Your research project involves the characterization of a recently identified transcription factor. As part of your project, you want to determine if this transcription factor binds directly to any of the general transcription factors. Unfortunately, you are having trouble expressing the full-length protein. What approach may help achieve your research goal?

Question

A.  You must find a way to express the full-length protein, since transcription factor functions do not lie in separate domains.
B.  Identify the RNA that mediates the protein-protein binding.
C.  Express and purify only the DNA-binding domain, and test for binding.
D.  Express and purify only the activation domain, and test for binding.

Clarify Question
· What is the key concept addressed by the question?
· What type of thinking is required?
· What key words does the question contain and what do they mean?

Gather Content
· What do you already know about specific transcription factors?

Consider Possibilities
· What other information is related to the question? Which information is most useful? · Consider the different answer options. Which can you rule out?
Choose Answer
· Given what you now know, what information and/or problem solving approach is most likely to produce the correct answer?
Reflect on Process
· Did your problem-solving process lead you to the correct answer? If not, where did the process break down or lead you astray? How can you revise your approach to produce a more desirable result?

Answer 1

D.  Express and purify only the activation domain, and test for binding.

Clarify Question
· What is the key concept addressed by the question?
        o This question addresses the structure of specific transcription factors.
· What type of thinking is required?
        o This question is asking you to analyze the information given, using logic, to dissect the problem and determine the answer.
· What key words does the question contain and what do they mean?
        o A recently identified transcription factor must refer to a specific transcription factor, since the general transcription factors have been well described.
        o The general transcription factors are the set of transcription factors that underlie transcription in all genes. By themselves, they can only support transcription at some low, basal rate.
        o Expressing a protein refers to the process of cloning the gene and producing large amounts of the protein using bacteria vitro.

Gather Content
· What do you already know about specific transcription factors?
        o Specific transcription factors constitute a huge variety of proteins that act in conjunction with the general transcription factors to increase transcription of specific genes in certain cells and times.
        o They typically have two separate domains: a DNA-binding domain that binds specific DNA sequence motifs, and an activating domain that interacts with the general transcription factors to stimulate transcription.

Consider Possibilities
· What other information is related to the question? Which information is most useful? · Consider the different answer options. Which can you rule out?
        o To answer your research question, must you find a way to express the full-length protein, since transcription factor functions do not lie in separate domains? No, this is not correct. Typically the DNA-binding and activation domains are separable.
        o To answer your research question, should you identify the RNA that mediates the protein-protein binding? No, because typically there is no RNA – the specific transcription factor binds the general TFs directly.
        o To answer your research question, should you express and purify only the DNA-binding domain, and test for binding? No, because that wasn’t the question you wanted to answer. You need to know how it bind the general TF proteins, not the DNA sequence.

Choose Answer
· Given what you now know, what information and/or problem solving approach is most likely to produce the correct answer?         o To answer your research question, should you express and purify only the activation domain, and test for binding? Yes, this strategy may very well work! It may be easier to express the shorter protein, and you can use the domain to test for protein binding to the general transcription factors.

Reflect on Process
· Did your problem-solving process lead you to the correct answer? If not, where did the process break down or lead you astray? How can you revise your approach to produce a more desirable result?
        o This question asked what approach could help to determine binding between a specific transcription factor and the general transcription factors.
        o The question required you to analyze the information given, using logic, to dissect the problem and determine the answer.
        o Did you recognize that the DNA-binding and activation functions use separate protein domains?