Term
| in general, how first determine whether translation is blocked? |
|
Definition
| you would ask whether ribosomes are associated with the mRNA |
|
|
Term
|
Definition
|
|
Term
|
Definition
| I've found the AUG, now we're getting started |
|
|
Term
|
Definition
|
|
Term
|
Definition
| when you reach the STOP codon, ribosomes come off & are recycled to translate another mRNA |
|
|
Term
| what does it mean to test the mechanism of regulation? |
|
Definition
| once you've decided which step is being affected, how is it being affected? |
|
|
Term
| 2 main groups of players in regulation of translation |
|
Definition
1. cis element(s) - sequences in mRNA
2. trans-acting factors |
|
|
Term
| trans-acting proteins or RNAs: how would you test if you have a candidate? or could it be anything? |
|
Definition
candidate - northern or western blot
anything is possible - RNA seq or mass spec |
|
|
Term
| 2 types of interactions that could cause regulation |
|
Definition
1. protein-protein
2. protein-RNA |
|
|
Term
| example of making a mutation to break an interaction & then assaying the step which it was affecting |
|
Definition
| mutate the part of the protein that would bind to the mRNA |
|
|
Term
| how would you get past a secondary structure in the mRNA that was blocking the ribosome? |
|
Definition
| use a helicase (enzyme!) to unwind it |
|
|
Term
| how to tell if translation is being regulated |
|
Definition
| is your mRNA bound to ribosomes or not? |
|
|
Term
| if attachment is blocked: _______ |
|
Definition
| ribosome would never associate with mRNA |
|
|
Term
| if scanning is blocked: ______ |
|
Definition
| 40S ribosome (60S hasn't joined yet!) would be present on the mRNA but would not have made it to the AUG |
|
|
Term
| if initiation is blocked: ______ |
|
Definition
| get to the AUG but be stuck |
|
|
Term
| if elongation is blocked: ______ |
|
Definition
| space between ribosomes is not what it should be |
|
|
Term
| if termination is blocked: ______ |
|
Definition
| ribosomes get to the end & there is a big pile-up there at the STOP codon because they are not being released |
|
|
Term
| 2 steps of gradient assays (low-tech method if you don't want to do ribosome profiling to determine ribosome location on mRNA) |
|
Definition
1. run a gradient & look to see where the peaks are
2. run a northern blot for your mRNA using a probe to see where it falls in the gradient |
|
|
Term
| what is in the complex that forms that recruits 40S to the mRNA? |
|
Definition
| cap, E (CBP), G, eIF3, poly-A tail, PABP |
|
|
Term
| a cis element sequence in the _______ would affect formation of the attachment complex |
|
Definition
|
|
Term
| 2 things that could block a scanning ribosome |
|
Definition
1. secondary mRNA structure
2. sequence in mRNA that binds a protein |
|
|
Term
| if you are looking to find a cis element that is preventing scanning: look in the ______ |
|
Definition
|
|
Term
| why would how long of a deletion is required to stop the scanning problem give you a hint as to whether it is binding a protein or making a secondary structure? |
|
Definition
| cis-acting sequences are usually short (unless they're forming some sort of complicated secondary structure) |
|
|
Term
| 2 ways to find the proteins that bind to the sequence to block ribosomes |
|
Definition
1. if you have a candidate: co-IP: tag your protein (epitope tagging) & when you pull down see which mRNA & other proteins come with it
2. if you know nothing: co-IP: tag your mRNA (biotin tagging is like epitope tagging) & when you pull down the mRNA the proteins will come with it --> mass spec the proteins to see what is there |
|
|
Term
| how do you prove that a cis element is causing repression? |
|
Definition
| mutate cis element --> is repression still happening? |
|
|
Term
| how do you prove that a trans-acting factor is causing repression? |
|
Definition
| mutate trans-acting factors --> is repression still happening? |
|
|
Term
| how do you know if proteins interact? |
|
Definition
| if you purify two proteins & put them in a tube & they dimerize --> they interact |
|
|
Term
| how would you test if protein X & protein Y are associated? |
|
Definition
| if you take a whole cell & purify out protein X, you look to see if protein Y is interacting with it |
|
|
