Lab 1 - Gene regulation part 1 Flashcards
Why do cells need to regulate the expression of their genes
external environmental conditions, developmental cues, in response to hormone signals, etc
Most common type of gene regulation in bacteria
transcriptional regulation
when does transcriptional regulation occur
during the first stage of gene expression, before significant amounts of mRNA are synthesized.
what does the ara operon contain
genes that encode proteins required to metabolize a sugar - L arabinose
what happens when L arabinose is present in bacterial growth mediums
the genes in the ara operon may be transcribed at high levels
What happens when glucose is present in bacterial growth medium
there is little transcription of the ara operon genes because glucose is more easily metabolized
pGLO plasmid
contains reporter gene that produces an easily visualized gene product
what do arrows in the plasmid map represent
open reading frames or genes
components in the plasmid map
araC, ori, bla, GFP, Pbad (not a gene but ara operon promoter)
ori
origin of replication for plasmid
bla gene
codes for a protein that makes the bacterial cell resistant to antibiotic ampicillin
GFP gene
codes for a green fluroescent protein, when cells are exposed to UV light the GFP emits bright green fluorescence.
Pbad
ara operon promoter
what does Pbad do under normal conditions
regulates the transcription of genes in the ara operon (responsible for the metabolism or arabinose)
in pGLO what relaces the regulatory genes (Pbad)
replaced by GFP gene
what is GFP controlled by in this experiment and what is it normally controlled by
GFP ormally has its own eukaryotic promoter but in this case it is controlled bu the bacterial promoter Pbad.
what happens when there is increased activity from the Pbad promoter
GFP will be produced
ara C protein
allosteric regulatory protein with 2 bidning sites. When arabinose is present in the environment AraCi binds to the sugar. this makes AraCi to AraC that permits recognition and binding to aral.
it is hypothesized that araC facilitates RNA pol binding to the Pbad promoter.
aral
activator sequence located just upstream of the Pbad promoter
what happens in the absense of arabinose
AraC is not activated and thus cannot bind to aral sequence
RNAP will not bind efficiently to Pbad sequence
very little transcription will occur (little GFP made)
How do we get the most efficient binding of RNAP to Pbad
two molecules ofAraC bind to aral sequence but CAP must bind to the cAMP-CAP binding site located just upstream of aral sequence.
2 forms of CAP
cAMP bound to CAP - active
cAMP not bound - inactive and cannot bind to CBS
what happens in the cell when theres high concentrations of glucose (in relation to CAP)
reduce synthesis of cAMP and thus CAP is less likely to be activated and bind to CBS sequence. Even in presence of arabinose, glucose will prevent formation of CAP.
what happens when there is arabinose and low levels of glucose
2 molecules of AraC will bind to the aral sequence, CAP will bind to CBS sequence and binding of RNAP to Pbad will efficient. transcription of GF will be high
