Term
| 2 ways to determine which proteins are present in a sample/cell/tissue/organ etc.? under what conditions? how much of each protein is present? |
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Definition
1. epitope tagging (1 protein/gene at a time) --> western blot
2. all proteins in a sample --> mass spectrometry |
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Term
| 3 kinds of "friends" a protein could have |
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Definition
1. close friend (one that actually binds to it)
2. a transient friend (one that comes by, does what it has to do, then leaves)
3. indirect friend (one that is not touching it, but is in a physical complex with it) |
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Term
| 4 examples of posttranslational modifications |
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Definition
1. phosphorylated
2. glycosylated
3. methylated
4. ubiquitylated |
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Term
| 3 things that give a good clues to protein function |
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Definition
1. location
2. friends
3. PTM |
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Term
| _______ tells you if your protein is present & you can use ______ to see if your RNA is present |
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Definition
| epitope tagging/mass spec; RNA-Seq |
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Term
| _______ tells you what is being translated (or not) at the time of harvesting the cells |
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Definition
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Term
| goal of ribosome profiling |
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Definition
| which mRNAs are being translated & how well (or how poorly) |
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Term
| steps to ribosome profiling |
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Definition
1. break cell open
2. load the extract on a gradient
3. spin it
4. RNAs that are light (no ribosomes) will be at top, RNAs with a few ribosomes will be in the middle, RNAs with a lot of ribosomes will be at bottom
5. extract layers of the gradient
6. measure optical density
7. collect fractions
8. treat with nuclease (enzyme that degrades RNA that isn't blocked by some molecule being bound to it)
9. extract protected RNA
10. make cDNA copies from RNA
11. sequence it
12. compare the sequence to that of the genome to see where the sequence came from (which genes)
13. tells you which sequence's RNA is protected by ribosomes (i.e. being translated) |
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Term
| ribosomes will deny ______ access to RNA to which they are bound (the ones being translated) |
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Definition
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Term
| the ratio of reads from ribosome profiling data to nucleotide reads from RNA Seq gives you ______ |
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Definition
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Term
| what does it mean that ribosome profiling is quantitative? |
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Definition
| you can learn how many RNAs are being profiled & how well |
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Term
| what does it mean that ribosome profiling is positional? |
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Definition
| you learn where exactly on the RNA the ribosome is |
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Term
| should ribosome profiling include 5' or 3' UTRs? |
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Definition
| no; something funky is happening if it does |
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Term
| what could cause a build up of ribosomes at one point in the RNA? |
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Definition
| ribosomes can hit a barrier as they go down the mRNA |
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Term
| when you sequence the RNA in ribosome profiling, it is pretty much always _____ nt |
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Definition
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Term
| can you see if there is +1 or -1 frame shifting with ribosome profiling? |
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Definition
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Term
| what would it mean if you see that ribosomes have read through a stop codon? |
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Definition
| sometimes they ignore there (either on purpose to make a longer protein or by accident) |
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Term
| can ribosomes use a non-canonical start codon? |
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Definition
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Term
| what would it look like on a ribosome profiling read-out if there is a uORF (upstream open reading frame)? |
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Definition
| a little blip on your read-out before the normal ORF in your plot of RNA |
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Term
| what would it mean if a longer RNA in one condition has upstream uORF that is lacking in shorter RNA in the other condition; differential transcription would allow different translation of protein (maybe only shorter RNA translates the protein)? |
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Definition
| you would think that uORFs are sequestering ribosomes & preventing them from going downstream until the right condition is met |
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Term
| how would you figure out where a protein is in the cell (not as good way)? |
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Definition
| you need an antibody that detects the protein & can be seen with a microscope (fluorescence!) |
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Term
| problem with this protocol: fix the cell --> put in antibody that is fluorescent --> see where proteins are located under the microscope |
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Definition
1. requires fixing the cell with a chemical though, so that could affect cell function
2. what if where the antibody connects to the epitope is in the middle of a protein complex & therefore inaccessible? |
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Term
| best way to figure out where a protein is in the cell |
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Definition
| insert GFP into frame onto C-terminus of RNA (right before STOP codon) |
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Term
| 5 steps for determining proteome "friends" |
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Definition
1. have an antibody for your protein
2. break cell open
3. add antibody
4. what can you immunoprecipitate?
5. see what proteins are there by mass spec (if no candidates for friends) or western blot (if you have candidates & antibodies for them too) |
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Term
| how do you tell a close friend vs an indirect friend? |
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Definition
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