DNA: Protein Interaction Methods:
– DNase I Foot printing: This method can
be applied towards determining the location that a protein binds to DNA in
vitro. This is accomplished by DNA extraction, followed by PCR amplification
using primers targeting the DNA region of interest and labeling of one strand of the DNA with a radioactive Phosphate
using a kinase. The protein of interest and the DNase I restriction enzyme are
added to the DNA, which will digest the DNA into fragments. Portions of the DNA
that are bound by the protein of interest will be protected from fragmentation.
Gel electrophoresis will then reveal a distinct cleavage pattern (as compared
with naked control DNA) that has a banding gap or “footprint” revealing exactly
where the protein was bound to the DNA. Binding strength can additionally be
examined through this method.
-Chromatin Immunoprecipitation: This
method can be applied to determine in vivo protein-DNA binding locations. First
DNA-protein complexes are crosslinked, which can be done using formaldehyde, and
then sonicated to fragment the DNA into shorter segments. Antibodies for the
target protein are attached to beads and then magnets, washing, and
purification steps are used to isolate the complexes, remove the protein, and
then isolate the DNA. The DNA can be processed through PCR, qPCR, or sequencing
(ChIP-seq) to find the location and
sequence of the DNA in which the protein binds by examining peaks in the data sets.
-Gel Mobility Shift Assay: This method
can be applied to determine if proteins or protein complexes bind DNA in vitro.
First the DNA is isolated and labeled with an isotope or marker then run
through a gel to establish the control band. Target protein or additional
proteins suspected to complex are added to the DNA. Each additional protein
added will “Shift” the bands in the gel as each additional protein that
complexes the DNA will slow the sample’s run through the gel.
Protein: Protein interaction Methods:
-GST pull down: This method can be
applied to determine protein-protein interaction in vitro. GST is fused to a
target protein using plasmids and restriction enzymes. The GST fused protein is
amplified in a bacterial vector and a protein of interest is added. The GST-fused
protein is pulled down using a GST affinity resin or agarose beads, centrifuged,
washed, and eluted. A western blot using antibodies against the GST-fused
protein will reveal changes in banding if the protein of interest is bound vs
GST protein alone. Additionally the
-Co-immunoprecipitation: This method can
be applied to detect protein-protein interaction in vivo. Cell lysate is added
to magnetic beads with antibodies against a protein that one wants to find
binding partners, centrifuged, washed, and eluted. A western blot is then
performed using antibodies against the protein and compared to the size of the
protein alone. Gel excision and Mass spec can be used to find the precise
identity of binding partners. The reverse western can be performed using
antibodies against the protein’s binding partner to confirm the original
binding results. Additionally, changes in binding under various treatment
conditions can be confirmed using Co-IP.
-Yeast 2-Hybrid System: This method can
be applied to identify protein-protein interaction in vitro. Three plasmids are introduced into
yeast cells, one plasmid has a gene for a protein inserted next to the DNA binding
domain from a known Transcription Factor. The second plasmid has a gene for a
different protein inserted next to the transcription-activating domain of the same
Transcription Factor, and the third has a promoter region linked to a reporter
gene such as lacZ.
-Protein microchip: This method can be
applied to identify protein-protein (or ligands) interaction in vitro. The chip has proteins bound to its
surface. The proteins can be selected for the specific experiment, or a
proteome for the organism can be used. Fluorescently labeled proteins (or
ligands) of interest are added to the chip (biotinylation or antibodies can
also be used as detection methods). Interaction between the surface bound
proteins and the added samples will fluoresce and be measured by a reader.
2. (4 points): How
would you distinguish I-, I-d, I-s and Oc mutants
by genetic experiments in the lac operon system?
Using a merodiploid system, insertion
of plasmid with desired mutations into E.coli, the wild type gene (WT), and
isopropylthiogalactoside (IPTG); an inducer that acts like allolactose.
I-: Is a
mutation in the repressor so that it cannot bind other repressors; however,
the wild type repressors still bind operators and therefore the mutation is
recessive and Lac operon target genes would be expressed only in the presence of inducer, but could still be
distinguished from WT, as WT should produce protein product at twice the rate
as I- due to WT having 2 copies of Lac genes.
I-s: Is a
mutation in the repressor where it can bind wild type repressors in
tetramer and shut off the operon by binding the operator, but the inducer
cannot bind and therefore both copies of the operon target genes are therefore
always off in all situations, or constitutive
I-d: Is a
mutation in the repressor where it can bind wild type repressors in
tetramer but cannot recognize the operator which means that the operon target
genes on both copies will be constitutively
active and not repressible.
Oc: Is a
mutation in the operator sequence and therefore
one copy of the operon target genes will always be active. Oc
is cis dominant, however, I-d is dominant for both DNA
strands in the merodiploid and therefore should produce protein product at
twice the rate as the Oc mutant.
3. (4 points): Use
riboswitch concept to design a new Trp sensor in the attenuation mechanism
that DOES NOT involve
TrpL (the leader that encodes a small peptide); explain how your sensor would
of using the speed of the ribosome as a proxy sensor for Trp (as the Trp leader
does), one could design an aptamer that would directly bind Trp in an
orientation that would cause the transcribed RNA to form a terminator hairpin,
whereas in the absence of Trp the RNA would have an anti-terminator in its
unbound configuration. Thus Trp levels would be more directly responsible for
termination at higher Trp concentrations.
4. (3 points) In the
control of lambda phage fate, what would happen in terms of lambda repressor
concentration and lambda phage fate if you replace OR3
with OR1 sequence?
CI lambda repressor protein binds OR3 to block the transcription of
CI by blocking polymerase transcribing from the PRM site, but only
when CI concentrations are very high (due to a weak binding affinity). OR1
on the other hand has a strong binding affinity for CI and would therefore shut
down transcription of CI at low concentrations of CI. This negative feedback
loop causing the low concentration of CI would thus lead to a loss of lysogenic
maintenance (as higher CI is needed to block cro) and cause an entry into Lytic