Lab 7 - Control of GFP gene expression

Question 1

What is an ‘operon’?

Answer 1

An operon is a region of DNA that consists of three key elements: a promoter, an operator and a set of related genes referred to as ‘structural genes’. Usually an operon includes 2-6 structural genes but can range from 1 to over 20

Question 2

Are genes within an operon under the control of same promotor or each have different promotor?

Answer 2

The genes within an operon are under the control of the same promoter and as such, are transcribed together when the promoter is turned ‘on’. Regulatory proteins bind to sequences within the operon allow for control of transcription.

Question 3

lac operon

Answer 3

The lac operon is a classic example of an inducible operon where the binding of a regulatory molecule increases gene transcription.

Question 4

trp operon

Answer 4

The trp operon is a repressible operon which means that gene transcription can be turned off or repressed by the binding of a regulatory molecule. It’s very important to note that different operons are regulated in different ways. That being the case, there are still some common elements

Question 5

What is the promotor?

Answer 5

A promoter is a section of DNA within the control region of the operon that RNA polymerase binds to. A promoter can be turned ‘on’ and ‘off’ by activators and repressors respectively in response to specific stimuli. A promoter is located upstream of the gene(s) it controls.

Question 6

Operator (O)

Answer 6

An operator is also a section within the control region of the operon. This section of DNA serves as a binding site for repressor proteins. The operator region overlaps with the promoter region such that when a repressor protein binds to an operator, it physically inhibits the binding of RNA polymerase. Depending on the operon, multiple operator regions may exist.

Question 7

Structural Genes

Answer 7

The structural genes are a series of related genes that encode proteins with a related function. For example, they could be individual subunits of a larger protein, or enzymes required for the metabolism of a particular catabolite such as lactose. These genes encode proteins that work together or towards a similar function within the cell. They are either all expressed or all turned off. They are not under individual control.

Question 8

Activator Binding Site

Answer 8

Activator binding sites or activator sites are specific region within the control region where activator proteins bind. These are located within or near a promoter. One common example of an activator site is the Catabolite Activator Protein (CAP) binding site.

Question 9

Regulatory Genes

Answer 9

Regulatory genes may be associated with an operon but are not technically part of it. They may be located in close proximity or in a completely different location. These genes are constitutively (constantly) transcribed to express repressor or activator proteins

Question 10

Control of Gene Expression

Answer 10

Gene expression can be controlled by regulatory molecules that interact with the control regions of the operon. This regulation can turn on (increase) or turn off (decrease) gene transcription. As mentioned above, an operon can be either inducible or repressible. These can each be positively or negatively regulated as outlined below.

Question 11

Negative Regulation

Answer 11

Negative regulation is modulated by a repressor protein. When bound to an operator site, a repressor protein inhibits the binding of RNA polymerase, effectively turning off the promoter. This results in a decrease in transcription.

Question 12

Negative regulation in inducible operons

Answer 12

In inducible operons, this is the default state. The repressor is constitutively expressed, bound to the operator and the expression is turned off. Repression stops when an inducer binds to the repressor and allows transcription to begin

Question 13

Negative regulation in a repressible operon

Answer 13

In a repressible operon, transcription remains active until a repressor protein binds to the operator Repression occurs when a co-repressor binds to a repressor protein, inducing a conformational change, allowing it to bind to the operator and prevent transcription. Until the co-repressor binds, a repressor protein is in an inactive state, unable to bind to the operator.

Question 14

Positive regulation

Answer 14

Positive regulation is modulated by activator proteins that bind to an activator binding site located within or within close proximity of a promoter region. When bound, activator proteins increase the efficiency of transcription and thus the amount of mRNA.

Question 15

Positive regulation for inducible operon

Answer 15

For an inducible operon, activator proteins remain unbound until an inducer molecule binds to the activator and increases its affinity for it’s specific binding site in the DNA

Question 16

Positive regulation for repressible operon

Answer 16

In a repressible operon, an activator protein is normally bound to DNA allowing gene transcription. Positive regulation of this system involves a signalling molecule that binds to the activator leading to its dissociation from DNA . This results in reduced transcription.

Question 17

Catabolite Repression

Answer 17

Many prokaryotes can use a number of different energy sources (catabolites) to generate ATP but need to express the relevant enzymes to do so. There is no point expressing all the enzymes for the catabolism of a particular energy source if it is not present.

Question 18

how is this controlled? How does a cell only transcribe the set of enzymes for the present or even preferred catabolite?

Answer 18

Catabolite repression. Some operons have a regulatory site known as a CAP-binding site that binds a protein called catabolite activator protein (CAP). When glucose is high, the levels of ATP in the cell are high. When glucose is low, ATP is converted to cAMP via an enzyme called adenylate cyclase and the cAMP levels in the cell rise. The specific mechanism for adenylate cyclase is beyond the scope of this unit but it relates to the way glucose is metabolised and transported within the cell. In the absence of glucose, cAMP binds to CAP. This complex then binds to a specific regulatory sequence in the operon known as the CAP-binding site, and increases the efficiency of transcription

Question 19

Under normal conditions, a repressible operon is turned on

true or false

Answer 19

True

Question 20

The binding of CAP to a CAP-binding site resulting in increased transcription is an example of:

Negative regulation
Positive regulation

Answer 20

Positive regulation

Question 21

Which of the following elements are common in a typical operon?

CAP binding site
Activator binding site
Structural genes
Promoter
Operator region

Answer 21

Structural genes
Promoter
Operator region

Question 22

If a regulatory protein binds to an operator and turns off gene expression, it is functioning as a(n):

Inducer
Co-repressor
Repressor
Activator

Answer 22

Repressor

Question 23

If a regulatory protein binds to an operon and activates gene expression, it is functioning as a(n):

Inducer
Activator
Repressor
Co-repressor
Check

Answer 23

Activator

Question 24

Under normal conditions, an inducible operon is turned on

True
False

Answer 24

False

Question 25

araC gene

Answer 25

Ara C The regulatory gene. This gene encodes the AraC protein that is involved in both repression and activation of the operon

Question 26

araO

Answer 26

This is the operator region for the arabinose operon. When the regulatory protein AraC binds here, gene expression is repressed

Question 27

CAP

Answer 27

This is the CAP protein binding site. When the CAP-cAMP complex binds here, it activates gene expression.

Question 28

araBAD

Answer 28

This is the promoter for the araBAD genes. When the operon is activated, RNA polymerase can bind at this points and gene transcription of the araBAD genes occurs.

Question 29

araBAD

Answer 29

These are the structural genes that encodes enzymes required for arabinose catabolism.