Lesson 14: Prokaryotic Gene Regulation

Question 1

Do different cell types have the same DNA?

Answer 1

Yes! The cells of an organism have the same DNA

Question 2

How do we get different cell types in our bodies?

Answer 2

Control of gene expression (turning genes ON and OFF) give cells their various properties

Question 3

What is gene expression?

Answer 3

This is how a gene directs the synthesis of a protein or a functional RNA molecule. This ultimately affects a phenotype of the cell/organism

Question 4

What does it mean for a cell to be differentiated?

Answer 4

This is when a stem cell changes to a specialized cell type (become a different “fate”).

Question 5

How do cell types become differentiated?

Answer 5

Two hypotheses:

1) The cell starts off as a stem cell and then certain pieces of DNA get lost from cells so they only retain the DNA that made that cell type.

2) CORRECT HYPOTHESIS: Each cell retains all the DNA but parts of the DNA are turned on or off so that only the right genes are expressed.

Question 6

How was the correct hypothesis developed?

Answer 6

Sir John Gurdon demonstrated that taking a nucleus from an adult frog cell and putting it in an unfertilized frog egg with the nucleus removed would create an entire new tadpole.
- This reveals that one cell has all the DNA needed to create different cells. There is NOT different DNA in each cell type.

Question 7

What is the main point of gene regulation in a cell?

Answer 7

While cells use exquisite control at almost every level to regulate the amount and types of protein produced by a cell, initiation of TRANSCRIPTION is the main point of control.

Question 8

How does prokaryotic gene regulation occur during transcription?

Answer 8

Regulatory sequences (ex: operators) are additional DNA elements that bind proteins and also control gene expression

Question 9

What are transcription regulators?

Answer 9

Aka transcription factors, are regulatory proteins that bind to these regulatory sequences

Question 10

How do transcription regulators interact with the operon?

Answer 10

Even though transcription regulators can look different from each other, they all make direct contact with DNA. They bind within the pockets. None of them break open the DNA but instead they bind to the sides.

Question 11

Describe how the transcription regulators make contact with the major and minor groove of DNA in particular.

Answer 11

The amino acid side chains make hydrogen, ionic, and hydrophobic bonds with nucleotide base pairs in the major (and minor) groove of DNA.
- Majority of transcription regulator proteins bind to the major groove.

Each of the four base pairs have unique patterns of hydrogen bond donors and acceptors so the transcription regulator can recognize a specific sequence in the DNA.

Question 12

Can transcription regulators tell the difference between T-A and A-T chemically?

Answer 12

Yes!
- Differences in hydrogen bond donors and acceptors in the major or minor groove can be “read” by amino acids in the transcription regulator.

Question 13

What is a consensus logo?

Answer 13

Shows the preferred nucleotides at each position in the DNA (coding strand written 5’ to 3’)
- NOTE: Letter height is proportional to the frequency of the base in the regulatory sequence. (the larger the letter, more important the preference)

Question 14

What is the function of the consensus logo?

Answer 14

Allows use to scan for specific DNA sequences and predict which genes will be regulated by the regulatory factor.

Question 15

What are the two different type of transcription regulators?

Answer 15

Repressors and activators

Question 16

What is a repressor in a prokaryotic cell?

Answer 16

prevents RNA polymerase from initiating transcription by binding to the operator

Question 17

What is an activator in a prokaryotic cell?

Answer 17

promotes RNA polymerase transcription initiation. Activators can help promote expression from inefficient promoters.

Question 18

What is the most important step to regulate, if you want to regulate gene expression?

Answer 18

Transcription!

You do not want to waste time transcribing and translating proteins.

Question 19

Why are bacterial mRNAs polycistronic?

Answer 19

Bacterial mRNAs have the ability to express multiple genes at the same time when they need to work together in response to the environment or to build a molecule for a biosynthetic process.

Question 20

How are bacterial genes organized?

Answer 20

Into operons!

Question 21

What is an operon?

Answer 21

A cluster of linked genes that are co-regulated (make enzymes that carry out the same function)
- NOTE: has one SINGLE promoter

Question 22

What is an operator?

