Unit 3.2: Prokaryotic Transcriptional Regulation:

Signals from the environment to E.coli for transition in metabolism from Glucose to Lactose Metabolism:

1) Changes in the availability of Glucose

2) The presence of lactose

As E. coli cells transition from glucose to lactose metabolism, what two protein levels increase?

• Beta-galactosidase and lactose permease

• These proteins are generally present at very low levels when glucose is the primary nutrient source.

• However, their expression increases when they are needed to transport and metabolize lactose.

• This means that the presence of lactose is what induces the expression of these 2 genes.

Lactose Permease:

• Transport protein embedded in the bacterial cell membrane. It enables lactose to enter the cell.

Beta-galactosidase:

• An enzyme located in the cytoplasm that breaks down lactose into glucose and galactose.

Operon Model:

• The basic model of bacterial gene regulation was developed by François Jacob and Jacques Monod in 1961

• A typical operon consists of:

1. Promoter: The DNA sequence where RNA polymerase binds to initiate transcription.

2. Operator: A regulatory DNA sequence that can act as an on-off switch for transcription.

3. Structural Genes: Genes that encode proteins involved in a common pathway or cellular response.

Operon:

• One advantage of the prokaryotic genome organization is that functionally related genes are often clustered together into structures called operons.

• in euks, each gene has their own promoter, but in proks, operons allow multiple genes to be controlled by one shared promoter.

• enables the gene expression of the entire cluster as a single unit.

• Whenever the operator in an operon isn’t bound by a repressor protein, the RNA polymerase can easily bind to the promoter and transcribe the genes in the operon.

Polycistronic mRNA:

• A single. long mRNA coding molecules that contains the coding sequences for multiple proteins.

• forms as a result of an operon being transcribed.

• During translation, separate polypeptides are produced because each coding region has its own translation initiation and termination signals.

lac Operon:

• A classic model of transcriptional regulation in prokaryotes

• In E. coli, it regulates the expression of proteins required for lactose metabolism, including beta-galactosidase and lactose permease.
  
  

Regulatory Components of lac Operon:

1. Contains a promoter: DNA sequence where RNA polymerase binds.

2. Contains lacO (operator): DNA sequence where repressor protein can bind

3. lacI gene: Encodes the LacI repressor protein, which inhibits transcription when bound to the operator.

Structural Genes of lac Operon:

1. lacY: Encodes lactose permease, which transports lactose into the bacterial cell.

2. lacZ: Encodes beta-galactosidase, which breaks lactose down into glucose and galactose.

Negative Transcriptional Regulation:

• A repressor protein binds to the operator region of an operon and inhibits transcription.

• The lac operon is negatively regulated because the LacI repressor, encoded by the lacI gene, can bind to the lac operator and prevent transcription of the genes required for lactose metabolism.

• The repressor is produced constantly, allowing it to be available whenever it is needed.

• When the repressor occupies the operator, transcription of lacZ and lacY is inhibited, so little beta-galactosidase or lactose permease is produced (the production is never completely stopped, just reduced)

Lac I Repressor Protein:

• tetrameric protein composed of four subunits.

• When it binds to operator sequences in the lac operon DNA, it can form a DNA loop that interferes with transcription.

• prevents effective transcription of the lac operon and reduces the production of proteins needed for lactose metabolism.

• When lactose is unavailable, the repressor remains capable of binding to the operator, keeping the operon switched off.

Negative Regulation using Allolactose:

• Used to relieve negative transcriptional regulation.

• When glucose is depleted and lactose is available, a lactose-derived molecule called allolactose acts as the natural inducer of the lac operon.

• Allolactose binds to the LacI repressor, causing a conformational change that reduces its ability to bind to the operator DNA.

• As a result, the repressor releases the operator, allowing RNA polymerase to bind to the promoter to transcribe necessary genes.

Positive regulation of the Lac Operon:

• Increases transcription when glucose levels are low.

• When glucose availability decreases, intracellular cyclic AMP (cAMP) levels increase.

•  The cAMP molecule helps activate a transcriptional regulator, enabling stronger expression of the lac operon.

• This positive regulation allows bacteria to increase the production of beta-galactosidase and lactose permease when lactose is available and glucose is scarce.

Impact of glucose levels on cAMP:

High Glucose = Adenylyl cyclase activity reduces = cAMP levels are lower.

Lower Glucose = Adenylyl cyclase activity increases = cAMP levels are higher.

Adenylyl Cyclase:

• the enzyme that catalyzes the production of cAMP from ATP.

Role of CRP/CAP:

• Positive regulation of the lac operon involves a transcriptional activator called CRP, also known as CAP.

• CRP stands for cAMP receptor protein, while CAP stands for catabolite activator protein.

• when glucose levels decrease, intracellular cAMP levels increase and cAMP binds to CRP.

• The resulting complex can bind to a specific DNA sequence near the lac operon promoter called the CRP–cAMP binding site.

• Binding of this complex helps stabilize RNA polymerase at the promoter, increasing transcription.