No glucose, high lactose

Negative control
Lac I will produce the repressor and lactose will come in an bind to the repressor
changing its shape through allosteric regulation. The repressor will release itself from the
operator allowing rna pol to bind to the promotor initiating transcription. Transcription
rate is slow. Lac z will produce B-gal, lac y will produce permease, and lac a produces
transacetylase.
ositive control
The presence of low glucose will activate !" which converts !T into c!#. c!# will
bind to the "! binding protein and change its shape through allosteric regulation during
post translational. The "! protein is now activated and binds to its $N! element on the
"! binding site upstream from the transcription %& site. "! will interact will 'N! pol
through protein protein intereaction to speed up transcription.
(verall ) fast
High glucose no lactose
Negative control
*ith no lactose present, lac I will synthesize the repressor which binds to the operator.
+ince there are no glucose to change the shape of the operator, the repressor will prevent
'N! pol from binding to the promoter to start transcription. There will be no
transcription at all.
Positive control
*ith the presence of high glucose, !" will remain inactive and cannot convert !T to
c!# to change the shape of "!. "! will remain inactive and not bind to the "!
binding site. to interact with 'N! in order to speed up transcription. Transcription will be
slow.
(verall, +low transcription
Extracting mRNA
m'N! contains the -.poly-!!! tail so we need to add oligo dT primers containing biotin
which have the -TTT tail bonded to it. Biotin will bind the m'N! poly !!! tail then we
also need to add streptividin can lin/ to biotin through a strong covalent bond.
+treptividin will also bind to magnetic particles in order to pull the m'N! out. Then a
high salt solution is added to bind the m'N! to the column while the remaining of
t'N!, rN! gets pushed down. *ater is then added to elute the m'N! out of the column
and completely isolated.
t'N! in vitro
$N! strand
ribonucleosome 0!1"23
'N! pol.
template --4
(nly t'N! can be visible on a get at 56s and &7s 4/b and 5/b
m'N! hypothesis "rac/ing the genetic code
add 58 a.a., cell e9tract, ribosomes to find out which amino acid will bind to which codon
Chromatin remodeling
In a non condensed chromatin, the :$!" is recruited in order to add a positive charged
to the :& histone. The :& histone is bounded to 6 positively charged histone and can
attract the negatively charged $N!. The :& protein will wrap around the 6 histones then
the lin/er $N! wraps around all of it. $N! wrapping around the histones are called
nucleosome and the wrapping of a bunch of nucleosomes are called scaffold protein.
Normal Eukaryote transcription
In an opened chromatin, 'N! pol. will interact with the TBT through protein protein
interaction bounded to the T!T! bo9 located on the basal promoter containing $N!
element for that gene to start transcription. The base promoter is located downstream of
the pro9imal promoter and upstream of the transcription start site.
Enhancer based transcription
In an opened chromatin, the T; will bind to enhancer region on introns, 4. 1T', -.1T',
and pro9imal promoter. The enhancers can also have a flipped orientation upon the
binding of T;. +ince enhacers are far away from the pro9imal promoter, $N! will loop
around itself allowing the T; bonded to a co-activator through protein-protein interaction
to lin/ with the TB bounded on the T!T! bo9 located on the basal promoter. The basal
promoter contain $N! element for that gene. 'N! pol. will then interact with the TB
on the basal promoter through protein protein interaction to speed up transcription. The
transcription will be fast.
RNA interference
;irst an unsual 'N! product is being transcribed and forms a loop around itself. !n
enzyme will come in an cut the hairpin $N! off then $I"<' enzyme cuts the $N! into
55 nucleotides which will be /nown now as si'N!. The 'I+" protein will now come in
and bind to the single stranded si'N! to process if the there is a match or not. If there is
a &88= match, the 'I+" will cut the protein in two, if there are no match, the 'I+" will
prevent that $N! from being transcribed.
Replica plating
Treatment &, glucose only
Treatrment 5, glucose % galactose
Treatment -, Lactose only
'esult B-gal is produced only in treatment - because it doesn.t produce B-gal in the
presence of lactose.
2lucose prevents the e9pression of the gene for B-gal.
dentifying gene under regulatory control
&. 2enerate large number of mutatns through radiation
5. +creen mutatns to find individual with defects in the process or biochemical pathway
-. +pread bacteria on the master plate with glucose
7. 1se a velvet to get a replica of the plate with glucose.
4. ress that velvet into a plate with only lactose.
>. "ells that can use lactose grow into a colony and cells that are not on the replica plate
cannot metabolize lactose.
ndicator plating
resence of B-2al ) yellow
"olonies defect in B-gal ) white
!itamin " path#ay
;irst the vitamin $ hormone is secrete and travels inside the nucleus because it is lipid
soluble. Then it will bind to ?$' that will either homodimerize with itself ?$' or
heterodimerize with '$@. Then it will bind to ?$'< which acts as a T; to bind to the
co-activator on $N! element and interact with 'N! pol. through protein protein interact
to ma/e the protein need for bone repair.
$ipid soluble steroid hormone
! steroid hormone will enter the cell and bind to its receptor on the cytoplasm. The
hormone bound to the receptor will act as a T; to access to nucleus then bind to its $N!
element to activate transcription.
$ipid insoluble hormone
! hormone will bind to its receptor on the transmembrane changing the shape of the
receptor. The receptor then enters the cytoplasm and gets amplified into many more
receptors that can enter the nucleus and bind to $N! element.
%&Protein path#ay'En(yme linked
! hormone will bind to its receptor on the transmembrane causing the receptor to change
shape. 1pon the conformation change, the receptor will stimulate 2$ to be converted
into 2T 0alpha, beta, gamma3. 2T alpha will unbind from beta and gamma then go
activate the transmembrane protein !". !" will convert !T into c!# which will act
as a second messenger to bind into the regulatory part of A!. The regulatory unbinds
from the catalytic and the catalytic can enter the nucleus to phosphorylate "'<B. *hen
"'<B gets phosphorylated, it will activate specific T; to illicit transcription.
R)* receptor path#ay
;irst a hormone binds to 'TA receptor. The receptor will change shape and
phosphorylate itself to form a dimer with another 'TA receptor. The dimer will
phosphorylate itself to activate +(+ bridge protein to lin/ to '!+ which is on the
peripheral part of the membrane. '!+ converts 2$ into 2T which will cause a
phosphorylation cascade of other protein in order to enter to nucleus to activate the T;.