Where does translation always start? correct answers cytoplasm
Do proteins destined for the nucleus (eg TF) also get translated in the cytoplasm? correct answers Yes, they must exit the nucleus to get translated and then get transported back into the nucleus
What strategies do you think the c...
NROS 310 Exam 2 || with 100% Correct Answers.
Where does translation always start? correct answers cytoplasm
Do proteins destined for the nucleus (eg TF) also get translated in the cytoplasm? correct
answers Yes, they must exit the nucleus to get translated and then get transported back into the
nucleus
What strategies do you think the cell can use to get proteins to the right place within the cell?
correct answers - protein tagging
What mechanisms do cells use to transport proteins to the right place within the cell? correct
answers - motor proteins (Kinesin and Dynein) that can drag vesicles around
- myosin can drag things on actin filaments
What is protein tagging physically? correct answers - imbedded within the amino acid sequence
of every protein that is targeted to a particular place is a signal sequence that says where to go
- its just an amino acid sequence
Do protein tags always stay part of the protein? correct answers No, sometimes tag is very big
- once protein is in the right place, it might cleave the tag off in order to do its function
Is the sequence that allows import into the mitochondria cleaved or not? Why? correct answers
cleaved - very long
Is the sequence that allows import into the ER cleaved or not? Why? correct answers - depends
on protein
- sequence is series of hydrophobic amino acids, which becomes the transmembrane domain in a
membrane for some of the proteins
- some are cleaved
Describe the structure of the nucleus. correct answers - two membranes
- contains nuclear pores
- nuclear pores have nuclear pore complex that regulate what goes in and out of nuclear pore
Do signal sequences that drive proteins into the nucleus get cleaved off? correct answers - never
get cleaved off
- relatively small
Why is it important that signal sequences that drive proteins into the nucleus never get cleaved
off? correct answers - they don't always stay in the nucleus, a lot of proteins go back and forth
- also cell division: nuclear envelope breaks down, so nuclear proteins must get back into nucleus
when nuclear envelope reforms
- signal sequence is never cleaved off because nuclear proteins often have to get retargeted
,Design an experiment to test to see if a protein has a signal sequence that can target it to the
nucleus. correct answers - find candidate amino acid sequence and knock it out to see if that
sequence prevents it from target to nucleus
- mutate / tag it
- watch and see what happens
How can you find candidate amino acid sequence that can target protein to nucleus? correct
answers - chop it up into bits and see which ones go to the nucleus
- forward genetics screen
How do you determine whether a candidate protein went into the nucleus? correct answers - tag
it with GFP (alive cells, but this causes a mutation that could cause a change)
- immunohistochemisty: make antibody that is specific to that protein that has flourescent tag on
it (but has to be in a dead cell)
If you mutated a candidate amino acid destined for nucleus and it stays in the cytoplasm, what do
you conclude? correct answers - that amino acid was necessary for nuclear targeting (doesn't
mean it is the entire sequence, doesn't prove sufficient)
What experiment was done to determine if sequence is necessary for nuclear targeting? correct
answers - normal: visualized in nucleus
- mutate one amino acid in the sequence: protein is now everywhere in the cytoplasm and not in
the nucleus
Once you've identified amino acids that are necessary for nuclear targeting, how do you know
that you've found all of the sequence? What can you do to show that you have a functional signal
sequence? correct answers - gain of function: stick it in another protein and see if it is sufficient
for nuclear targeting
- if protein goes into nucleus, you have identified set of amino acids that is sufficient for nuclear
targeting
What is a problem you have to think about when determining if a protein is sufficient for nuclear
targeting? correct answers - there could be a another sequence on the protein that assists the
signal sequence, without which the sequence is not sufficient
What experiment addressed the problem:
- there could be a sequence on the target that assists the signal sequence, without which the
sequence is not sufficient correct answers - coated little gold beads with candidate sequence
amino acids
- that is enough for gold bead to enter the nucleus, without ANY OTHER amino acid interactions
Where do the signal sequences for nuclear targeting actually have to be? correct answers - must
be next to each other in folded protein, not unfolded protein
- this means they can be spaced along the unfolded protein.
- proteins often have signal PATCHES, not just signal sequences that come together when
protein is folded in the right way
, Why is it an advantage to have signal patches that come together only when they fold in the right
way? correct answers - regulation
- phosphorylation can change the folding of the protein, which can stop or start the nuclear
signals when the patches dissociate or come together
Design an experiment to show that a given signal sequence is both necessary and sufficient to
target a protein to the nucleus correct answers - identify candidate sequence
- knock it out (necessary)
- add it somewhere (sufficient)
What does it mean if your results show that a protein sequence is necessary but not sufficient?
correct answers - amino acid is part of a string and you have not identified the entire thing. It is
necessary because the sequence won't function on its own but if you add it somewhere alone it is
not sufficient
What does it mean if your results show that a protein sequence is sufficient but not necessary?
correct answers - redundant signal sequences that both do nuclear targeting
What are the origins of the mitochondria? correct answers - anaerobic cell derived from an
archaeon took up an aerobic bacterium that had bacterial outer membrane and bacteral plasma
membrane
- loss of membrane derived from archael cell
- result: mitochondria with double membrane from the bacteria
What is the structure of mitochondria? correct answers - 2 membranes, an outer and inner
- contains its own genomic DNA
What are the 4 destination targets for proteins going to the mitochondrion? correct answers 1.
outer membrane
2. inner membrane
3. mitochondrial matrix space
4. intermembrane space
Describe the first signal sequence discovered that brings proteins to the mitochondria? Where
does it go? correct answers - amphipathic (one part hydrophobic, one part hydrophilic)
- alpha helix
- goes to mitochondrial matrix
What is the problem with the amphipathic protein going to the mitochondrial matrix? correct
answers Hydrophobic part will not be stable in the cytoplasm of the cell as it is transported to the
mitochondria
Since hydrophobic part of amphipathic protein won't be stable in cytoplasm , what do you need?
What process is triggered? correct answers - a protein that mediates folding: chaperones
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