-
Transcription control is relatively ______.
slow
-
Post transcriptional regulation control is for _______ response to the environment quickly.
fast
-
Because _____ is already around, you can use post-transcription.
mRNA
-
Using post-transcriptional control can allow you to do what to things?
- You can change translation of mRNA
- Change the stability of mRNA
-
cis elements regulates..
mRNA
-
trans factors regulate...
disfusable factors (Protein/RNA)
-
If you have too little, or too much fe in the blood what happens?
death
-
Too much fe in the blood can cause ____ rxns.
redox
-
Fe that is in the blood is bound by...
transferrin
-
If cells need iron, what do they do?
take in iron by transferrin receptors (TFR) on cell membrane
-
When there is iron in the cell, we either do what two things with it?
- used
- stored in a protein called ferritin
-
High Iron means ____ TFR
low
-
High Iron means _____ ferritin
high
-
Low TFR means _____ ferritin
high
-
Low iron means _____ levels of TFR
high
-
Low iron means _____ levels of ferritin
low
-
High TFR means _____ levels of ferritin
low
-
The changes of iron levels in the bloodstream are ....
fast
-
The changes in iron had no effect on .....
ferritin mRNA levels
-
What was the experiment regarding regulation of ferritin levels?
- There was a polysome loading of ferritin mRNA
- One mRNA with mutiple ribosomes bound
- If The fe levels were down, the ferritin mRNA on polysomes were down
- If the fe levels were up, the ferritin mRNA on polysomes were up
-
Changes in mRNA increases ______ and decreases ______.
-
ferretin protein levels ARE NOT controlled by...
the abundance of mRNA
-
Ferritin protein levels are controlled by ...
cis elements and transfactors
-
Ferritin does what?
stores iron and releases it in a controlled fashion.
-
What are Transferrin receptors needed for?
to get iron into the cell
-
What are TFRs (transferrin receptors)
A carrier protein for transferrin
-
changes in transcription leads to changes in .....
RNA
-
What was the result of the Ferritin RNA abundance expeiriment?
Turned out it was the same with or without iron ( so RNA doesn't affect it)
-
When iron was present, the ferritin was ______, meaning lots of polysomal _________ were present, meaning that there was lots of ________.
- polysomal
- ribosomes
- translation
-
When iron was absent, the ferritin was ______, meaning no polysomal _________ were present, meaning that there was no ________.
- absent
- ribosomes
- translation
-
What effetcs where ferritin is translated?
Iron
-
What is IRE?
An Iron response element, An RNA sequence that is recognized by the iron regulatory protein.
-
What is IRP?
An iron regulatory protein, A translational regulatory protein that recognizes IRE that are found in specific mRNAs.
-
The IRP may inhibit ______ or stabilize the _______.
-
What is the 2nd experiment that can be used to help us find where an IRE is located?
reporter analysis
-
What is reporter analysis?
Common reporter w/ green flourescnt protein (GFP)
-
The reporter analysis is easy to assay because...
floureses bright green
-
What is the control of the reporter analysis experiment?
5'_GFP_UTR__[GFP]__GFP_UTR_3'
-
5'_GFP_UTR__[GFP]__GFP_UTR_3'
the "-" and "+" results for iron was?
- -Fe = Greenish
- +Fe = Greenish
-
5'_Ferritin__[GFP]__GFP_3'
the "-" and "+" results for iron was?
- -Fe = No Green
- +Fe = Very Green
-
The iron elements in 5' UTR of ferritin are very....
responsive
-
5'_GFP__[GFP]__Ferritin_3'
the "-" and "+" results for iron was?
- -Fe = Greenish
- +Fe = Greenish
-
Ferritin 5' UTR has the...
iron responsive element (IRE)
-
IRP is a ____ factor
trans
-
If you have +Fe, IRP ______ bind to RNA
can't
-
If you have -Fe, IRP ______ bind to RNA
does
-
When the IRP cannot bind to RNA what occurs?
translation
-
When +Fe, ferritin takes the _____up so the cell doesn't die when it takes in the excess iron.
TFR
-
The IRP binds______ and is released from the ____, translation occure
-
IRP blocks...
translation
-
When iron levels are low, you don't have _________, so you secrete whats left in the cells.
ferritin
-
RNA abundance is a balance between ....
transcription and RNA stability
-
Absent Fe = ____mRNA
lots of
-
Present Fe = ____ TFR mRNA
a little bit
-
Transcription of TFR is not Fe _________.
responsive
-
TFR mRNA stability is Fe ________.
responsive
-
If there is +Fe, the TFR mRNA is ______.
Unstable
-
if there is -Fe, the TFR mRNA is _____.
