B: Transcription prokaryotes (lac operon)

Question 1

CAP protein

Answer 1

• CAP protein can bind to cAMP
• CAP-cAMP complex can bind to cap binding site
• CAP-cAMP complex activates lac promotor
• In the absence of cAMP, CAP cannot bind the cap binding site effectively
• Lac promoter is a weak promoter in the absence of CAP-cAMP complex
• When glucose is high in the cell, cAMP is low.

Question 2

LACI protein

Answer 2

• Can bind to lacO
• LACI protein is a repressor, so represses lac promoter
• Lactose can bind to LACI protein
• LACI-lactose complex does not bind lacO effectively
• Thus in the absence of lactose the LACI repressor is active, in the presence of lactose the LACI repressor is inactive.

Question 3

E. Coli promoter features

Answer 3

• TATA box: DNA sequence of TATAAT found 10 bases upstream from +1 site
• TTGACA found in -35 region –> -35 box
• Gap of 17-19bp consistently found between TATA box and -35 box.

Question 4

What is sigma factor 70?

Answer 4

Sigma factor 70 required for promotor recognition by RNA polymerase in E. coli

Question 5

Sigma factor 70 has 4 main sections:

Answer 5

• Section 4, subsection 4.2 has alpha helix-turn-alpha helix which recognizes -35 region
• Section 2, subsection 2.4 has alpha helix which recognises -10 TATA box region
• Section 1 has autoinhibitory domain which stops sigma 70 binding to promoters in absence of RNA polymerase.

Question 6

LacZ

Answer 6

Encodes beta galactosidase –> hydrolyses lactose

Question 7

LacY

Answer 7

Encodes permease –> transports lactose into the cell

Question 8

LacA

Answer 8

Encodes beta galactoside transacetylase –> acetylates lactose

Question 9

Scenario if Glucose and lactose are high, cAMP is low?

Answer 9

CAP-cAMP complex does not form, LACI-lactose complex forms - no repression of lac promoter, lacZYA transcription is low

Question 10

Scenario if glucose is high and lactose is low, cAMP is low?

Answer 10

CAP-cAMP complex does not form - no activation of lac promoter, LACI-lactose complex does not form - strong repression of lac promoter, lacZYA transcription is very low.

Question 11

Scenario if glucose and lactose is low, cAMP is high?

Answer 11

CAO-cAMP complex forms, high activation of lac promoter, LACI-lactose complex does not form - strong repression of lac promoter, little transcription of lacZYA genes as lac promoter is a weak promoter

Question 12

Scenario if glucose is low and lactose is high, cAMP is high?

Answer 12

CAP-cAMP complex does form - high activation of lac promoter, LACI-lactose complex forms - strong repression of lac promoter, high transcription of lacZYA genes.