what are the 2 types of negative and positive regulation?
negative regulation - turn gene on via removing a repressor
- addition of ligand to turn gene on (removing repressor)
- removal of ligand to turn gene on (removing repressor)
positive regulation - turning gene off via removal of activator
- addition of ligand to turn gene off (removing activator)
- removal of ligand to turn gene off (removing activator)
what is negative regulation for prokaryotic gene regulation
Competition between RNA
polymerase and repressor protein for promoter binding
what is positive regulation for prokaryotic gene regulation
Activator protein recruits RNA
polymerase to the promoter to activate transcription
where are gene regulatory elements found
- regulatory elements are found close to the transcriptional start site of prokaryotic genes
BUT regulatory elements can also be found:
* Far upstream of gene
* Downstream of gene (eukaryotes)
* Within gene (introns; eukaryotes)
- these elements are found far from the transcriptional start site but can still influence transcription
how does the NtrC protein - transcriptional activator activate transcription
- NtrC protein is a transcriptional activator found downstream of the gene
- DNA looping allows NtrC to directly interact with RNA polymerase to activate transcription even from a distance
what is bacteriophage lambda
it is a virus that infects bacterial cells
- the lambda virus attaches to the host cell and injects the lambda dna
- lambda dna circularizes
- can exists in one of two stages of bacteria
- the Positive and negative regulatory mechanisms work together to regulate
the lifestyles of bacteriophage lambda
bacteriophage lambda can exists as one of two states in bacteria: explain the two
- under favourable bacterial growth conditions
- under damaged host cell conditions
- under favourable bacterial growth conditions
- integration of lambda dna into the host chromosome
- the dna joins the host chromosome continuously
- integrated lambda dna replicates along the host chromosome
- prophage pathway - when the host cell is damaged
- from bacterial outcome one (prophase pathway) an induction event may occur (host response to dna damage)
- synthesis of viral proteins needed for the formation of new viruses
- rapid replication of lambda dna and its packaging into complete viruses
- cell lysis releases a large number of new viruses
-lytic pathway
Two gene regulatory proteins are responsible for initiating this switch and actually repress each other’s synthesis…the lambda repressor protein (cl) and lambda cro protein
what are the two gene regulatory proteins that are responsible for initiating the switch between prophage and lytic pathways
- Lambda repressor protein from prophage state (cI) and lambda Cro protein from lytic state
They repress each other’s synthesis, giving rise to the two stable states
explain the molecular biology that occurs in prophage state 1 with its gene regelatory protein
slide 11 DIAGRAM
- lambda repressor is made by a cl gene
Lambda repressor occupies the operator next to the same gene its made from:
* blocks synthesis of Cro
* activates its own synthesis (loop)
* most bacteriophage DNA not transcribed
explain the molecular biology that occurs in lytic state 1 with its gene regulatory protein
slide 12 DIAGRAM
- lambda Cro protein is made by a Cro gene
Cro occupies the operator next to the same gene its made from:
* blocks synthesis of lambda repressor
* allows its own synthesis (loop)
* most bacteriophage DNA is
extensively transcribed
DNA is replicated, packaged, new bacteriophage released by host cell lysis
What triggers the switch between prophage and lytic states in bacteriophage lambda?
what is the prophage-lytic control an example of?
It’s host response to DNA damage!
An induction event occurs only when the host/DNA is damaged.
➢ DNA damaged? switch to lytic state inactivates lambda repressor
Under good growth conditions (ie. DNA is not damaged), lambda repressor protein blocks Cro and activates itself in a positive feedback loop
➢ maintains prophage state
the prophage-lytic circuit is an example of a transcriptional circuit – other types of circuits controls various biological processes
what are 4 types of transcriptional circuits? list examples, and how it is conducted
- positive feedback loop - ex. lambda repressor protein - production of protein A from gene A. positive loop so it effects transcription of itself (making A protein)
- negative feedback loop - production of protein A from gene A. negative loop so it effects transcription of itself (repressing A protein)
- flip-flop device (indirect positive feedback loop) ex. Cro repressor switch - a protein is made from gene a, and inhibits DNA B. B gene is made and inhibits DNA A. But these CANNOT work at the same time since repression is occuring.
