The antiviotic drug teteracycline kills bacteria by inhibiting ribosomes. Bacter

Question

The antiviotic drug teteracycline kills bacteria by inhibiting ribosomes. Bacteria that are resistant to tetracycline have the TetA gene, which encoeds a membrane protein that pumps tetracycline out of the cell. These bacteria also express a repressor protein called TetR, which binds a specific 15 base pair DNA sequence element named TetO that is present just downstream of the TetA core promoter.

a) If TetR is bound to tetracycline, then it can no longer bind to TetO. Predict TetA expression (on,off) and cell growth (live, die) with the mutations shown in the table below.

c) Sketch a LacZ reporter gene that can be "turned off" in human cells by the TetR-SP1 activation doman fusion protein with the addition of tetracycline.

ll shown in the shown in the table below. (10 pts) Mutation tetracycline tetracycline TetA? cells? Teet A? cells? None (wild type) Mutate TetO sequence Delete TetA core promoter Disables tetracycline binding to TetR Disables DNA binding ability of TetR

Explanation / Answer

This question has multiple questions. I am answering the first question as per Chegg’s policy.

None (wildtype)

-tetracycline:

TetA is off. Because tetR will be bound to tetO. There is no tetracycline to sequester tetR. So, it is free to bind to tetO

Cells survive because they are not placed in medium containing tet.

+tetracycline:

TetA is on. In the presence of tetracycline, TetR will not be able to bind to tetO. Cells survive because tetA is on. It will pump tetracycline out of cell.

Mutate tetO sequence:

-tetracycline:

TetA is on.

If tetO is mutated tetA expression is constitutive because tetR cannot bind to tetO even in the absence tetracycline.

Cells survive.

+tetracycline:

TetA is on (reason is same as stated above)

Since, tetA expression is constitutive, cells survive.

Delete TetA core promoter:

-tetracycline:

TetA is off.

Core promoter is required for transcription (for binding of RNA polymerase and other transcription machinery). If promoter is deleted, transcription is off always.

Cells survive even though tetA is off because tetracycline is not present.

+tetracycline:

TetA is off. (Reason is same as stated above).

Cells die because TetA is off. Tetracycline enters cell and inhibit ribosomes.

Disable tetracycline binding to tetR:

-tetracycline:

TetA is off.

In the absence of tetracycline, tetR binds to tetO. So, tetA will be off.

Cells survive because tetracycline is not present.

+tetracycline:

TetA is off.

Tetracycline enters cell but it will not sequester tetR. So, tetR can still bind to tetO. So, tetA will be off.

Cells die because tetA is off.

Disable DNA binding ability of TetR:

-tetracycline:

TetA is on.

If tetA cannot bind to DNA, it means it cannot bind to tetO. So, tetA expression will be constitutive.

Cells survive as there is no tetracycline. Even if it is present, they would survive because TetA is on.

+tetracycline:

TetA is on. As said above, tetA is constitutively expresses if tetR does not bind to tetO.

Cells survive because TetA is on. Tetracycline will be pumped out.

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