Answer 22

Regulatory DNA sequence that is found in the promoter region (but is different from the -35 and -10 sequences)
- Where the repressor binds

Question 23

How do cells (ex: E. coli) control the tryptophan operon?

Answer 23

The cell responds to the AMOUNT of tryptophan present

Question 24

What happens when tryptophan is low in the cell?

Answer 24

Turn the operon ON
- Since there’s no tryptophan to bind to the repressor and activate it. The repressor will not be able to bind to the operator, thus allowing RNA polymerase to be able to transcribe the operon.

Question 25

What happens when tryptophan is high in the cell?

Answer 25

Turn the operon OFF
- The tryptophan binds to the repressor and changes its shape (activates it). The repressor then binds to the operator and blocks RNA polymerase from transcribing the operon.

Question 26

How is the Trp repressor an example of an allosteric enzyme?

Answer 26

When tryptophan binds to the Trp repressor, it changes the shape of the repressor allowing the tryptophan to bind to the operator.

Question 27

How is the Trp repressor an example of feedback inhibition?

Answer 27

As the enzymes that are made from the operon start making trp, the levels of trp will rise, causing the repressor to bind to the operator.

Once the trp gets used up, the trp repressor will change its shape to the inactive repressor and will unbind from the operator allowing more trp to be made again.

• This cycle will continue to repeat itself

Question 28

How does tryptophan binding to the Trp repressor allow for the repressor to bind to the operator?

Answer 28

When tryptophan binds to the Trp repressor, it increases the width of the repressor, allowing the repressor to bind to the operator more easily

Question 29

How do cells control the lac operon?

Answer 29

By responding to the amount of lactose AND glucose in a cell

Question 30

What is the function of the genes on the lac operon?

Answer 30

Lac Z (Beta-galactosidase): breaks down lactose
Lac Y (Permease): allows lactose to get in the cell
Lac A (Transacetylase): promotes lactose break down

Question 31

What happens when there is NO lactose in a cell?

Answer 31

Turn the operon OFF
- The LacI gene codes for the LacI repressor which binds to the operator blocking RNA polymerase from transcribing the operon (no transcription happens)

Question 32

What happens when there is HIGH lactose in a cell?

Answer 32

Turn the operon ON
- The LacI gene codes for LacI repressor but it cannot bind to the operator because allolactose binds to the repressor deactivating it. Therefore, RNA polymerase can transcribe the operon

Question 33

Which results in greater expression: high glucose or low glucose?

Answer 33

LOW GLUCOSE
- When glucose levels are low, cAMP levels are high which then bind to the catabolite activator protein (CAP), allowing the CAP to bind to the CAP binding site, which facilitates transcription by helping RNA polymerase attach to the promoter.

Question 34

What is combinatorial control?

Answer 34

Promoter and regulatory elements can act like boolean logic gates to control gene expression

Question 35

What happens when glucose is HIGH and lactose is HIGH?

Answer 35

There is a LOW level of transcription

• There is some transcription since lactose is present to make the repressor inactive (allolactose) and allow RNA polymerase to transcribe the operon. However, since glucose is also present, there is no cAMP to bind to the CAP, so the CAP will not be able to bind to the CAP binding site and help facilitate transcription.

Question 36

What happens when glucose is HIGH and lactose is LOW?

Answer 36

There is NO level of transcription

• Since there is no lactose, the repressor remains active preventing the RNA polymerase from transcribing the gene.
• Whether glucose is high or low in this case does not make a difference because transcription cannot be facilitated if it is not happening in the first place.

Question 37

What happens when glucose is LOW and lactose is LOW?

Answer 37

There is NO level of transcription

• Since there is no lactose, the repressor remains active preventing the RNA polymerase from transcribing the gene.
• Whether glucose is high or low in this case does not make a difference because transcription cannot be facilitated if it is not happening in the first place.

Question 38

What happens when glucose is LOW and lactose is HIGH?

Answer 38

There is a HIGH level of transcription

• Since lactose is present, transcription will occur because the allolactose will make the repressor inactive allowing for RNA polymerase to transcribe the gene. Also, since glucose is low, cAMP will be produced. And thus it will bind to the CAP allowing the CAP to bind to the CAP binding site which will help RNA polymerase better attach to the promoter.