Question

When the gene encoding a certain cytoskeleton protein is deleted, the resulting mutant cells round up and do not form their normal appendages. These mutants can be rescued when a gene encoding an N-terminal green fluorescent protein (GFP) fusion of the protein is expressed, but not when a gene encoding a C-terminal GFP fusion is expressed. Which fusion protein is appropriate to use in studying cellular localization and activity

Answer 1

Answer: N-terminal green fluorescent protein (GFP)

Explanation:

Protein synthesis takes place in ribosomes located in the cell cytoplasm, in which amino acids are transported by transfer RNA corresponding to each amino acid to the messenger RNA where they bind in the appropriate position to form new proteins. The messenger RNA has a sequence of nucleotides that are translated into protein, as each codon (set of three amino acids) codes for one amino acid.

Genes are the storage units of genetic information, so they are segments of DNA that contain the information on how the cells of the organism should function. Each gene codes for a protein, so if a gene is damaged or absent, the protein will not be obtained. In this case, mutant cells with a deleted gene, will round up and wont form their normal appendages. This mutation can be rescued or repaired with a gene that encodes an N-terminal green fluorescent protein (GFP) fusion of the protein. The N-terminal end is the amino-terminal end and it refers to the end of a protein that ends with an amino acid that has a free amino group. The C-terminus or carboxyl-terminus is the end of a protein that ends in a carboxyl group. The convention for writing peptides is to place the C-terminal end to the right and write the sequence from the N- to the C-terminal end. So, when an N-terminal green fluorescent protein (GFP) fusion of the protein is expressed, the gene will produce the protein with a GFP tag.

GFP is Green Fluorescent Protein, and it is a protein produced by the jellyfish Aequorea victoria that emits bioluminescence in the green region of the visible spectrum. When a gene is fused to another gene (at either the N- or C-terminus, although in this case it is the N-terminus), the entire messenger RNA is translated together as if it were a single fused protein. Thereby, since the protein will be produce with a GFP tag, it can be seen under the microscope and it will be apropiate to use in studying cellular localization and activity.

There are some considerations that this problem does not question, such as that there must be a start codon for protein synthesis and a stop codon, and this stop codon must not be in the middle of the gene or between the gene and the GFP tag.