BSCI 170 - The Regulation of Prokaryotic Gene Expression

Prokaryotic Genes

• What are genes that are always actively expressed?

• Why may some genes be on or off?

• How do the expression of genes meet metabolic needs?

• constitutive expression; e.g. genes for glycolytic enzymes

• depending on environmental conditions

• they are very variable and highly coordinated

Bacterial Gene Expression

• What kind of pressure are bacteria under to survive?

  • What does this lead to?

• Bacteria are under extreme competitive pressure to survive

  • small selective advantages are the difference between life and death

What can gut-living bacteria do?

• make whatever they need

• if they can get nutrients from environment, then no reason to make them from scratch

  • save energy for reproduction, etc.

Levels of Gene Control

• What is “gene expression” related to?

the amount of functional product in a cell

All 3 Levels Are Active in Cells

• What are characteristic of translational and posttranslational controls?

• What is characteristic of transcriptional control?

• rapid, but energetically costly; all resources already generated

• slower, but more energetically efficient

Prokaryotic Gene Organization

• In E. coli, how is amino acid tryptophan synthesized?

• in a multi-step process

  • each step is catalyzed by a different enzyme coded for by one or more of 5 genes

  • All 5 genes are clustered on the chromosome in the order they are used

What are the gene clusters clustered on the chromosome in the order they are used called?

operons

What does one promoter do?

coordinately regulate the transcription of all 5 genes

Operon Control

• Is the operon on/off control switch the promoter?

• What does the operator overlap? What does it lie between?

• no; the switch is a short DNA sequence called the operator

• the promoter; it lies between the promoter and the transcription start site

How does the operator control accress to the promoter?

by RNA polymerase

What comprises the operon?

the promoter, the operator, and the transcription unit

What are the two general operon types?

• negatively regulated: the genes are normally on, and binding of a repressor protein to the DNA turns transcription off

• positively regulated: the genes are normally off, and binding of an activator protein to the DNA turns transcription on

What is an example of negative control?

the repressible operon

What is the repressible operon?

• a repressible operon is normally active but is turned off when a specific repressor protein binds to the operator

• the repressor blocks access by RNA polymerase to the promoter or to the genes

What is the trp operon?

• it is normally active

• RNA polymerase has access to the promoter

What is the trp Repressor?

the product of a gene (trpR) located outside and upstream of the operon

What is characteristic of the trpR gene?

it is constitutively active at a low level; there is always some repressor protein available

If the repressor is always available, then why isn’t the operon turned off all the time?

• binding to operator is reversible

• the repressor is an allosteric protein

What are the two conformations that allosteric proteins exist in? Designate these as functional or nonfunctional.

• active conformation: functional

• inactive conformation: nonfunctional

What does binding a regulator do? What are the types of regulators; what do they do?

stabilize the protein in one state or the other

• activator: stabilizes in active conformation

• inhibitor: stabilizes in inactive conformation

Action of the trp Repressor

• What kind of protein is the trp repressor?

• What is it mostly present as?

• What is tryptophan?
  

• an allosteric protein

• in the inactive form

• an active allosteric effector (corepressor)

Summary:

• What state is the trp operon in normally?

• What does the expression of the operon lead to?

• What occurs when tryptophan is present in the environment?
  

• the trp operon is normally active

• expression of the operon leads to production of enzymes for making tryptophan (tryptophan is necessary to make protein)

• if tryptophan present in the environment:

  • tryptophan binds to the repressor

  • trp-repressor complex binds to operator

  • operon transcription repressed

Negative Control Example 2: The Inducible Operon

• What state is an inducible operon normally in? Why?

• How can an operon be turned on? Why?

• What is a classic example of an inducible operon?

• an inducible operon is normally off because of an active repressor

• the operon can be turned on in the presence of an inducer; the inducer inactivates the repressor

• classic example if the lactose operon

  • regulates metabolism of lactose

  • lactose is available to E. coli cells if host consumes milk or milk products

Metabolism of Lactose (Milk Sugar)

• What is lactose a disaccharide of?

• What is the first step of the metabolism of lactose?

• Lactose is a disacchride of B-Glucose and B-Galactose

• Step 1 is cleavage of the B-glycosidic bond

  • B-Galactosidase (B-Gal)

Metabolism of Lactose

• Without lactose, only a few molecules of permease and B-Gal are present

• With lactose, what happens to the amount of B-Gal?

The amount of B-Gal increases 1000X in 15 minutes

lac Operon

• What is the lac operon comprised of?

• What is the lac Regulatory gene? Where is it located?

  • What does it encode?

  • What is it essentially?
    

