Assignment 4 – Functional Assays

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Conceptual Questions

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The following figure is a poorly drawn depiction of a hypothetical yeast 2-hybrid experiment.

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Circles in the above figure indicate protein domains and are numbered 1 to 4. Dotted lines indicate
protein-protein interactions. Unbroken lines between circles indicate covalent bonds. The DBD and AD of
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the GAL4 TF are indicated. You may assume that the assay works normally and correctly.
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1. (3 points) What is/are the bait(s) of this experiment? Explain your reasoning logically in three
grammatically correct sentences at most. Protein domain 1 is the bait because it
is fused with DBD (DNA binding domain) in 2-D hybrid the bait will
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make bond with prey.

2. (3 points) What is/are the prey molecule(s) whose interactions are detectable by this assay?
Please explicitly identify this/these interaction(s). Explain your reasoning logically in three
grammatically correct sentences at most.

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 Protein domain 3 is a prey and it is fused with the AD (activating domain) because we can see
there is covalent bond between domain3 and activating domain.
3. (3 points) Are there any protein-protein interactions that are undetectable by this assay? Explain
your reasoning logically in three grammatically correct sentences at most.

Protein-protein interaction between domain 1 and domain 3 also between AD and domain 4
remain detectable. Because only fusion of prey and bait will produce transcriptional signalling.

4. (3 points) Identify the molecule most likely to be RNA polymerase in the figure. Explain your
reasoning logically in three grammatically correct sentences at most.

The number 4 is the RNA polymerase in this figure. The RNA polymerase will bind to AD

Research Preamble

It is largely unknown how the glyceollin biosynthetic genes are turned on (i.e. regulated) and made in

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soybean. Since glyceollins and related phytoalexins appear to be of major pharmaceutical value, there is

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interest in determining how plants turn on these genes. Dr. Kovinich’s research group has identified

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several candidate transcription factors that may be responsible for this, but whether these transcription

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factors truly do (and if they do, which specific gene promoters they bind to and activate) is currently

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unknown.
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Over the course of the past several weeks, you have gone through the process of generating a construct
that fuses the GmMYB103 gene (one of the identified candidate transcription factors) to the GAL4-AD
for use in a yeast-1-hybrid assay. To conduct the assay, Dr. Kovinich provides you a strain of yeast that
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possesses the promoter from the IFS2 gene (one of the genes that encode an enzyme in the glyceollin
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biosynthesis pathway). This promoter has been engineered into the yeast genome to direct the
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transcription of a gene that gives yeast the ability to grow on histidine-deficient media.

Research Questions

5. (4 points) Before doing the experiment, Dr. Kovinich asks you in a lab meeting to think about
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potential experimental outcomes and what they may mean. Consider the following scenario:
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Imagine that you see growth of cells on histidine-deficient media in the yeast-1-hybrid assay.
Assume you know that this is a false positive result. Provide a mechanistic explanation for this
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false positive result that does not rely on experimental error. Your explanation can be anything,
but you will be heavily penalized if your reasoning is not plausible, logically sound, or if you
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invoke the presence of other molecules that are not required for the assay to work. You may
assume the yeast cells you are using are normal. Provide your answer in four sentences max.
False positives are usually caused by high recognition of
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the bait sequence by TFs so that the reporter gene

which it causes activated in
the absence of prey.

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 6. (4 points) Since this is an experiment in an ongoing research project, we still are not certain what
this GmMYB103 gene does (e.g. what the protein looks like, what it binds to). However, let us
assume that this candidate TF (without the fused activation domain) can directly bind to and
recruit RNA polymerase. If this is the case, will this assay still work as intended, or will the assay
be unreliable? Explain your answer in four grammatically correct sentences.

TF have directly ability to bind RNA polymerase. One way to fuse TF to DNA binding domain
and adding core promoter including upstream activating regions from yeast into promoter
sequence. If the UAS are for specific DNA binding domain, so TF will bind to UAS and recruited
RNA polymerase.
7. You conduct the assay. The experimental result is shown below.

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a. (2 points) What do the results of the yeast 1-hybrid assay tell you about the protein encoded
by the GmMYB103 gene? Explain in two sentences max. the result of the yeast one – hybrid
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assay is a genetic method to identifying a protein that can interact with a DNA sequence of
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interest. When TF interacts with the DNA the AD activates reporter expression in yeast.

b. (1 bonus point) Given our discussion on controls over the past two modules, I think there is
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something concerning about the experiment above. Explain why in two sentences max.
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c. (1 bonus point) What is the most logical control experiment that could improve this
experiment? Describe in three sentences max.
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8. (6 points) You are asked to start writing up your results for a manuscript. Write a summary
paragraph that describes your findings, in a style suitable for manuscript submission to the
journal Nature. The full editorial guidelines can be found at
https://www.nature.com/nature/for-authors/formatting-guide . An annotated example can be
found at the URL. Please consider the following excerpt specifically:

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 Articles are original reports whose conclusions represent a substantial advance in understanding of an
important problem and have immediate, far-reaching implications. In print, they do not normally exceed
5 pages of Nature. Articles start with a fully referenced [yes, you need references from primary
literature] summary paragraph ideally of no more than 200 words, which is separate from the main text
and avoids numbers, abbreviations, acronyms, or measurements unless essential. It is aimed at readers
outside the discipline. This summary paragraph should be structured as follows: 2-3 sentences of
basiclevel introduction to the field; a brief account of the background and rationale of the work; a
statement of the main conclusions (introduced by the phrase 'Here we show' or its equivalent); and
finally, 2-3 sentences putting the main findings into general context so it is clear how the results
described in the paper have moved the field forwards.

The yeast one hybrid measures protein – DNA interactions. In the yeast one hybrid system, detection is
based on the interaction of single protein (the prey) with bait DNA sequence. In the yeast one hybrid we
need the bait sequence this will act as the promoter sequence and also bait plasmid and prey plasmid.
The yeast one hybrid is able to detect protein-DNA interactions that are no actually activating

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transcription. The yeast one hybrid has some disadvantages. Yeast one hybrid does not provide any

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information about functional consequences of a DNA- protein interaction. The rates of false- positive

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results are high. Expression of prey protein fused to an activation domain can result in improper folding

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protein.

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9. (4 points) You are asked by Dr. Kovinich to present a research poster with one of his Ph.D.
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students at the annual “Mechanisms of Eukaryotic Transcription” Conference at Cold Spring
Harbor Laboratory on Long Island, NY. Details of the upcoming meeting in 2021 can be found
here:
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https://meetings.cshl.edu/meetings.aspx?meet=MOET&year=21
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As you present your yeast 1-hybrid work during the happy hour/poster session, a semi-drunk,
rude graduate student at your poster tries to quiz you:
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“I am not sure about your results, because I’ve seen other yeast 1-hybrid experiments with lots of
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false negatives in the past. Do you know what could cause a false negative in this assay?”

[On the spot, you] provide a mechanistic explanation that answers the query above in four
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grammatically correct sentences at most. Your explanation can be anything, but you will be
heavily penalized if your reasoning is not plausible, logically sound, or if you invoke the presence
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of other molecules that are not required for the assay to work. You may assume the yeast cells
you are using are normal.

There is two possible reason in false negative results in the hybrid:

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1. Lack of post translational modifications: it means that prey unable to cause the expression of the
reporter gene even though it will bind to bait because of the absence of the certain post
translational modifications in the activator domain.
2. Steric hindrance: the transcription factor might not able to bind to the bait sequence

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