What makes me tick…tock? June 2012
Lesson 3: How can genetics change your clock?
DNA synthesis, replication, and mutation
DNA holds the code for every protein assembled in the body. InPart 1 of this activity, you will work in groups to construct a double-stranded model of a circadian gene, per2, replicate the per2 gene, and mutate the per2gene.
Constructing your DNA model
For the DNA model, you will be using pipecleaners and colored Velcro. The pipecleaners represent the backbone of the DNA and the colored Velcro pieces are base pairs. In this model, each base pair is represented by a different color of Velcro:
Adenine –red
Thymine –yellow
Guanine – green
Cytosine – blue
1. Before you start assembling your DNA model, use masking tape to label one end of the pipe cleaner 5’ coding strand. The desired protein product corresponds to the gene on the coding strand. That is, the coding strand represents the gene that is being transcribed and later translated into a protein.
2. Once you have this end labeled, start assembling your DNA model by sliding colored Velcro pieces onto the pipecleaner. The genetic code for the gene is: 5’ GCC GAG AGT GTG GCG TCC CTC 3’. Label the other end of the strand 3’ coding strand. Use the table below to write out the sequence of the template strand with the corresponding base pairs.
Coding Strand / 5’ end / GCC / GAG / AGT / GTG / GCG / TCC / CTC / 3’ endTemplate Strand / 3’ end / 5’ end
3. Now that you have one coding strand assembled, construct the template strand with the sequence you determined above. Make sure to also label the 3’ and 5’ ends of your template strands you did in step 2. Remember that the template strand is the strand of DNA that will be transcribed. This is because mRNA transcription must occur in the 5’ to 3’ direction and must have a complementary base pair sequence with the template DNA strand, in the 3’ to 5’ direction.
· Looking at your double-stranded DNA models, what do you notice about the structure? For example, do you see any patterns that may make it easier or harder to replicate (copy) genes?
DNA mutation
1. Carefully un-Velcro your two strands of DNA.
2. Now that you have two single strands of DNA, get a set of Gene Mutation cards from your teacher and break into pairs.
3. Each pair should take a Gene Mutation card and follow the instructions on the card. Each group should work separately when mutating the per2 gene.
4. After your group and your partner group have mutated your single strand of per2DNA, build the other strand so you have a double-stranded per2DNA model.
***Note: make sure you are mutating the correct strand. One card has instructions for the coding strand and the other has instructions for the template strand.
5. When both pairs have completed the instructions on the Gene Mutation card, compare your double-stranded mutated gene sequence to the other group’s model.
· When you compare the two models, is there anything about the mutation that surprises you? What does this tell you about the replication of DNA mutations?
· Do you think this gene mutation will have a noticeable effect on the animal’s ability to function? Explain your answer.
Transcription of DNA to RNA
In part 2 of this activity, you will transcribe theper2 DNA to mRNA, the second step in the process of making proteins from DNA.
1. Before starting the transcription of DNA to RNA, one group of students will be assigned to transcribe the wild type DNA and their partner group will be assigned to transcribe the mutant DNA.
2. Make sure to use a different colored backbone pipecleaner so that it is easy to tell the difference between DNA and mRNA.
3. To transcribe your gene, unzip your double-strandedper2 DNA model.
4. Using the template strand, build your corresponding RNA sequence.
***Note: Since uracil replaces thymine in the RNA strand, you now have a new Velcro color. Use the orange Velcro for uracil in your per2 mRNA model.
Wild type DNA / 5’ end / GCC / GAG / AGT / GTG / GCG / TCC / CTCWild type
RNA
Mutant DNA / 5’ end / GCC / GAG / GGT / GTG / GCG / TCC / CTC
Mutant RNA
5. When both partner groups have completed transcribing the genes, compare your RNA sequence to the other partner group’s model.
· When you compare the two mRNA sequences, is there anything that surprises you? What similarities/differences do you notice between the two sequences?
· Do you think this gene mutation will have a noticeable effect on the animal’s ability to function? Explain your answer.
Translation of RNA to protein
In part 3 of this activity, you will translate the per2 mRNA into amino acids, the third step in the process of making proteins from DNA.
1. Work in groups to build amino acid chains.
2. The groups who transcribed the wild type DNA will translate the wild type gene sequence and the other groups who transcribed the mutant DNA will translate the mutated gene sequence.