Stable
-
If the TFR mRNA is stable, it results in what?
more Fe being brought into the cells
-
When using TFR ans GFP to test for iron, what is the control?
5'_GFP__[GFP]__GFP_3'
-
5'_GFP__[GFP]__GFP_3'
the "-" and "+" results for iron was?
-
5'_TFR__[GFP]__GFP_3'
the "-" and "+" results for iron was?
-
5'_GFP__[GFP]__TFR_3'
the "-" and "+" results for iron was?
-
If you have no color in the reporter analyses, that means...
you have no iron
-
The TFR cis elements are....
IRE -> 3' UTR
-
Cis elements and IRE are the same, just in....
different places
-
If you have no IRP, the RNA is...
Not stable
-
If IRP binds, RNA is ...
stable
-
When IRP binds, it makes RNA stable resulting in making more....
TFR
-
-
When there is +Fe, _____ binds, Fe4-S4, but not RNA.
IRP
-
When there is -Fe, IRP _____Fe4-S4, So binds RNA
has no
-
Low iron levels affect these how?
IRP
____IRE__Ferretin
_TFR___IRE____
- No (Fe-S)4, binds IRE (RNA)
- No Translation
- Stabilizes RNA to make more translation
-
High iron levels affect these how?
IRP
____IRE__Ferretin_
TFR___IRE____
- Has (Fe-S)4, cannot bind RNA
- Translation occurs
- Destabilizes mRNA, makes less translation
-
If IRP binds IRE , the iron levels are _____ and the TFR mRNA is ________.
-
suppose a person is homozygous for a mutation in the IRP gene that changed the structure of the iron regulatory protein in such a way that it could not bind iron, but it could still bind to IREs. How would this mutation affect the regulation of ferritin?
This person would be unable to make ferritin, because the IRP would always be bound to the IRE.
-
suppose a person is homozygous for a mutation in the IRP gene that changed the structure of the iron regulatory protein in such a way that it could not bind iron, but it could still bind to IREs. How would this mutation affect the regulation of transferrin receptor mRNAs?
The amount of transferrin receptor mRNA would be high, even in the presence of high amounts of iron,because the IRP would always remain bound to the IRE and stabilize the transferrin receptor mRNA.
-
Suppose a person is homozygous for a mutation in the IRP gene that changed the structure of the iron regulatory protein in such a way that it could not bind iron, but it could still bind to IREs. Do you think such a person would need more iron in his or her diet than normal individuals?
Such a person would not have any problem taking up iron into his/her cells. In fact, this person would take up a lot of iron via the transferrin receptor, even when the iron concentrations were high.
-
Suppose a person is homozygous for a mutation in the IRP gene that changed the structure of the iron regulatory protein in such a way that it could not bind iron, but it could still bind to IREs. Do you think that excess iron in the diet would be more toxic than it would be for normal individuals?
Yes
-
Why would excess iron in the diet be very toxic?
- For two reasons. First, the person cannot make ferritin, which prevents the toxic buildup of iron in the cytosol.
- Second, when iron levels are high, the person would continue to synthesize the transferrin receptor,which functions in the uptake of iron.
-
What prevents the toxic buildup of iron in the cytosol?
ferritin
-
What are the disadvantages of mRNAs with a short half-life compared with mRNAs with a long half-life?
A disadvantage of mRNAs with a short half-life is that the cells probably waste a lot of energy making them
-
What are the advantages of mRNAs with a short half-life compared with mRNAs with a long half-life?
An advantage of short-lived mRNAs is that the cell can rapidly turn off protein synthesis.
-
eukaryotic primary transcripts are non-___________.
functional
-
mRNA is responsible for...
Coding, so that things can be translated.
-
-
Promoters are transcribed at the ...
interior promoters of tRNAs in RNAPIII (5s rRNA)
-
RNAP II consists of (specifically)
-
URNAs are U-rich RNAs that consists of what two components?
- snRNA (small nuclear)
- snoRNAs (small nucleolar)
-
snRNA (small nuclear) aids in...
mRNA splicing
-
snoRNAs (small nucleolar) aids in
rRNA modification
-
what is an miRNA
a "micro" RNA that is less than 25 nucleotides when mature
-
What are the three rRNA operons that lead to maturation.
-
What do RNA operons require for maturation?
cleavage and modification
-
What is gRNA?
RNAs that guide the insertion or deletion of uridine residues into mitochondrial mRNAs by RNA editing
-
RNA editing is....
Changes the sequence of the RNA (no longer matches the gene)
-
Lnc stands for...
