L13: Transcriptional Regulation in Prokaryotes I (lac operon and mercury resistance) Flashcards
what are the types of regulatory proteins in transcription?
- activators
- repressors
types of regulatory proteins - activators
positively influence gene expression
types of regulatory proteins - repressors
negatively influence gene expression
types of regulatory proteins - how do they typically work?
- they are usually DNA-binding proteins that recognize sites near the genes they regulate
- they influence the activity of RNA Pol
explain the control of RNA Pol Binding
- RNA Pol only weakly binds to promoters that deviate from the consensus sequence
- when RNA pol binds, it results in basal level of expression
control of RNA Pol Binding - basal level of expression
low levels of constitutive expression due to occasional RNA Pol binding
control of RNA Pol Binding - repressor
- binds to the site overlapping with RNA Pol binding site
- cis-acting element is called an operator
- it phsycally prevents the binding of RNA Pol to the promoter
control of RNA Pol Binding -activator
- usually uses one surface to bind DNA near the promoter and another surface to recruit the RNA Pol
- this mechanism is a type of cooperative binding called recruitment
signal integration between a repressor and activator - lac Operon
- in E. coli the preferred carbohydrate is glucose
- but it can use lactose if necessary
- lactose is broken down by beta-galactosidase and converted into galactose and glucose
what is the lac operon in E. coli
- involves the polycistronic messages composed of three genes
1. lacZ
2. lacY
3. lacA
lac operon in E. coli - lacZ
- encodes beta-galactosidase
- cleaves lactose into galactose and glucose
lac operon in E. coli - lacY
- encodes lactose permease
- a transporter that transports lactose into the cell
lac operon in E. coli - lacA
- encodes thiogalactoside transacetylase
- rids cells of toxic thiogalactosides also imported by lacY (due to it not being selective)
lac operon in E. coli - when is the operon expressed?
- its only expressed at high levels only when:
1. glucose is absent
2. lactose is present
lac operon in E. coli - what are the two regulators
- repressor: lacI (i for 1 not l) gene encodes the Lac repressor
- activator: CAP (Catabolite Activator Protein)
lac operon in E. coli - repressor
- the Lac repressor is transcribed from a constitutive promoter
- it then binds to the operator sequence upstream of lacZ, lacY, and lacA
- it uses a helix-turn-helix motif for binding DNA
- it prevents the RNA Pol from binding
lac operon in E. coli - activator
CAP (Catabolite Activator Protein) binds to the CAP site and recruits RNA Pol
lac operon in E. coli: activator - what is CAP
- it binds to DNA as a dimer
- it contains a helix-turn-helix motif
lac operon in E. coli - what is the helix-turn-helix motif
- two alpha helices separated by a short irregular region called “turn”
- one helix binds to the DNA major groove, while other helix binds to the DNA backbone
lac operon in E. coli - helix-turn-helix motif in regards to activation
- CAP requires cAMP to bind to DNA (allosteric regulation)
- cAMP binding creates an alpha-helix in CAP which realigns the DNA-binding domain into functional orientation
- cAMP is only present in high-concentrations in the absence of glucose
lac operon in E. coli: helix-turn-helix motif activation - what is allosteric regulation
regulation of a protein through the binding of an effector molecule
lac operon in E. coli - why is the Lac repressor absent if lactose is present
to save energy and not make things the cell does not need
lac operon in E. coli - presence of glucose and lactose with a basal level of transcription
- no activator or repressor
- RNA Pol binds to promoter
- glucose and lactose are both present
lac operon in E. coli - presence of glucose and lactose with no transcription
- no CAP and no RNA Pol
- repressor is present
- glucose is present
- lactose is absent
lac operon in E. coli - presence of glucose and lactose with a activated level of transcription
- no repressor on mRNA
- CAP is present
- RNA Pol binds and mRNA changes shape (helix-turn-helix motif)
- glucose is absent
- lactose is present
lac operon in E. coli - what happens to the repressor when lactose is present
- the lactose directly binds to the Lac repressor and induces a confirmational change
- the Lac repressor is no longer able to bind to DNA
- this is due to allosteric regulation
explain signal integration in the lac operon
- lac operon expression is controlled by two signals (glucose and lactose)
- each signal is communicated to the genes via a separate regulator:
1. glucose (via cAMP): CAP
2. lactose: Lac repressor
signal integration - Gene layout of the lac operon
- from 5’ to 3’
1. lacl →
2. CAP binding site
3. Promoter
4. operator
5. lacZ →
6. lacY →
7. lacA →
signal integration - low glucose and lactose available
- high cAMP due to low glucose
- CAP binds to CAP binding site
- repressor is not bound bc lactose inactivates it
- RNA Pol binds to the promoter efficiently with CAP’s help (cooperative binding/recruitment)
signal integration: low glucose and lactose available - result
lac genes are strongly expressed
signal integration - high glucose and lactose unavailable
- low cAMP and low CAP bc high glucose
- repressor is bound bc there is no lactose to remove it
signal integration: high glucose and lactose unavailable - result
lac genes not expressed
signal integration - low glucose and lactose unavailable
- high cAMP levels and high CAP bc low glucose
- repressor binds bc there is no lactose to inactivate it
signal integration: low glucose and lactose unavailable - result
lac genes not expressed (repressor is bound even is CAP is bound)
signal integration - high glucose and lactose available
- no repressor bc lactose inactivates it
- low cAMP and no CAP binding bc of high glucose
signal integration: high glucose and lactose available - result
very low (basal) level of gene expression bc no CAP or repressor
mercury resistance - how can activators use allosteric regulation
allosteric regulation can affect protein or DNA confirmation
mercury resistance - what is MerR?
an activator that induces the mercury resistance gene merT in the presence of mercury
mercury resistance - what is merT?
a mercury transport protein
mercury resistance: MerR and merT - how does it work?
- merT has -35 and -10 promoter (separated by 19bp) elements that are not aligned on the same face of DNA
- the binding of Hg^2+ to MerR causes a confirmational change that twists the DNA
- the promoter than becomes a strong sigma (σ) promoter with -35 and -10 separated by 17bp
- this brings the merT gene closer so RNA Pol can initiate transcription of the gene
