TMC 8

Question 1

What is the function of the lac ZYA genes?

Answer 1

To transcribe proteins that metabolise lactose

Question 2

What is LacZ and what is its function?

Answer 2

LacZ = beta galactosidase which cleaves lactose into galactose and glucose

Question 3

What is LacY and what is its function?

Answer 3

LacY = lactose permease which transports lactose into the cell

Question 4

What is LacA and what is its function?

Answer 4

LacA encodes transacetylase which acetylates lactose

Question 5

What is the CAP protein?

Answer 5

Cap protein is encoded by the CAP gene
Its not a strong activator of the lac operon on its own but when bound to cAMP it is.

Question 6

What is the relationship between glucose and cAMP concentrations?

Answer 6

When glucose is high cAMP is low and therefore little activation of the lac operon happens.
When glucose is low then cAMP is high therefore it binds to the CAP protein and the CAP binding site on the lac operon and stimulates the transcription of LacZYA proteins

Question 7

What is the LAC repressor?

Answer 7

LACl protein binds to lac operator
This binding blocks RNA polymerase transcription of lacZYA from the lac promoter even in the presence of the CAP-cAMP complex

Question 8

What is the lac promoter?

Answer 8

Region upstream of the LacZYA gene between -10 - -35. The promoter is weak on its own but the CAP binding site is immediately upstream of it, from -35 to -80
CAP-cAMP complex binds to the binding site and is a strong activator of the promoter

Question 9

What is the lac operator?

Answer 9

Binding site of the LACl protein which when binded blocks RNA polymerase transcription of lacZYA from the lac promoter

Question 10

What is the CAP binding site?

Answer 10

Site immediately upstream the lac operon which the CAP-cAMP complex binds to, forming a strong activator of the lac promoter

Question 11

Describe the lac operon.

Answer 11

-80 to -35
CAP binding site
CAP protein can bind to cAMP. The CAP-cAMP complex can bind to the CAP binding site, it then activates the promoter. In the absence of cAMP, CAP can not bind to the CAP binding site effectively. The lac promoter is very weak promoter in the absence of the CAP-cAMP complex. When glucose is high cAMP is low and visa versa.

-35 to-10
Lac promoter
Very weak promoter. Does not support transcription of LacZYA genes by itself. Activation by CAP-cAMP complex.

Roughly -12 to +1
Lac operator

Question 12

Describe the gene regulation of the lac operon.

Answer 12

Glucose high and lactose high: CAP-cAMP complex doesn’t form, no activation of lac promoter, LACl-lactose complex forms high repression of lac operator, low transcription and lac promoter is weak
Glucose high and lactose low: CAP-cAMP complex doesn’t form, no activation of lac promoter, LACl-lactose complex doesn’t form low repression of lac operator very low transcription
Glucose low and lactose low: CAP-cAMP complex forms, activation of promoter, LACl-lactose complex doesn’t form meaning there’s repression, 0 transcription
Lactose high and glucose low: CAP-cAMP complex forms, activates promoter, LACl-lactose complex forms repressing operator meaning there’s high transcription

Question 13

List 5 points about gene promoters

Answer 13

They can be strong/weak
Upstream
RNA pol binds to them
TATA box 10 bp upstream
-35 box upstream TTGACA
17-19bp gap between -35 box and TATA box
Sigma factor 70 used to recognise promoters with RNA pol (RNA polymerase holoenzyme)
If the promoter is removed there’ll be no transcription

Question 14

Describe the sequence and structure of a strong E. Coli promoter

Answer 14

TATAAT -10bp from +1 site
TTGACA -35bp
Gap of ~17-19bp found between the TATA box and -35 box

Question 15

Explain the relationship between the gap found between the -10 and -35 regions in a strong E. coli promoter and
sigma factor 70

Answer 15

Sigma factor 70 has 2 alpha helices that form a HTH in subdomain 4.2, First helix inserts into the major groove of DNA and interacts with -35 box
Subdomain 2.4 binds the -10 box where melting occurs - assisted as it is easier to melt A:T in the TATA box
The spacing between these regions is the 17-19 bp between the -35 box and TATA box - 7.5 nm

Question 16

Explain how RNA Pol recognises the promoter in E. Coli

Answer 16

Extremely inefficient at recognising and the presence of sigma factor 70 is essential as RNA pol holoenzyme can recognise them well

Question 17

Explain the relationship between RNA polymerase and sigma factor 70 in recognising promoters in E. coli.

Answer 17

Sigma factor 70 cannot bind promoters by itself even though it has binding regions for the -35 and -10 promoter region.
In the absence of RNA polymerase, sigma
factor 70 DNA binding regions are occluded with an autoinhibitory domain in the N-terminal of the sigma factor.
When sigma factor 70 associates with the
RNA polymerase core enzyme, the conformation of sigma factor 70 changes and the DNA binding domains are exposed.

Question 18

Explain how sigma factor 70 binds to the promoter.

Answer 18

RNA polymerase must be present- RNA polymerase holoenzyme
N-terminal end is autoinhibitory part must be in the presence of RNA pol for the binding site to be revealed. Domain 4 – in the 4.2 region alpha HTH binding motif.
The first H binds to the major groove of the DNA at the TTGACA consensus and the second H binds to the backbone of the DNA.
In domain 2 there is a 2.4 subunit with a HTH. The H here enables the melting of the TATA box- helped by the fact that they are made of AT.
Sigma factor 70 has several aromatic amino acids that stabilizes the melted DNA. Space between region 2 and 4 – 7.5 nm same distance as between the -10 and -35 regions.

Question 19

What is negative regulation?

Answer 19

Repression of genes by repressors

Question 20

What is positive regulation?

Answer 20

Activation of gene transcription by activators

Question 21

Describe the structure of sigma factor 70.

Answer 21

2 alpha helices in subdomain 4.2
Alpha helix in subdomain 2.4
Several aromatic amino acids that stabilise melted DNA
~7.5nm space between region 2 and region 4
DNA binding regions are occluded with an autoinhibitory domain in the N-terminal and only activates when it becomes RNA polymerase holoenzyme