CAP Regulation

Question 1

what binds the dimers in CAP?

Answer 1

leucine zipper

Question 2

CAP bends DNA more than 90 degrees, what is the purpose of this?

Answer 2

• may facilitate contact with RNA polymerase
• causes the DNA to curl around the polymerase
• allows for broader contact between CAP and the core polymerase

Question 3

what binds to CAP as an activator?

Answer 3

• rotation of the monomer subunits induced by cAMP binding
• cyclic AMP binds to the N terminal domain and causes the two C helices to reorient and move the DBD apart

Question 4

what do CAP dependent promoters not have?

Answer 4

• a good -35 binding site

Question 5

what does CAP do to the gal promoter do?

Answer 5

• CAP increases the transition from the closed to open promoter complex

Question 6

what does CAP do to the lac promoter?

Answer 6

• CAP increases formation of the closed promoter complex

Question 7

CAP class 1 promoters bind to what site?

Answer 7

lac
- binds at -61, -71, etc.
- binds to the same helical face to make contact with the core enzyme

Question 8

CAP class 2 promoters bind to what site?

Answer 8

• gal promoter
  exactly 41.5

Question 9

CAP class 3 promoters bind to what site?

Answer 9

• arabinose
• has two trans-activators, atleast one of which is CAP
• can bind in any order and at any position

Question 10

what is the binding of pBAD?

Answer 10

• class 3
• CAP then arabinose

Question 11

what is the binding of pFGH?

Answer 11

• class
• arabinose then CAP

Question 12

how many promoters does CAP activate transcription for?

Answer 12

100

Question 13

what is the molecular mass of CAP?

Answer 13

• 45 kDA and binds DNA as a dimer

Question 14

what was the first transcriptional activator to be purified in 1970 and the first to have its 3D structure determined?

Answer 14

CAP

Question 15

CAP Class 1 definition

Answer 15

require only CAP for activation and have single site for CAP binding located upstream from promoter. The CAP site can be at various distances, but must be on the same helical face as RNA pol. Example: lac
- CAP makes contact with the alpha-CTD

Question 16

activation region 1

Answer 16

AR1
- makes contact with the alpha-CTD at the lac promoter
- only contact with a single alpha-CTD is required

Question 17

what is the mechanism of Class 1?

Answer 17

interactions between CAP and a-CTD increase binding constant for formation of the closed promoter complex. Increases initiation by facilitating recruitment of holoenzyme to the promoter.
- does not affect ability to melt or open promoter transition directly
- alpha CTD binds to the DNA immeiatly downstream of CAP
- DNA bending is not though to play a role

Question 18

Class 2 definition

Answer 18

only require CAP for activation and have a single CAP site that overlaps the promoter replacing the -35 site. Example: gal P1 promoter
- alpha CTD binds upstream of the CAP

Question 19

what is the difference between class 1 and 2 CAP dependent promoters?

Answer 19

location of the CAP binding site
- for class 1 promoters, the CAP binding site is upstream of the UP site
- for class 2 promoters, the CAP binding sites is in the between the UP and core promoter

Question 20

Class 2 AR1 and 2

Answer 20

• make contact with the alpha-CTD and the alpha NTD at the gal promoter
• AR2 is in the N terminus near the cAMP binding domain and carries a net positive charge of +2
• CAP makes contact with a single alpha-subunit. the alpha contracts beta works better than the one that contracts beta prime

Question 21

class 2 upstream action

Answer 21

CAP makes contact with the alpha CTD with AR1

Question 22

class 2 downstream action

Answer 22

CAP makes contact with the N terminal domain through AR2`

Question 23

Class 3 definition

Answer 23

These require two or more CAP dimers, or one or more CAP molecules with one or more regulon-specific activators. Examples: araBAD and malK.
- may use class 1 or 2 mechanism for activation

Question 24

what is mechanism that is sometimes associated with Class 3?

Answer 24

• anti-activation
• A negative regulator may block the action of CAP as seen in the case of the CytR promoter. CAP is prevented from contacting the a-CTD and the CTD is blocked from binding the DNA.

Question 25

how do class 3 CAP dependent promoters differ from class 1 and 2

Answer 25

class 3 requires two activators, at least one of which must be CAP

Question 26

regulation of the arabinose operon

Answer 26

1- At the araBAD and araFGH promoters, both CAP and AraC are required for activity.
2- At both promoters, the binding of RNA pol is extremely tight due to the simultaneous contact of the a-CTD with AraC and with CAP
3- AraC contacts with RNA pol prevent polymerase from binding to the “dead end” promoter”.

Question 27

what are the two requirements for activity at the lac and ara operons?

Answer 27

• repression has been lifted
• CAP is present

Question 28

what is the activation state with glucose and lactose is in the media?

Answer 28

off

Question 29

what is the activation state if glucose is not present in the media but lactose is?

Answer 29

on

Question 30

what is the activation state if glucose is present in the media but lactose is not?

Answer 30

off

Question 31

what is the activation state if glucose is not in the media and lactose is not in the media?

Answer 31

on