BCH5413 ACTUAL EXAM 5 || NEWEST EXAM WITH
COMPLETE QUESTIONS AND CORRECT DETAILED
ANSWERS\VERIFIED 100% ALREADY GRADEDC A+
Briefly describe how the S1 nuclease protection assay works
AND describe what it shows regarding the function of TATA
box in a eukaryotic promoter.
In the S1 nuclease protection assay, hybridize the RNA that is being
produced with a DNA probe labeled on 5' end. The probe
needs to be hybridized close to where you think the
transcription start site might be.
Also, the probe need to be longer than where you think the
transcription start site should be. Then, you will treat the sample
with S1 nuclease. The probe will be degraded to the point
where hybridization to the RNA begins. The RNA will also be
degraded down to the
beginning of the probe. Then, you denature the RNA, now
you're only left with the probe, which is the transcription start
site. You will run the probe using gel electrophoresis to
determine its size.
When the TATA sequence is deleted, the RNAP doesn't know
where to start, so you will have multiple transcription sites.
,What is an enhancer? Briefly describe how a research might
investigate whether a transcription element is an enhancer?
An enhancer is a tissue-specific, position- and orientation-
independent DNA element that stimulates transcription. To
investigate if a
transcription element is an enhancer, researchers can delete
areas within potential enhancer regions then radiolabel amino
acids so that proteins will be labeled. When an
immunoprecipitation is done and run on a gel, the area where
there is a less protein is the enhancer region.
This is because when the enhancer is not present (because the it
was
deleted), less transcription and subsequently less translation takes
place and less protein is produced.
,Describe the method by which investigators can determine what
sequences in DNA are necessary for the binding of transcription
factors.
To determine where transcription factors will bind in the DNA
sequence, investigators can utilize the method of DNA
Footprinting. From our
earlier lectures regarding DNA Footprinting, we know that protein
bound DNA is generally protected from digestion from DNase
(enzyme that catalyzes the cleavage of the phosphodiester
bonds in DNA). As such, when DNA sequences are ran on gel
electrophoresis, protein
bound DNA will leave a footprint (no bands present on
autography). For the experiment, investigators started with
radioactively labeling DNA sequences they believed to contain
transcription factor binding sites. They next incubated the DNA
with protein (transcription factors) and without (control) and
subjected the DNA to limited DNase I digestion to create DNA
fragments. Lastly, DNA fragments are
separated by size using gel electrophoresis.
During PIC formation, how does TBP bin and carry out its
function if a TATA box is present?
TBP binds the TATA box brings the rest of TFIIB and all its
associated with. If some subunits are taken away, the two TAFs
can still bind to other elements next to the TATA box to help
stabilize and recruit other to the promoter.
, During PIC formation, how does TBP bin and carry out its
function if an Inr and DPE are present with NO TATA box?
If TBP is associated with all of its TAFs, then TBP can make
contact with the initiator element and other TAFs that are able to
bind to the initiator and the downstream promoter. That is
enough binding to start recruiting other elements for
transcription.
During PIC formation, how does TBP bin and carry out its function
if GC boxes are present without a TATA box?
The GC can bind the protein Sp1 and TFIID can bind due to the TAF
#4 associating with the Sp1. This association is enough to hold the
rest of
TFIID in place and allows other proteins to bind and begin
transcription.
What are the two domains of TFIIB AND how to they assist in
determining the position and direction of transcription?
TFIIBn and TFFBc are the two domains. TFIIBn binds to TPB and DNA
while TFIIBc binds to the RNA polymerase. These two domains give TFIIB a shape
and configuration that can wrap around and bind to the DNA
and still reach and bind to RNA polymerase to put it into the correct position. This
allows TFIIB to function as a bridge between TBP and RNA polymerase to ensure it
is positioned correctly and moves in the right
direction. IF TFIIB is absent, RNA polymerase could begin transcription in either
direction.
COMPLETE QUESTIONS AND CORRECT DETAILED
ANSWERS\VERIFIED 100% ALREADY GRADEDC A+
Briefly describe how the S1 nuclease protection assay works
AND describe what it shows regarding the function of TATA
box in a eukaryotic promoter.
In the S1 nuclease protection assay, hybridize the RNA that is being
produced with a DNA probe labeled on 5' end. The probe
needs to be hybridized close to where you think the
transcription start site might be.
Also, the probe need to be longer than where you think the
transcription start site should be. Then, you will treat the sample
with S1 nuclease. The probe will be degraded to the point
where hybridization to the RNA begins. The RNA will also be
degraded down to the
beginning of the probe. Then, you denature the RNA, now
you're only left with the probe, which is the transcription start
site. You will run the probe using gel electrophoresis to
determine its size.
When the TATA sequence is deleted, the RNAP doesn't know
where to start, so you will have multiple transcription sites.
,What is an enhancer? Briefly describe how a research might
investigate whether a transcription element is an enhancer?
An enhancer is a tissue-specific, position- and orientation-
independent DNA element that stimulates transcription. To
investigate if a
transcription element is an enhancer, researchers can delete
areas within potential enhancer regions then radiolabel amino
acids so that proteins will be labeled. When an
immunoprecipitation is done and run on a gel, the area where
there is a less protein is the enhancer region.
This is because when the enhancer is not present (because the it
was
deleted), less transcription and subsequently less translation takes
place and less protein is produced.
,Describe the method by which investigators can determine what
sequences in DNA are necessary for the binding of transcription
factors.
To determine where transcription factors will bind in the DNA
sequence, investigators can utilize the method of DNA
Footprinting. From our
earlier lectures regarding DNA Footprinting, we know that protein
bound DNA is generally protected from digestion from DNase
(enzyme that catalyzes the cleavage of the phosphodiester
bonds in DNA). As such, when DNA sequences are ran on gel
electrophoresis, protein
bound DNA will leave a footprint (no bands present on
autography). For the experiment, investigators started with
radioactively labeling DNA sequences they believed to contain
transcription factor binding sites. They next incubated the DNA
with protein (transcription factors) and without (control) and
subjected the DNA to limited DNase I digestion to create DNA
fragments. Lastly, DNA fragments are
separated by size using gel electrophoresis.
During PIC formation, how does TBP bin and carry out its
function if a TATA box is present?
TBP binds the TATA box brings the rest of TFIIB and all its
associated with. If some subunits are taken away, the two TAFs
can still bind to other elements next to the TATA box to help
stabilize and recruit other to the promoter.
, During PIC formation, how does TBP bin and carry out its
function if an Inr and DPE are present with NO TATA box?
If TBP is associated with all of its TAFs, then TBP can make
contact with the initiator element and other TAFs that are able to
bind to the initiator and the downstream promoter. That is
enough binding to start recruiting other elements for
transcription.
During PIC formation, how does TBP bin and carry out its function
if GC boxes are present without a TATA box?
The GC can bind the protein Sp1 and TFIID can bind due to the TAF
#4 associating with the Sp1. This association is enough to hold the
rest of
TFIID in place and allows other proteins to bind and begin
transcription.
What are the two domains of TFIIB AND how to they assist in
determining the position and direction of transcription?
TFIIBn and TFFBc are the two domains. TFIIBn binds to TPB and DNA
while TFIIBc binds to the RNA polymerase. These two domains give TFIIB a shape
and configuration that can wrap around and bind to the DNA
and still reach and bind to RNA polymerase to put it into the correct position. This
allows TFIIB to function as a bridge between TBP and RNA polymerase to ensure it
is positioned correctly and moves in the right
direction. IF TFIIB is absent, RNA polymerase could begin transcription in either
direction.
