Protein;Protein Interactions Flashcards
What program can you use to investigate the primary structure of a protein?
EXPASY
What program can you use to predict the secondary structure of a protein?
JPRED
How does magoh interact with Y14?
Interact to form a heterodimer involved in exon junction binding in yeast
What extra factor was included when designing Y14 or Magoh (or UAP56) expressing plasmids?
Plasmids were designed to express the proteins fused to a tag (his6 or GST)
What is the name of the genetic approach used to study Y14 interactions?
Bacterial-2-Hybrid assay
What is the bait protein fused to in a bacterial 2 hybrid assay?
A full length bacteriophage repressor protein with a DNA binding domain in N terminus (lamdacl)
What is the prey protein fused to in a bacterial 2 hybrid assay?
Fused to N terminal domain of RNA polymerase(alpha) (transcriptional activator region)
What happens if the bait and prey protein interact?
If the bait and prey protein interact they will activate transcription of the reporter gene (by stabilizing RNApol at promoter region)
What is an example of the reporter gene used in our experiments?
lacZ - b-galactosidase activity
When choosing restriction sites for cloning the plasmids containing the tagged fusion protein - what must be excluded?
T7 tag
How can you ensure that the reporter gene cassette will not be induced by IPTG (when bait and prey plasmid not present)
remove CRP binding site
how do you transform engineered plasmids into E.coli cells?
Heatshock
What must the reporter gene cassette contain in order to be activated by bait/prey complex?
lamda operator site - which will bind lamdacl
What is the prey plasmid activating region under control of?
IPTG induced expression
What is the advantage of bacterial 2 hybrid assay over a yeast 2 hybrid assay?
yeast 2 hybrid can only capture directly pairwise pathways.
If studying bacterial protein system - yeast may not have the correct ligands or chaperone proteins
