L13: Transcriptional Regulation in Prokaryotes I (lac operon and mercury resistance)

Question 1

what are the types of regulatory proteins in transcription?

Answer 1

1. activators
2. repressors

Question 2

types of regulatory proteins - activators

Answer 2

positively influence gene expression

Question 3

types of regulatory proteins - repressors

Answer 3

negatively influence gene expression

Question 4

types of regulatory proteins - how do they typically work?

Answer 4

• they are usually DNA-binding proteins that recognize sites near the genes they regulate
• they influence the activity of RNA Pol

Question 5

explain the control of RNA Pol Binding

Answer 5

• RNA Pol only weakly binds to promoters that deviate from the consensus sequence
• when RNA pol binds, it results in basal level of expression

Question 6

control of RNA Pol Binding - basal level of expression

Answer 6

low levels of constitutive expression due to occasional RNA Pol binding

Question 7

control of RNA Pol Binding - repressor

Answer 7

• 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

Question 8

control of RNA Pol Binding -activator

Answer 8

• 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

Question 9

signal integration between a repressor and activator - lac Operon

Answer 9

• 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

Question 10

what is the lac operon in E. coli

Answer 10

• involves the polycistronic messages composed of three genes
  1. lacZ
  2. lacY
  3. lacA

Question 11

lac operon in E. coli - lacZ

Answer 11

• encodes beta-galactosidase
• cleaves lactose into galactose and glucose

Question 12

lac operon in E. coli - lacY

Answer 12

• encodes lactose permease
• a transporter that transports lactose into the cell

Question 13

lac operon in E. coli - lacA

Answer 13

• encodes thiogalactoside transacetylase
• rids cells of toxic thiogalactosides also imported by lacY (due to it not being selective)

Question 14

lac operon in E. coli - when is the operon expressed?

Answer 14

• its only expressed at high levels only when:
  1. glucose is absent
  2. lactose is present

Question 15

lac operon in E. coli - what are the two regulators

Answer 15

1. repressor: lacI (i for 1 not l) gene encodes the Lac repressor
2. activator: CAP (Catabolite Activator Protein)

Question 16

lac operon in E. coli - repressor

Answer 16

• 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

Question 17

lac operon in E. coli - activator

Answer 17

CAP (Catabolite Activator Protein) binds to the CAP site and recruits RNA Pol

Question 18

lac operon in E. coli: activator - what is CAP

Answer 18

• it binds to DNA as a dimer
• it contains a helix-turn-helix motif

Question 19

lac operon in E. coli - what is the helix-turn-helix motif

Answer 19

• 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

Question 20

lac operon in E. coli - helix-turn-helix motif in regards to activation

Answer 20

• 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

Question 21

lac operon in E. coli: helix-turn-helix motif activation - what is allosteric regulation

Answer 21

regulation of a protein through the binding of an effector molecule

Question 22

lac operon in E. coli - why is the Lac repressor absent if lactose is present

Answer 22

to save energy and not make things the cell does not need

Question 23

lac operon in E. coli - presence of glucose and lactose with a basal level of transcription

Answer 23

• no activator or repressor
• RNA Pol binds to promoter
• glucose and lactose are both present

Question 24

lac operon in E. coli - presence of glucose and lactose with no transcription

Answer 24

• no CAP and no RNA Pol
• repressor is present
• glucose is present
• lactose is absent

Question 25

*lac* operon in *E. coli* - presence of glucose and lactose with a **activated level** of transcription

Answer 25

- no repressor on mRNA - CAP is present - RNA Pol binds and mRNA changes shape (helix-turn-helix motif) - glucose is **absent** - lactose is **present**

Question 26

*lac* operon in *E. coli* - what happens to the repressor when lactose is present

Answer 26

- 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

Question 27

explain signal integration in the *lac* operon

Answer 27

- *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

Question 28

signal integration - Gene layout of the *lac* operon

Answer 28

- from 5' to 3' 1. *lacl* → 2. CAP binding site 3. Promoter 4. operator 5. *lacZ* → 6. *lacY* → 7. *lacA* →

Question 29

signal integration - low glucose and lactose available

Answer 29

- 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**)

Question 30

signal integration: low glucose and lactose available - result

Answer 30

*lac* genes are strongly expressed

Question 31

signal integration - high glucose and lactose unavailable

Answer 31

- low cAMP and low CAP bc high glucose - repressor is bound bc there is no lactose to remove it

Question 32

signal integration: high glucose and lactose unavailable - result

Answer 32

lac genes not expressed

Question 33

signal integration - low glucose and lactose unavailable

Answer 33

- high cAMP levels and high CAP bc low glucose - repressor binds bc there is no lactose to inactivate it

Question 34

signal integration: low glucose and lactose unavailable - result

Answer 34

lac genes not expressed (repressor is bound even is CAP is bound)

Question 35

signal integration - high glucose and lactose available

Answer 35

- no repressor bc lactose inactivates it - low cAMP and no CAP binding bc of high glucose

Question 36

signal integration: high glucose and lactose available - result

Answer 36

very low (basal) level of gene expression bc no CAP or repressor

Question 37

mercury resistance - how can activators use allosteric regulation

Answer 37

allosteric regulation can affect protein or DNA confirmation

Question 38

mercury resistance - what is MerR?

Answer 38

an activator that induces the mercury resistance gene **merT** in the presence of mercury

Question 39

mercury resistance - what is *merT*?

Answer 39

a mercury transport protein

Question 40

mercury resistance: MerR and *merT* - how does it work?

Answer 40

- *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