Long non-coding RNA
-
Lnc RNA is when you have a ...
non-protein coding that is more than 200 nucleotides when mature
-
the five steps of mRNA processing
- 1) Synthesis of the 5' cap
- 2) Synthesis of the 3' poly-A tail
- 3) Splicing (removal of useless sequences//Spliced together for an ORF)
- 4) Base modification (methylation of some A)
- 5) RNA editing (sequence change of the RNA)
-
the largest subunit of RNAPII is....
The CTD
-
What does CTD stand for?
Carboxyl terminal domain
-
What is the CTD (carboxy terminal domain) consists of?
A capping enzyme, spliceosomal subunits, poly-A polymerase
-
What is the function of the CTD?
cappin enzyme, spliceosomal subunits and poly-A polymerase all bind to the CTD and that is how the RNA is recognized
-
The CTD aids in the recognition of?
the RNA
-
What is the heptapeptide?
A repeating sequence found in CTD sequence
-
What is the CTD heptapeptide sequence?
Tyr Ser(-OH) Pro Thr Ser(-OH) Pro Ser(-OH)
-
heptapeptide can be phosphorylated on the....
Ser OH group
-
The RNAII A is not phosphorylated on the....
CTD
-
What protects the RNA from being degraded by enzymes that degrade RNA from the 5' end?
The cap
-
What are the functions of the poly-A on the mRNA?
- 1) RNA stability
- 2) Enhances Translation
-
there is some _______ between stability and tail length
correlation
-
on a histone mRNA there is no...
Poly-A
-
The cis element in polyadenelyation is...
-
poly A polymerases are...
trans factors
-
what are the three steps of poly adeneylation?
- 1) Cleavage
- 2) addition of Poly A
- 3) termination
-
Polyadenylation does not happen with _______mRNAs
histone
-
Polyadenylation does not happen with histone mRNAs
because they dont have A2UA3
-
what are the 2 types of transfactors involved in splicing?
- SR proteins (Ser-Arg rich)
- snRNPs (small nuclear ribonuclear proteins)
-
What are the components of the snRNPs (small nuclear ribonuclear proteins)?
U1, U2, U4/U6, U5 and proteins
-
transfactors are necessary for specific ________.
splicing
-
The average size of exons in humans are...
about 150 nucleotides (small)
-
the size of an intron can be....
small to huge
-
What are the three steps to exon definition?
- 1) U1 snRNP binds to 5' splice site
- 2) U2 snRNP binds to 3' splice site
- 3) SR proteins bind to U1/U2 snRNPs and coat the exon, making it "defined"
-
When is an exon defined?
SR proteins bind to U1/U2 snRNPs and coat the exon
-
What are the steps to alternative splicing?
- 1) define exon
- 2) nucleophilic attack of 2' OH of branch point A on the 3' Phosphate of the 5' exon
-
A spliceosome consists of...
SR proteins, snRNPs
-
what holds RNA fragments together?
splicesosome
-
splicing is two...
transesterifications
-
What are two types of RNA editing?
- 1) Substitution editing
- 2) Insertion/Deletion
-
Subsititution editing is usually found in...
humans
-
Insertion/deletion editing is usually found in...
parasites
-
When apoliprotein B is edited by a single C -> U, what happens?
you get an early stop codon
-
Short apo B -> ______lipids (_________)
-
Short apo B -> _______lipids (________)
- import (liver)
- (non-edited)
-
What is alternative splicing?
In alternative splicing, variation occurs in the pattern of splicing, so the resulting mRNAs contain alternative combinations of exons.
-
What is its biological significance of alternative splicing?
The biological significance is that two or more different proteins can beproduced from a single gene.
-
After the intron (which is in a lariat configuration) is released during pre-mRNA splicing, a brief moment occurs before the two exons are connected to each other. Which snRNP(s) holds the exons in place so they can be covalently connected to each other?
U5
-
What are the four step to the splicing of pre-mRNA?
- 1) U1 binds to 5' splice site, U2 binds to branch site
- 2) U4/U6 and U5 trimer binds, intron loops out and exons and brought close together
- 3) 5' splice site is cut, 5' end of intron is connected to the A in the branch site to form a lariat while U1 and U4 are released
- 4) 3' splice site is cut, Exon are connected, The lariat intron is relased with U2, U5 and U6, intron will be degraded
-
Explain how you would use a poly-dT column to obtain a purified preparation of mRNA from eukaryotic cells. 7 steps
- 1) mRNA molecules bind to the column b/c they have a Poly A tail.
- 2) The A and T hydrogen bond
- 3) To purify, get a sample of cells and break them open by homogenization
- 4) remove large cellular structures by centrifuge (structures in pellet, RNA in supernatant)
- 5) pour high-salt supernatant over poly dT column
- 6) to break bonds pour low salt concentration over column, to get excess material off.
- 7) mRNAs will stay due to hydrogen bonding
|
|
