Question

Analysis and Prediction of Binding Order in Cascade/Cas3 Immunity.
Molecular biology challenge by Frostbite

Answer 1

In the \(\href{/users/frostbite#/updates/5396fb56e4b01c619df25040}{\sf last~challenge}
\), we looked at how Cascade is able to specifically bind RNA-complementary DNA.
It has now come our knowledge that Cascade is not only to bind DNA, but also a protein we call Cas3 (CRISPR associated) protein. As we now got two "substrates" for the protein we want to investigate the binding order of this system \(\it in~vivo\).

In order to analyse this problem there will be done a BiFC (Bimolecular fluorescence complementation) analysis with the half non-florescence protein Venus attached to Cas3 and Cascade. The results are attached to the question and contains a picture of the test and a diagram of the relative florescence intensity.

A) Outline the principles in a BiFC (Bimolecular fluorescence complementation) expriment.

B) Analyze the data, focusing on the bounding between the two "substrates" and Cascade.

C) Propose a conclusive binding order model that can explain data.

C - EXTRA) Suggest a molecular mechanism that don't violate your model and can explain data.

Data obtained from Westra et al. (2012)
Source: Hansen. S (2014), \(\it Review~of~Cascade/Cas3~interferes~cycle~in~CRISPR~type~I-E\)

Last times main contributors:
@Somy

Answer 2

NOTE: the questions follow Bloom's taxonomy.

Answer 3

alright
everything is clear
but labeling in pic is not <.<

Answer 4

i remember from previous work and the diagrams i made that cascade cuts DNA by binding to it

Answer 5

i see so it also binds to Cas3 protein
so that means either its shape in like Cas3's active site or it binds to other side of Cas3 other than its active site

Answer 6

so now 2 substrates are DNA and Cas3

Answer 7

i don't really know what does Bimolecular fluorescence complementation do
but im reading here and it says its used to 'validate protein interactions' meaning prove/ show

Answer 8

i don't quite get what is 'infected' and 'uninfected' and by what?

Answer 9

Well Cascade did not cut DNA (at least not alone). But we saw from the last experiment that Cascade is able to bind RNA-complimentary DNA which usually comes from viruses or pasmids.

So when it say infected we mean that our cells has been infect with pasmid DNA. :)

Answer 10

So I suggest. Start by reading about BiFC then do B) :)

Answer 11

ah! i remembered! it was blocking active site thus preventing the cut by i don't remember what

Answer 12

But this time we simply want to find out what is happening: If we say Cascade = A, DNA = B, and Cas3 = C. Then we try find out what is working:
A + B + C -> AB + C -> ABC
A + B + C -> AC + B -> ABC
A + B + C -> ABC

We just want to find the binding order :)

Answer 13

That is why the experiment is gonna reveal :)

Answer 14

hmm i see

Answer 15

first pic uninfected so its cascade alone?

Answer 16

Yes :) Cascade alone.

Answer 17

The whole first row

Answer 18

yes

Answer 19

idk whats phase lambda
but i see the color yellow
i suppose there is protein interaction

Answer 20

Or wait sorry Cascade AND Cas3

Answer 21

In the first row

Answer 22

oh i see

Answer 23

Phage you know what is... that is a virus :)

Answer 24

So it is virus DNA.

Answer 25

yes i know

Answer 26

hmmm

Answer 27

alright so first row is Cascade+Cas3

Answer 28

second row doesn't look like its 3 things
it looks like they got separated

Answer 29

or im getting a wrong idea

Answer 30

Second row is virus DNA (RNA complimentary), Cascade and Cas3

Answer 31

Third row is virus DNA (not RNA complimentary), Cascade and Cas3

Answer 32

yes but not together thats what i was thinking

Answer 33

What do you mean together?

Answer 34

ah nvm lol i don't get those pics

Answer 35

Okay. Lets start from the top:
What is BiFC? :)

Answer 36

Then I help you understand

Answer 37

(look at the wiki page)

Answer 38

bimolecular fluorescence complementation
shows protein-protein interaction

Answer 39

How can you see that two proteins interact from BiFC?

Answer 40

The BiFC assay is based on structural complementation between two non-fluorescent N-terminal and C-terminal fragments of a fluorescent protein.

Answer 41

its not written or im missing it
but im guessing - color?

Answer 42

'emit its fluorescent signal'

Answer 43

Exactly! :)
So when two proteins interact they fluorescent :) (see drawing below)