• The lac operon comprises a promoter, an operator, and 3 genes

• the lac Regulatory gene, lacI, is located upstream from the operon

  • encodes an allosteric protein

  • lac Repressor

lac Repressor

• What does the repressor bind to?

• What does the repressor prevent?

• How does the repressor bind?

• The repressor binds to the operator

• The repressor prevents RNA polymerase from binding to the promoter and moving forward; conceptually identical to trp repressor

• Repressor binds operator without a corepressor

  • different from the trp repressor

What happens to some lactose in the cell when lactose is present?

some lactose in the cell is converted to an isomer of lactose (allolactose)

When lactose is present what does allolactose bind to? What does this cause? What does this make allolactose?

• allolactose binds to and inactivates lac repressor; this makes allolactose an inducer

Summary

• In the absence of lactose, where does the lac repressor bind?

• When lactose is available, what happens to some of the lactose?

• What does allolactose bind to?

• How does the lac repressor shift in its conformation?

• What occurs after this shift in conformation occurs?

• lac repressor binds the operator and keep operon inactive

• some of the lactose is converted to allolactose

• allolactose binds to lac repressor

• lac repressor shifts to inactive allosteric conformation and detaches from the operator

• transcription of the operon commences

lac Operon vs. trp Operon

• lac operon is _______ - what does this mean?

• trp operon is ____________ - what does this mean?

• inducible

  • repressor normally bound and then unbinds

  • inducer converts repressor to inactive form

  • operon transcribable

• repressible

  • repressor normally unbound and then binds

  • corepressor converts repressor to active form

  • operon not transcribable

lac Operon vs. trp Operon

• what do inducible operons generally function in? Why?

• what do repressible operons generally function in? Why?

• inducible operons generally function in catabolic pathways

  • you break things down only if they are present

• repressible operons generally function in anabolic pathways

  • you build things up only when they are not available

Positive Control: Operon Regulation

• How are both the lac and trp operons controlled?

• What does it mean if an operon is negatively controlled?

• What does it mean if an operon is positively controlled?

• How can the lac operon be regulated?

• both are negatively controlled

• operon transcription is switched off in the presence of an active form of a repressor

• operon transcription is switched on in the presence of an active form of an activator

• the lac operson can be regulated both positively and negatively

Positive Gene Regulation

• What do E. coli cells prefer to use as their energy source?

  • Why?

• When will E. coli activate genes for lactose metabolism? What is this called?

• E. coli cells prefer to use glucose as their energy source when it is available

  • glycolytic enzymes are always present, always active, and very efficient

• E. coli will activate genes for lactose metabolism only when lactose is present AND glucose is absent (or low).

How does E. coli sense the presence of glucose?

• a regulatory protein interacts with a small signaling molecule

  • regulatory protein is catabolite activate protein (CAP)

  • signaling molecule is cyclic AMP (cAMP)

• [cAMP] in cell is inversely related to [Glucose] outside the cell

  • High [Glucose]_out correlates with low [cAMP]_in

Positive Regulatory Protein

• How is CAP regulated?

• What does the active CAP-cAMP complex bind to?

• CAP is regulated allosterically

  • cAMP binding to CAP stabilizes CAP in the active conformation

  • cAMP is allosteric activator

• Active CAP-cAMP complex binds to DNA upstream of the lac promoter

  • presence increases efficientcy of RNA polymerase binding to the promoter

lac Promoter

• Are all promoters equal?

  • Why?

• What is polymerase binding related to?

• How does the lac promoter bind RNA polymerase even in the absence of repressor?

• all promoters are NOT equal

  • some bind polymerase efficiently

  • some bind polymerase inefficiently

• polymerase binding is related to amount of transcription

• the lac promoter binds RNA polymerase poorly even in the absence of repressor

Catabolite Repression

• When CAP is inactive, what occurs to RNA polymerase?

RNA polymerase binding is weak and transcription activity is low

Dual Control of the lac Operon

• When lactose is present and glucose is also present, what is the state of the operon?

• When lactose is present and glucose is absent, what is the state of the operon?

• the operon is weakly active

• the operon is highly active

Dual Control of the lac Operon

• _________ control by the lac repressor

  • What does the state of the lac represoor determine?

  • What does an active repressor do?

  • What does an inactive repressor do?

  • What is a repressor like?

• negative

• whether the operon is transcribed

• turns gene transcription “OFF”

• turns gene transcription “ON”

• an on/off switch

Dual Control of the lac Operon

• _________ control by CAP

• What does the state of CAP determine?

  • What does an “inactive” activator do?

  • What does an “active” activator do?

  • What is CAP like?

  • Many way to fine tune gene expression

• Positive

• the level of transcription

  • turns level of gene transcription “DOWN”

  • turns level of gene transcription “UP”

  • a volume control