HASPI Medical Biology Lab 0 Purpose Create a model to simulate the process by which a protein is produced, and how a mutation can impact a protein s function. Background http://mssdbio.weebly.com/uploads/1//7/6/17618/970_orig.jpg DNA contains the directions to create the proteins that allow our bodies to function. Portions of the DNA that contain directions for a single protein are called genes. Because DNA is delicate we DO NOT want to remove it from the nucleus and instead it makes a copy of the directions using RNA. RNA polymerase is a protein that copies the DNA and the finished copy is made of messenger RNA, or mrna. This copying process is called transcription. After transcription, the copy leaves the nucleus and is in the nucleus with special organelles called ribosomes. The ribosomes translate the directions from the copy to build a protein in a process called translation. Lastly, the protein must fold into its final shape in order to be able to perform a function. In this activity, your team will be making a copy of the CFTR gene that contains the directions for creating the CFTR protein. This protein is a transport protein that embeds itself in the cell membrane and regulates certain substances (Na+ and Cl-) moving in and out of the cells in the skin, pancreas, and lungs. If this protein is not built correctly, and therefore not able to function correctly, these substances cannot move in/out of the cell and cause a thickening and build-up of mucus. This causes the condition known as cystic fibrosis. Materials Scissors Tape Plastic bags Normal CFTR Gene template Mutated CFTR Gene template mrna Nucleotides template trna template RNA Polymerase template Ribosome template Pipe cleaners Glue dots sheet (1) Bowl 10 Glycine beads Methionine beads Alanine beads Arginine beads Asparagine beads Aspartic acid beads Cysteine beads Glutamic acid beads Glutamine beads Histidine beads http://learn.genetics.utah.edu/content/disorders/whataregd/cf/images/cfchannel.jpg Isoleucine beads Leucine beads Lysine beads Phenylalanine beads Proline beads Serine beads Threonine beads Tryptophan beads Tyrosine beads Valine beads Cell Membrane template
Procedure/Directions Your lab team will be given tasks, or directions, to perform on the left. Record your questions, observations, or required response on the right when space is available. 1 Obtain the following supplies: Scissors Tape small plastic bags Normal CFTR Gene sheets ( pgs) Mutated CFTR Gene sheets ( pgs) mrna Nucleotides sheets ( pgs) trna Templates sheets ( pgs) RNA Polymerase sheets (1 pg) Ribosome sheets (1 pg) Part A: Set-Up Cut out the Normal CFTR Gene and Mutated CFTR Gene strips along the dotted line and tape each end together. Make sure to tape the correct ends to one another to create a single DNA strand of the normal CFTR gene AND the mutated CFTR gene (see image.) Cut out the four sheets of mrna nucleotides and put each of the four nucleotides into a different plastic bag (see image). Cut out the trna Templates and put them in a plastic bag. Cut along the dotted lines on the RNA Polymerase and Ribosome sheets.