- feed forward loop - (ex. the repressilator) A protein is made, promotes DNA B and DNA Z. B protein is made and ALSO promotes DNA Z. Z protein is made (requires signals from both A and B)
- Positive feedback loops
how are positive feedback loops used to create cell memory
- there is a parent cell which has gene A but never creates protein A because it is required for the transcription of its own gene
- then there is an initial transient signal which turns on the expression of gene A
- this results in the A to produce more and more A as the positive feedback loop acts on itself
- now when this cell divides, every daughter cell will be turned on to produce more and mor A via the positive feedback loop
- this is always because of that one initial signal that was sent – cell memory is created forever because of one transient signal
what are feed forward loops? how do they measure the duration of a signal?
- a feed forward loop is based on sensitivity
- A protein is made, promotes DNA B and DNA Z. B protein is made and ALSO promotes DNA Z. Z protein is made (requires signals from both A and B)
- you need a prolonged input of B or A to stimulate the production of protein Z. if there is only a brief stimulus it will not cause protein Z to be produced.
what is synthetic biology?
- Combinations of regulatory
circuits combine in eukaryotic
cells to create exceedingly
complex regulatory networks - Scientists can construct
artificial circuits and examine
their behavior in cells. This is
called synthetic biology
what is the repressilator example from synthetic biology?
- what are the three proteins produced in the repressilator
- what did the authors predict what would happen if the flip-flop transcriptional circuit took place?
- where did they introduce this circuit to in order to test it?
scientists wanted to construct an artifical circuit for the sake of creating something (aka synthetic biology) and thus created a simple gene oscillator using a delayed negative feedback circuit
the three proteins produced:
A: Lac repressor
B: Tet repressor (response to antibiotic)
C: Lambda repressor
Predicted: delayed negative feedback gives rise to oscillations
Introduced this circuit into bacterial cells and
observed expression of the repressor genes
how does the repressilator work ?
slide 21 DIAGRAM
- A repressor protein is expressed
- A repressor protein binds to the A binding site on gene B, thus repressing the synthesis of B repressor protein
- Since nothing is bound to the repressor B binding cite on gene c, c repressor protein is produced
- c repressor protein loops back to gene A and represses A protein expression (note the A protein produced in step 1 is degraded by this time)
- since A protein repressor is not produced, nothing binds to repressor binding site A on gene B, thus protein repressor B is expressed
- B repressor protein binds to gene C, and represses C repressor protein synthesis
- repeat 1-4
its a loop!
did the repressilator gene circuit work as the researchers predicted?
- no
- the researchers predicted that the genes would oscillate perfectly
- in actuality, the genes somewhat oscillated but also increased with amplitude at time went on
- this was due to increasing bacterial growth in the system through time – delayed feedback loop
what is transcriptional attenuation
- In both prokaryotes and eukaryotes there can be a premature termination of transcription called transcription attenuation
- RNA adopts a structure that interferes with RNA polymerase so transcription may be stopped earlier
- Regulatory proteins can bind to RNA and interfere with attenuation
- Prokaryotes, plants and some fungi also use riboswitches to regulate
gene expression
what are riboswitches and the different types?
- Short RNA sequences that change conformation when bound by a small molecule
- different types: some riboswitches conduct transcription attenuation and some dont.
eg. prokaryotic riboswitch that regulates purine biosynthesis is a riboswitch which does regulate genes via transcription attenuation
what are pyrimidines and purines - review of hs bio
- what bases
- how many rings
pyrimidines
- U, C, T
“you see the pyramids”
- 1 ring
purines
- A, G
- 2 rings
explain how the prokaryotic riboswitch works to regulate purine biosynthesis
Low guanine levels
- Transcription of purine biosynthetic genes is on
High guanine levels
* Guanine binds riboswitch
* Riboswitch undergoes
conformational change which changes a portion of the RNA into a transcription terminator
* Causes RNA polymerase to
terminate transcription before it even gets to the AUG start codon (premature transcription termination)
* Transcription of purine
biosynthetic genes is off
