Ubiquitin Enzymatic Cascade: Role of E3 Ligase

Our laboratory studies E3 ubiquitin ligase enzymes. This video puts these E3 enzymes into the context of the ubiquitin enzyme cascade that consists of E1, E2, and E3 enzymes as well as ubiquitin and target proteins. The goal for our lab is to identify proteins that the E3 enzymes interact with to inform future studies and form hypotheses about the potential functional roles of the proteins we are attempting to characterize.

Questions to be answered using Y2H

When you clone a gene into pGBKT7 and have confirmed that it is the exact sequence you think it is, the next thing you’ll want to do is transform it into yeast.  We have a kit for that and the manual is here: PT1172-1.

Some immediate needs are as follows:

  • Make a glycerol stock.  Just like bacterial freezing media, yeast freezing media consists of 50% glycerol in the typical
  • Test whether the gene causes autoactivation of the Y2H system (see if it grows on yeast media lacking tryptophan (confirms that the plasmid is in the yeast) as well as histidine and adenine (tests whether the gene product alone can lead to reporter gene expression).  Growth under these conditions means that you can’t use Y2H for your particular TRIM protein.

Next set of experiments:

  • Perform a Y2H screen by mating the yeast with your gene in pGBKT7 with the yeast containing a library of human gene cDNA.
  • Perform pairwise Y2H assays by mating the yeast with your gene in pGBKT7 with the yeast containing other TRIM and MAGE genes in pGADT7.

Tool: Finding Splice Variants

Previously, we have used a laborious method to identify splice variants of the various TRIM proteins.  Moreover, we have not had a good method to quickly figure out what protein isoforms are possible from the splice variants.

Our lives just got easier with the discovery of SpliceMiner.  Just go to this website and type in the name of the TRIM you’re working with.  To try it out, I tried “TRIM6”.  Immediately I was given the information to 10 splice variants and 4 protein isoforms.