
• Positive control: Transcription occurs only when a regulator molecule directly stimulates RNA production.
• Systems can use either or both types of control to induce or repress gene expression.

Allosteric Activation and Repression

• Enzymes can reversibly interact with another molecule, causing a conformational change.
• This change affects the enzyme's shape and chemical activity.

The Operon Model: Negative Control

• An operon consists of a group of genes expressed and regulated as a single unit.
• Transcription occurs only when a repressor fails to bind to the operator region.
• The repressor is allosteric.

Operons

• Transcription is under the control of a single regulatory region in an operon.
• Genes coding for enzymes with similar functions are organized in clusters with their regulatory sequences (operons).
• Regulatory regions are located upstream of the gene cluster.
• Regulatory region on the same strand is cis-acting.
• Trans-acting elements binding at a cis-acting site regulate the cluster negatively or positively.

Structural Genes

• Structural genes code for the primary structure of enzymes.
• The lac (lactose) operon has three structural genes: lacZ, lacY, and lacA.

Structural Genes - lacZ

• The lacZ gene encodes β-galactosidase.
• β-galactosidase converts the disaccharide lactose into the monosaccharides glucose and galactose.
• This conversion is necessary for lactose to serve as the primary energy source in glycolysis.

Structural Genes - lacY and lacA

• The lacY gene encodes permease.
• Permease facilitates the entry of lactose into the bacterial cell.
• The lacA gene encodes transacetylase.
• Transacetylase may be involved in removing toxic by-products of lactose digestion from the cell.

Lac Operon Structural Genes

• The lac operon structural genes (lacZ, lacY, and lacA) are all transcribed as a single unit.
• This results in polycistronic mRNA.
• A cistron is the part of the nucleotide sequence coding for a single gene.
• The single mRNA is simultaneously translated into three gene products.
• Glucose is the regular source of energy, while lactose is an alternative source.
• Using lactose requires energy consumption and protein synthesis.

Lac Operon Components

• The I gene produces the repressor protein (trans-acting).
• The operator (O) region is the repressor binding site.
• The promoter (P) regions are the RNA polymerase binding sites.
• There is also a CAP binding site.

Lac Operon - Negative Control

• The lac operon is subject to negative control.
• Transcription occurs only when the repressor fails to bind to the operator region.
• The repressor normally binds to the DNA sequence in the operator region.
• This inhibits RNA polymerase and represses transcription of structural genes.

Lac Operon - Negative Control (No Lactose)

• When no lactose is present, enzymes are not needed.
• The expression of genes encoding the enzymes is repressed.

Lac Operon - Negative Control (Lactose Present)

• When lactose is present, it indirectly induces activation of the genes by binding to the repressor.
• The repressor is allosterically inactivated.

Lac Operon - Negative Control (All Lactose Metabolized)

• When all lactose is metabolized, none is available to bind to the repressor.
• Transcription is repressed.

Lac Operon - Positive Control

• When the cell has ample amounts of glucose, β-galactosidase is not made.
• There is no need to break down lactose, as glucose is the preferred carbon source for E. coli.

CAP

• Catabolite-activating protein (CAP) exerts positive control over the lac operon.
• It binds to the CAP-binding site, facilitating RNA polymerase binding at the promoter and facilitating transcription.
• cAMP (cyclic adenosine monophosphate) is a co-activator for CAP.
• CAP must be bound to cAMP to bind to the promoter.
• Glucose inhibits the activity of adenylyl cyclase, which catalyzes the conversion of ATP to cAMP.
• In the absence of glucose, cAMP production rises in the cell.
• This prevents CAP from binding when glucose is present.
• Tryptophan (trp) operon is another important example of gene regulation in bacteria.

Tryptophan (trp) Operon

• Tryptophan is the corepressor for the inhibitor in the trp operon.
• Repressor is allosterically activated.

Tryptophan (trp) Operon

• Five contiguous genes on the E. coli chromosome encode enzymes for tryptophan synthesis.
• The model of gene regulation is analogous to the lac system.
• When tryptophan (corepressor) is present:
  • The repressor and tryptophan complex attain a new conformation.
  • This complex binds to the operator, repressing transcription.
  • Enzymes are not made.

Regulation by RNA

• Three types of regulation involve RNA:
  • Attenuation
  • Riboswitches
  • Small noncoding RNAs (sRNAs)
• These types of regulation help fine-tune prokaryotic gene regulation.
• They are used in addition to the regulation of transcription initiation.

Transcription Attenuation

• When the trp operon is repressed, initiation of transcription still occurs at low levels.
• Transcription is weakened or impaired.
• Transcription is terminated.
• When tryptophan is absent or present, transcription is initiated but not terminated.

Transcription Attenuation

• Transcription is coupled with translation.
• The leader region located between the operator and structural genes is transcribed into leader RNA.
• Leader mRNA contains an attenuator consisting of four separate regions.
• These regions can fold and form three different stem-loop structures.

Transcription Attenuation

• Translation begins as the RNA polymerase transcribes the attenuator.
• The position of the ribosome on the leader RNA plays a role in regulating transcription.
• After transcribing regions 1 and 2, the RNA folds and forms a stem-loop between these two regions, causing RNA polymerase to pause.
• This allows the ribosome to catch up with translation.

Transcription Attenuation and Tryptophan Levels

• There are two tandem tryptophan codons at the beginning of the leader mRNA.
• In the absence of tryptophan, the ribosome stalls (covering region 1), allowing regions 2 and 3 to form the antitermination loop.
• This allows RNA polymerase to continue transcription of the tryptophan operon.
• In the presence of tryptophan, the ribosome proceeds with translation and reaches the stop codon at the end of the leader mRNA, covering region 2.
• This allows regions 3 and 4 to form the termination hairpin loop and terminate transcription of the tryptophan operon.

Transcription Attenuation - Summary (Absence of Tryptophan)

• Transcription from the tryptophan operon continues.
• This makes polycistronic mRNA that will be translated into five enzymes necessary for tryptophan synthesis.

Transcription Attenuation - Summary (Presence of Tryptophan)

• A short leader peptide will be synthesized.
• Transcription will be terminated.