Question

I need help with questions numbers 20 and 21

Lider 12 0 p54 | hTra2B pinin 20. Researchers analyzed RNPS1, an mRNA splicing activator which contains three domains: serine-rich (S), RNA recognition motif (RRM), and arg/ser/pro-rich (RS/P). Each domain interacts with one of the splicing factors p54, hTra2p, and pinin. RNPS1's domains were expressed in yeast and analyzed for interactions with these proteins. Based on the yeast two-hybrid (left), to which domain of RNPS1 does each of these proteins bind? Recall "A" indicates a deletion. RNPS1 S RRM HRS/P WT ARS/P RRM+RS/P 21. Researchers studied protrudin (ZEYVE 27), a protein involved in axon elongation that is mutated in hereditary disorders, by performing a yeast two-hybrid assay. It was hypothesized that protrudin self-interacts to form a dimer. To test this, wildtype protrudin (in a PGADT7 prey vector) was mixed with various constructs of protrudin in a PGBKT7 bait vector) as shown below. The bait and prey must interact for the cells to survive on the selective-LTHA media ("lacking leucine, tryptophan, histidine, and adenine"). GAMYEWE GADT CE FVE RADHIRI HR OVO WT OKTI ZEYVE Yeast two hybrid constructs 110-15 1-335 AHR3 110-335 150-250 174-218 1-184 208-404 INN 2 . . G LT UTHA a. Which amino acids and domain(s) are responsible for protrudin's self-interaction? Explain your answer.

Answer 1

The image involves request for answering two questions. However, as per Chegg guidelines, I am answering the first question i.e. Q20.

Yeast two hybrid system is an extensive method used to study protein-protein interactions. It involves a simple system of three plasmids. One plasmid has a prey protein conjugated to a DNA-binding protein. The second plasmid has a bait protein conjugated to a Transcription Factor. If the bait and prey were absent, the DNA binding protein would sit on DNA. The transcription factor would bind to it and recruit an RNA Polymerase. However, due to the presence of bait and prey conjugated to these proteins, they cannot interact directly. However, if the prey can bind to the prey protein, the transcription factor can bind indirectly to the DNA-binding protein thereby allowing the recruitment of RNA polymerase and hence the transcription of the downstream gene. The transcribed mRNA can then undergo translation to form a reporter protein which can then be detected using various techniques. Below is a brief summary of yeast two-hybrid system in play.

Bait Transcriptional Activation Domain Prey RNA Polymerase DNA-binding Domain The guion) Tres com A: No binding of bait to prey Bait Transcriptional Activation Domain Prey DNA-binding Domain RNA Polymerase Transcription Translation> B: Bait binds to the prey

In the question we have a protein with three domains and we know that each of p54, hTra2$\beta$ and pinin binds to one of the domains. The goal is to determine which protein binds to which domain. As mentioned in the question, yeast two-hybrid system can be employed for this. The binding proteins can be conjugated to the TAD (Transcriptional Activation Domain) and cloned into the yeast. Simultaneously combination of the domains can be conjugated to the DNA binding domain. The downstream gene can be chosen whose protein product can be detected via Western blot. Upon cloning all three plasmids into the yeast, the test can be performed.

In the experiment mentioned, they have cloned following three scenarios:

WT RRM RS/P ARS/P ARSIP © RRM RRM asp RRM + RS/P RRM RS/P

WT has the presence of all three domains. Hence as expected all three baits bind to this prey as can be seen by the band in the WB.

$\Delta$RS/P has two domains, namely S and RRM. It lacks the domain RS/P. As can be seen from the gel image, p54 and pinin bind to $\Delta$ RS/P, but hTra2$\beta$ doesn't bind. Thus hTra2$\beta$ requires the domain RS/P to bind to the protein RNPS1. Hence hTras2$\beta$ binds to the domain RS/P.

RRM + RS/P has the domains RRM and RS/P but lacks the domain S. As can be seen from the gel image, hTras2$\beta$ binds and pinin binds this prey. However, p54 fails to bind. Thus, p54 requires the presence of the domain S. Thus, p54 binds to the domain S.

From the above two statements, we can safely that pinin binds to RRM domain.

To summarize, the domains and the binding proteins can be depicted as:

p54 pinin hTras2B RRM RS/P