Lecture 2 - Prokaryotic Transcriptional Regulation Flashcards
Define the interactome
All the protein interactions occurring inside the cell.
Define the metabolome
All the metabolites occurring inside the cell.
Name two reasons why regulation of gene expression is important.
- Responses to extracellular stimuli (both multicellular and unicellular). - It needs to change depending on the environment in order to conserve energy 2. Defining cell types (multicellular organisms) -All have a different finger print, can compare transcriptomes to identify the disease.
Which enzyme is responsible for transcription? (DNA to RNA)
RNA polymerase
Describe what is happening in the picture below
DNA enters the active site where it is unwound, ribonucleoside triphosphates are added one by one to create a complementary strand of RNA.
What forms an RNA polymerase holoenzyme?
RNA polymerase + sigma factor
What is a promoter?
It is the region of DNA that indicates the transcription start site.
What is abortive initiation?
The initial transcription can start and stop over and over again.
When is the sigma factor released ?
When 10 nucleotides are synthesized.
What differs in transcription elongation?
It occurs without the sigma factor
Describe what is happening in the photo.
Transcriptional Regulation
The sigma factor binds to the promoter at the transcription start site forming an RNA polymerase holoenzyme. The RNA polymerase unwinds the DNA and transcription begins. Abortive initiation occurs until 10 nucloetides are synthesized and the sigma factor is released. The transcription elongation occurs and transcription is terminated.
How is gene expression regulated in prokaryotes and eukaryotes?
By gene regulartory proteins (transcription factors)
What do gene regulatory proteins (transcription factors) bind to?
Regulatory regions of DNA (cis elements)
Gene regulatory proteins that turn genes ON are called:
Positive regulators (activators)
Gene regulatory proteins that turn genes OFF are called?
Negative regulators (repressors)
-Eg. Trp operon
Describe E.coli
- unicellular prokaryote
- one chromosome of circular DNA
- many are transcriptionally regulated by food availability
- replicate rapidly (20 minute doubling time)
What is an operon?
Multiple genes that can be transcribed into a single RNA molecule.
Describe the tryptophan operon
- Five genes
- Transcription regulated by a single promoter
- Encodes enzymes for tryptophan biosynthesis
Describe what is happening in the image.
All the genes are being transcribed on the one RNA molecule. There is one promoter initiating the expression of all the genes. This process is only require if there is not enough tryptophan in the environment.
What are the two protein bound states of the Tryptophan (Trp) operon promoter?
1) Bound by RNA polymerase
- Trp gene expression in ON
2) Bound by a tryptophan repressor protein
- Trp gene expression is OFF
How does the tryptophan repressor protein turn OFF the Trp gene expression?
The protein binds to the operon (specific DNA sequence of the promoter). As a result RNA polumerase cannot bind to the promoter.
How is the Tryptophan repressor DNA binding activity regulated?
Two moleucles of tryptophan must bind to DNA
What allows from simple switch control of tryptophan biosysthesis?
Repressors and operators
What occurs when tryptophan is high?
The repressor binds to the operator, it turnsoff the TRP operon so that tryptophan is not produced.
What occurs when tryptophan is low?
The RNA polymerase can bind to the promoter and turn on Trp gene expression. The repressor becomes inactive.
Study this photo
OK
What kind of binding motif does the Tryptophan repressor have?
Helix-turn-helix DNA binding motif (most common)
What comprises the Helix-turn-helix DNa binding motif? Where does it bind?
It is made up of two alpha helices connected by a turn.
It binds in the major groove of DNA double helix.
In the functional form the tryptophan repressor cannot fit together due to the orientation. The binding induces what?
Conformational change and it ends up fitting in the major groove.
Describe the Lac operon
- three genes required for transport of lactose into the cell and catabolism
- enables use of lactose in the absence of glucose
- dual regulation : positive and negative control
What is the activator for the Lac operon?
Catabolite activator protein (CAP)
What promotes Lac expression?
Low glucose/high lactose. (Activator)
What is the repressor in the Lac operon?
Lac repressor protein
What inhibits Lac expression?
Low lactose (repressor)
Where does the Lac repressor bind?
At the operator
What is the job of the first gene of Lac operon?
It encodes Beta-galactosidase; breaks down lactose to glucose and galactose.
Why do lactose levels become low?
The Lac repression is sound to the operator.
What is required to increase lactose and allolactose?
Beta-galactosidase
What happens when the Allolactose binds to Lac repressor?
- Conformational change
- Decreases DNA-binding activity
- Release from the operator
Why is an activator required for Lac promoter?
- RNA polymerase binding is not enough for the Lac promoter
- For it to be efficient CAP it required to be bound
- CAP has a helix-turn-helix DNA binding domain
Why is CAP DNA binding activity activated by low glucose?
Decreasing glucose increases levels of cyclic AMP (cAMP, a signaling molecule). cAMP binds CAP protein.
What happens when cAMP binds CAP protein?
- Conformational change
- Increases DNA-binding activity
- Bonds to CAP-binding site
Cap in turn recruits RNA polymerase to the Lac promoter.
What are the conditions that the Lac operon will be expressed?
high lactose/ low glucose