1 Part B: Transcription Get into a team of. Within your team separate into pairs. One pair will be following the procedure with the Normal CFTR Gene and the other pair will be following the EXACT same procedure with the Mutated CFTR Gene. In this part of the activity, your team will be making a copy of the normal and mutated CFTR gene that contains the directions for creating the CFTR protein. Don t forget TRANSCRIPTION OCCURS IN THE NUCLEUS Mutated a. Where does transcription occur? Normal RNA polymerase is the protein that functions to unzip, or open, the DNA double helix and allow RNA nucleotides to match up to the DNA nucleotides. Remember Cytosine bonds to Guanine and Adenine bonds to Thymine. In RNA, Thymine becomes Uracil. Notice on your DNA and RNA nucleotides that the base ends match the base to which they bond (see image). a. What are the DNA nucleotides? b. What are the RNA nucleotides? c. Which nucleotides match or bond to one another? Place your RNA Polymerase template on the table (each pair needs a template). Feed the START end of your CFTR gene into and through the cut you made in the RNA polymerase sheet (see image). Your team will be sharing the RNA nucleotides and tape. 6 Starting at the START end of the CFTR gene, match an RNA nucleotide to the first DNA nucleotide. For example, the Adenine DNA nucleotide will match with a Uracil RNA nucleotide (see image). You are NOT taping the DNA sequence to the RNA nucleotides, ONLY TAPE THE RNA NUCLEOTIDES TOGETHER
7 Move to the next DNA nucleotide and find the matching RNA nucleotide. Once you have found the correct RNA nucleotide, tape it to the first RNA nucleotide (see image). Try to tape the RNA nucleotides together as straight as possible. 8 As you move down the CFTR gene, slide it through the RNA polymerase. Continue this process of copying the DNA nucleotides with the matching RNA nucleotides until you reach the end of the CFTR gene. Make sure you are taping the RNA nucleotides together as you move down the CFTR gene. 9 Remove the CFTR gene from the RNA polymerase. You have completed an RNA copy of the CFTR gene. This copy is called messenger RNA, or mrna. In an actual cell, this process would be repeated with the same CFTR gene and RNA polymerase many times, to create multiple mrna copies. a. What is the function of RNA polymerase? b. What is mrna? Why is it important to create mrna rather than use actual DNA for the next step? c. Summarize the transcription process.
1 Part C: Translation In this part of the activity, your team will be using the mrna copy of the CFTR gene you created in transcription to decode the order of amino acids that make up the CFTR protein. a. What is the purpose of translation? Obtain two pipe cleaners and a paper bowl for your team. To simulate the structure of proteins, you will be using plastic beads to represent the 0 amino acids that make up protein. Using the paper bowl, collect of every amino acid (colored beads) EXCEPT glycine and methionine. Collect 10 glycine (clear beads) and methionine (clear star beads). First, the mrna copy must leave the nucleus and move to a ribosome in the cytoplasm of the cell. Your team will be sharing the trna templates and beads/amino acids. Place your Ribosome on the table. A ribosome is actually made up of ribosomal RNA (rrna) and protein. Feed your mrna copy into the ribosome with the nucleotides facing up (see image). Only the first three mrna nucleotides should be visible in the window of the ribosome. a. Why do you think it is important for the mrna copy to leave the nucleus? 6 Every three mrna nucleotide bases are called a codon. Each codon is actually a -base code for a specific amino acid. There are 6 possible codons that code for 0 amino acids. This means some amino acids have more than one codon.
7 8 9 Your instructor MAY provide you with an Amino Acid Chart that contains all of the 6 codons and the amino acid that each one codes. If not, you will only be using the trna A A G A A molecules to determine the amino acids. Transfer RNA (trna) float around in the cytoplasm near ribosomes and the endoplasmic reticulum. amino One end of a trna molecule has an amino acid acid Isoleucine attached and the other end has a set of bases that can match up to an mrna codon. This -base trna code located on trna is called the anticodon. A A U A G A Using the trna molecules that were cut out earlier, find the match for each codon on your mrna copy. For example, the first mrna codon is AUG so the anticodon is UAC, which codes for the methionine amino acid (see image). anticodon A A A A 10 11 1 Once you have a trna match, look at the amino acid (pictured at the top of the trna). Find the bead that matches in your weighing boat. Slightly bend the end of the pipe cleaner to prevent the amino acids/beads from sliding off the end. Place the amino acid/bead on your pipe cleaner and slide it to the end (see image). Slide the mrna copy through the ribosome until the next bases, or codon, is visible. Find the trna match to determine the amino acid. Find the corresponding bead and slide it onto the pipe cleaner. 1 Continue this process until you reach the end of the mrna copy and hit a STOP codon. Fold the end of the pipe cleaner to prevent the amino acids/beads from falling off. You have just created an amino acid chain. a. Summarize the translation process.
