Question

You are studying two proteins (A and B) that you think are involved in regulating the expression of Gene Y. You perform a gel shift (EMSA) assay using a regulatory DNA sequence from Gene Y. Each question below represents a hypothesis that you are testing. For each hypothesis, determine whether it is supported or rejected by the results of your EMSA experiment, or whether the results do not provide enough information.

Answer 1

A gel shift assay (EMSA) is used to detect protein-DNA interactions, which can reveal if certain proteins are involved in the regulation of gene expression. For the proteins A and B in question, the presence of a DNA mobility shift in the assay would support the hypothesis that they are involved in regulating Gene Y; no shift suggests no binding or an inconclusive result.

Step-by-step explanation:

The student is concerned with a gel shift assay (EMSA) to investigate whether proteins A and B are involved in regulating the expression of Gene Y. EMSA is a technique used to study protein-DNA interactions and is a powerful tool for understanding gene expression regulation.

Gene expression is a critical process where information from a gene is used to synthesize a functional gene product, usually a protein. This process can be regulated at multiple levels, including transcriptional and post-transcriptional stages. Regulation at the transcriptional level often involves proteins that bind to regulatory DNA sequences to either promote or inhibit transcription.

To examine if proteins A and B regulate Gene Y's expression, we would expect a shift in the mobility of the DNA band in the presence of these proteins if they indeed bind to the regulatory sequence. If the hypothesis is that Protein A or B binds to DNA and affects gene expression, and the EMSA shows a shift upon addition of these proteins, then the hypothesis is supported. If there is no shift, either the proteins do not bind to the DNA or the assay may not have been conducted correctly, which means the results are inconclusive without further investigation.

Answer 2

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The electrophoretic run or EMSA change test for its initials in English (electrophoretic mobility shift assay) is a technique used to investigate interactions between proteins and DNA or RNA. The technique is based on an electrophoretic run between labeled DNA or RNA fragments and the presence of cell extracts. The basic DNA assay consists of a lane only with the DNA fragment of labeled interest, a second lane with the same DNA plus proteins that, under the given conditions, do not interact with it, and the third lane containing the same fragment of DNA plus proteins with supposed interaction with that sequence. Depending on the result of the third lane, it can be determined whether or not there was interaction. A DNA fragment interacting with proteins tends to travel less distance through the gel at the same time as in lanes 1 or 2, and therefore should be delayed. This technique is based on the works of Garner and Revzin and Fried and Crothers

Due to their physicochemical properties, proteins can be classified into simple proteins (holoproteids), formed only by amino acids or their derivatives; conjugated proteins (heteroproteides), formed by amino acids accompanied by diverse substances, and derived proteins, substances formed by denaturation and unfolding of the above. They consist of structural units called polymers4. Proteins are necessary for life, especially for their plastic function (they constitute 80% of the dehydrated protoplasm of every cell), but also for their bioregulatory functions (they are part of the enzymes) and defense (antibodies are proteins). 5

Proteins play a fundamental role for life. They represent about 50% of the dry weight of the tissues.6 They are the most versatile and diverse biomolecules. They are essential for the growth of the organism and perform a huge number of different functions, among which are:

Structural. This is the most important function of a protein (eg, collagen)

Contractile (actin and myosin)

Enzyme (Ex .: sucrose and pepsin)

Homeostatic: collaborate in maintaining pH (since they act as a chemical buffer)

Immunological (antibodies)

Scab production (eg, fibrin)

Protective or defensive (Ex .: thrombin and fibrinogen)

Signal transduction (eg, rhodopsin).

Proteins are composed of amino acids. The proteins of all living beings are mostly determined by their genetics (with the exception of some antimicrobial peptides of non-ribosomal synthesis), that is, genetic information largely determines what proteins a cell, a tissue and an organism have.

Proteins are synthesized depending on how the genes that encode them are regulated. Therefore, they are susceptible to external signals or factors. The set of proteins expressed in a given circumstance is called a proteome.

Through a family of methods called peptide synthesis it is possible to chemically synthesize small proteins. These methods depend on organic synthesis techniques such as ligation to produce peptides in large quantities.10 Chemical synthesis allows the introduction of unnatural amino acids into the polypeptide chain, such as amino acids with fluorescent probes linked to their side chains.11 Methods are useful in laboratories of biochemistry and cell biology, but not so much for commercial applications. Chemical synthesis is inefficient for polypeptides of more than 300 amino acids, and synthesized proteins may not readily adopt their native three-dimensional structure. Most chemical synthesis methods come from the C-terminal end to the N-terminal end, in the opposite direction to the biological reaction.