Lecture 22: Promoter Recognition, Strength and Specificity, Regulation of Eubacterial Transcription

Question 1

Differential protein expression

Answer 1

• high mRNA levels lead to high protein levels

Question 2

Regulated Transcription rules

Answer 2

1. RNA polymerase containing different sigma factors will transcribe different genes by recognizing distinct promoter sequences
2. Activators can bind promoters for specific genes and stimulate transcription
3. Repressors can bind operators (promoters) for specific genes and repress transcription

Question 3

Bacteriophage lytic cycle (process)

Answer 3

• process

1. early: host shut off, mRNA synthesis
   
   • serine threonine kinase: phosphorylates the host RNA polymerase - deactivates it
   • RNA polymerase
   • recognized by sigma 70 of host RNA polymerase
2. middle: DNA synthesis
   
   • host RNA poly inhibitor: further shuts down host mRNA synthesis
   • recognized weakly by T7 RNA polymerase
3. late: Capside proteins
   
   • terminase: puts replicated gene into phage’s head
   • recognized strongly by T7 RNA polymerase

Question 4

Promoter specificty

Answer 4

• determined by consensus sequence
• eg. sigma 70 (rpoD) - unregulated activity
• eg. sigma 32 (rpoH) - activate with stimulus (heat shock)
  *

Question 5

Consititutive

Answer 5

synthesis at a constant level in the presence or absence of inducer

Question 6

Inducible

Answer 6

synthesis that is switched on by an inducer

Question 7

lac operon

Answer 7

• positive control
  
  • CAP-cAMP complex: turn on expression
  • only expressed when CAP is binded to CAP site
    
    • recognized by alpha subunit
• negative control
  
  • lac repressor: turn off expression
    
    • coded by I gene
    • binds to operator
      
      • binds with helix turn helix motif (dimeric alpha helices structure)
      • palindromic sequence
    • allolactose/IPTG binds to repressor to cause allosteric conformational shift making it dissociate from operator - allow transcription
• both are allosteric control
  *

Question 8

Why are genes organized into operons?

Answer 8

• Genes encoding enzymes in a common pathway can all be induced simultaneously.
• This type of control is called coordinate control. One mRNA expresses multiple proteins.

Question 9

lac repressor

Answer 9

• coded by I gene
• binds to operator
• binds with helix turn helix motif (dimeric alpha helices structure)
  
  • palindromic sequence
• allolactose/IPTG binds to repressor to cause allosteric conformational shift making it dissociate from operator - allow transcription

Question 10

CAP-cAMP complex

Answer 10

• cAMP high when glucose low
• CAP-cAMP dimer
  
  • binds to specific DNA sequences
    
    • causes bending
  • interacts with RNA polymerase to stimulate binding ot weak promoter sequences

Question 11

Lac Operon Activity

Answer 11

• no glucose, no lactose: CAP-cAMP bound, lac repressor bound: repressed transcription
• glucose, no lactose: CAP-cAMP not bound, lac repressor bound: repressed transcription
• glucose, lactose: CAP-cAMP not bound, lac repressor not bound: normal/basal transcription
• no glucose, lactose: CAP-cAMP bound, lac repressor not bound: activated transcription

Question 12

cooperative DNA binding proteins (distance of binding, effect, example)

Answer 12

• can interact:
  
  • adjacent binding
  • distant binding
• effect is greater than sum of individual proteins
• eg. positive control of glnA gene (glutamine synthetase)
  
  • activator proteins bind enhancers upstream (80-160bp) of +1
  • high glutamine/nitrogen, unphosphorylated NtrC (activator) does not bind cooperatively with RNA polymerase
  • low glutamine/nitrogen, NtrB activates NtrC by phosphorylation - cooperative binding