1 Part D: Protein Folding So far you have made an mrna copy of the CFTR gene and decoded the copy to determine the amino acid order. You have an amino acid chain, but it is not yet a functional protein. Different amino acids interact and bond with each other causing the amino acid strand to fold and create a protein that can perform a function. Obtain a sheet (share among your team). Place a in each of the 6 spaces of the methionine amino acid/bead (see image). http://www.piercenet.com/media/prostructurefig1.gif For your amino acid strand, methionine and glycine will bond to each other. Starting at methionine, follow the amino acid chain until you find a clear bead (glycine). Fold the amino acid strand and connect glycine to a space in methionine at one of the s (see image). glycine methionine Continue down the amino acid chain, folding and connecting any glycine to the next open space on methionine, until all of the glycine amino acids on the chain have been attached. You now have a completed protein Mutated 6 Normal
1 Part E: Protein Function Now that your team has completed a normal and mutated CFTR proteins, compare their structures. CFTR functions to move salts in/out of cells. You have only created a portion of this protein that attaches and anchors the remaining protein to the cell membrane. The complete CFTR protein is actually more than 1,00 amino acids long The part of the normal CFTR protein that you created is responsible for connecting the CFTR protein to the cell membrane. The green portion of your normal protein would be the active site that binds to a portion of the cell membrane. a. Compare and contrast the structure of your normal and mutated CFTR proteins. Your instructor has taped up a simulated cell membrane. Take your normal and mutated CFTR proteins to the simulated cell membrane and see if the green active site binds and stays attached to the green attachment point on the cell membrane (see image). You have completed this activity DNA RNA protein is known as the central dogma of genetics, and you now have first-hand experience as to how this process happens. In actuality, your cells can transcribe a gene that is 1,00 base pairs and produce the protein it codes for within 1. seconds Med Bio Lab 0: The Cell Membrane The cystic fibrosis transmembrane conductance regulator (CFTR) protein creates a channel for chloride ions (Cl -) to move through the cell membrane of epithelial cells lining the lungs, pancreas, skin, and other surfaces of the body. NORMAL PROTEIN: If your CFTR protein is normal and attaches the cell membrane, it will allow Cl to move through the cell membrane and maintain a balance of ions on the outside and inside of the cell. As a result of this balance, mucus within the airways hydrated and functioning correctly. MUTATED PROTEIN: If your CFTR protein is mutated and DOES NOT attach or open Cl cannot move through the cell membrane. This creates an imbalance in the amount of ions on the outside and inside of the cell membrane and affects the amount of water in the cell. As a result, mucus becomes extra sticky and is difficult to remove from the lungs. This includes any bacteria trapped in the mucus, which can cause numerous respiratory infections. This is only ONE example of a symptom of cystic fibrosis. Cl#$# Cl#$# Cl#$# Cl#$# Cl#$# Outside Lung Airway mucus mucus mucus Cell Membrane Inside Lung Airway
Analysis & Interpretation Analysis Questions answer questions on a separate sheet of paper 1. Compare and contrast how the normal and mutated proteins adhered to the cell membrane? How did the mutation impact the function of the CFTR protein?. What happened to the green active site of the protein in the mutated CFTR protein? Why is this a problem?. Hypothesize what might happen if the deletion mutation was at the end of the gene rather than towards the beginning.. Explain how the CFTR gene leads to the creation of the CFTR protein?. Approximate how many minutes it took to complete this activity (a close estimate is fine). The complete CFTR gene is actually 0,000 base pairs long. In this activity, you transcribed and translated a section that was 96 base pairs long. Use a ratio to determine how long it would take you to transcribe and translate the entire CFTR protein. 6. There are actually many different types of mutations that can occur in the CFTR gene, any of which can cause cystic fibrosis. More than 70% of cystic fibrosis cases are caused by a -nucleotide deletion that results in the loss of phenylalanine at the 08 th amino acid. Research and determine the second and third most common types of mutations that cause cystic fibrosis. Cite your source(s). 7. Construct an explanation based on evidence for how the structure of DNA determines the structure of proteins, which carry out essential functions of life through systems of specialized cells. Research and cite at least one source for your explanation.