3. In the chart below, the per2 mRNA sequence you created is written. Translate the mRNA sequence below by writing the corresponding amino acid below the RNA codon.
Wild type / 5’ end / GCC / GAG / AGU / GUG / GCG / UCC / CUCAmino acid sequence
Mutant / 5’ end / GCC / GAG / GGU / GUG / GCG / UCC / CUC
Amino acid sequence
4. Once you have translated your per2 mRNA sequence, start building your assigned amino acids. For this activity, you will be using a longer amino acid chain than what you have translated. The code below is a shorthand to record the name of proteins: each protein is assigned a three letter code.
Wild type Per2:
Ser- Leu-Ala-Leu-Pro-Gly-Lys-Ala-Glu-Ser-Val-Ala-Ser-Leu-Thr-Ser-Gln-Cys-Ser-Tyr
Mutant Per2:
Ser- Leu-Ala-Leu-Pro-Gly-Lys-Ala-Glu-Gly-Val-Ala-Ser-Leu-Thr-Ser-Gln-Cys-Ser-Tyr
5. Write down your group’s assigned amino acids ______
6. Each amino acid will be built using candy, wire, and pipe cleaners. The candy amino acids are built on wires that you will string on a pipe cleaner that represents the peptide bonds between the amino acids. Use the attached amino acid guide to build the amino acids. The peptide chain that you create is a small piece of the larger Per2 protein.
7. After all groups have constructed their candy amino acids, combine the amino acids into a long peptide chain. The sequence of amino acids in a peptide chains referred to as primary structure.
· When you compare the two peptide chains, is there anything that surprises you? What are the noticeable differences between the two models?
· Do you think this amino acid mutation will have a noticeable effect on the animal’s ability to function? Explain your answer.
· Does the gene mutation lead to a bigger difference in amino acid sequence than you expected? Explain your answer.
Protein Folding
1. After you have compared your amino acid primary structures (sequence of amino acids), fold your amino acid chains into a three-dimensional protein, using the following rules of thumb:
· Hydrophobic amino acids will be on the inside of proteins away from the cytosol. They tend to be close to other hydrophobic amino acids.
· Hydrophilic amino acids will be on the outside of proteins near the cytosol. They tend to be close to other hydrophilic amino acids.
· Amphipathic amino acids will do whatever the surrounding amino acids are doing (hydrophobic if surrounded by hydrophobic amino acids, hydrophilic if surrounded by hydrophilic amino acids).
· Proline will induce a β(beta)-turn in the protein structure. β sheets consist of β-strands connected laterally by at least two or three backbone hydrogen bonds, forming a twisted, pleated sheet.
The manner in which a strand of amino acids takes shape is referred to as secondary and tertiary structure. Secondary structure refers to local patterns (such as helices and sheets), whereas tertiary structure refers to the protein as a whole unit.
***Note: Some proteins have a defined structure that helps determine its biological function. However, in essence, a protein is a strand of amino acids (like beads on a string) and the three-dimensional structure is determined by the interactions of the amino acids with one another. There is inherent variability in protein shape and even when scientists know the amino acid sequence (primary structure) of a protein, they can often only make educated predictions about the protein’s ultimate shape (secondary and tertiary structure). Note areas of potential variability as you fold your peptide chain.
2. After both groups have folded their candy amino acid sequences, compare your proteins.
· When you compare the two protein structures, is there anything that surprises you? What is a major difference between the two structures?
· Do you think this protein mutation will have a noticeable effect on the animal’s ability to function? Explain your answer.
A final level of protein structure is quaternary structure. This level refers to the biophysical and chemical interactions among proteins. Some proteins assemble into groups and act together. Mutations, even single amino acid mutations, may influence the ability of one protein to bind to another by altering the protein’s structure. Change in structure may ultimately lead to change in function.
· Some mutations do not cause large changes in protein structure or folding, but change how the protein interacts with other proteins. Now that you know that point mutations like the one you created can change how the protein interacts with other proteins, how has your answer to the previous question changed?
· Now that you have created the known Per2 mutation, create a new mutation that causes a greater change in the structure of the Per2 gene. How might this mutation you have created affect the function of the protein?
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