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One Gene - One Polypeptide Background The genes for each of our traits are located on our chromosomes, which in turn are made up of DNA. Each chromosome represents a single molecule of DNA tightly wound around sets of proteins called histones. Each DNA molecule (chromosome) is made up of sequences nucleotides in varying orders and lengths. A molecule of DNA may be hundreds of thousands of nucleotides long, but is broken up into sequences of several hundred to several thousand nucleotides called genes that each code for a single polypeptide. Polypeptides are chains of amino acids that are eventually folded or joined together in the cell to form proteins. Recall that most proteins usually consist of between 2 and 4 polypeptide chains bonded together. These proteins form the molecular basis of our phenotypes; structural proteins are the building blocks of the body and enzymes control all of our metabolic processes. The process that a cell uses to convert a sequence of nucleotides into a specific polypeptide involves a variety of enzymes, protein factors, ATP, amino acids, and cellular organelles. This process can be summarized in two general steps; transcription and translation. During transcription the sequence of nucleotides in a gene in DNA is copied into a sequence of nucleotides in RNA. During translation tRNA serves as an interpreter between nucleotide language of nucleic acids and the amino acid language of proteins. We will be using paper models to simulate transcription and translation and learn how proteins are built from the DNA code. You will work with a partner to model the actual steps that a cell follows to carry out transcription and translation. Remember, cellular enzymes like RNA polymerase that do not have a brain, hands, or eyes are carrying out many of these steps. Thus, transcription and translation must proceed in a step-by-step chemical process that adds one nucleotide or one amino acid at a time. Pre-Lab Questions Answer the following questions in complete sentences. 1. Describe the relationship between DNA structure and genes. 2. What is the relationship between a gene and a protein (be specific)? 3. How does DNA ultimately determine our phenotype? 4. What are the base pairing rules that RNA polymerase follows during transcription? 5. Complete the table shown below. Original instructions in the form of : Molecule that is being synthesized: Location where this takes place: Transcription Translation Procedures: 1. Today your desk represents a cell. To set up your cell for protein synthesis you will need several structures and molecules. Get a piece of DNA and place it in the nucleus of your cell. Place a ribosome in the cytoplasm of your cell. Scatter 9 tRNA molecules throughout the cytoplasm. Finally scatter a set of amino acids throughout the cytoplasm. You will not use all of the amino acids. 2. Obtain a strip of paper labeled mRNA . 3. One partner in the group will model the role of the mRNA molecule and should transcribe the DNA code from the gene whose sequence is shown on the DNA, onto the mRNA molecule. Using a dry erase marker, this partner will write down the codons onto the spaces provided on the mRNA strip of paper, three letters per underlined space. The transcribed mRNA will then leave the nucleus and travel to the ribosome and bind to the small subunit of the ribosome. 4. Now you will begin translation. tRNA is the cellular interpreter. The other partner in the pair will interpret the first mRNA codon and write a complimentary anticodon onto the bottom of one laminated tRNA molecule using a dry erase pen. Meanwhile the mRNA student uses the codons in the mRNA to look up the amino acid that the tRNA student will get from the cytoplasm and attach to the tRNA with a small piece of tape. 5. Write the sequence of nucleotides from the DNA in the first row of the table below (3 letters per box). Write the corresponding mRNA codons in the second row of the table on the next page. List the corresponding sequence of nucleotides that represent the anticodons from each of the 9 molecules of tRNA you have created in the third row of the table. List the corresponding amino acids, determined by the mRNA codons, in the fourth row of the table. 1 2 3 4 5 6 7 8 9 10 DNA mRNA tRNA Amino Acid . 5. The mRNA should still be positioned in the ribosome. Now, the first tRNA molecule with its attached amino acid goes to the ribosome / mRNA complex and the tRNA anti-codon pairs with the first mRNA codon at the P site of the ribosome. 7. The next tRNA along with its amino acid molecule enters the A site of the ribosome. Only the tRNA anti-codon matching the next mRNA codon in the sequence can bind to this site. 8. The original amino acid is then transferred to the amino acid of the tRNA in the A site and the two amino acids are taped together. The tape represents a peptide bond. The empty tRNA leaves the P site and goes back into the cytoplasm. 9. The tRNA, mRNA and the attached chain of amino acids move to the left and into the P site. The amino acids should point vertically away from the tRNA, with the first amino acid being farther from the P site. The next matching tRNA arrives in the A site and delivers its amino acid. 10. Continue repeating this process until you have linked together four of the amino acids coded for by the gene on your DNA and have the fifth amino acid in the A site of the ribosome waiting to join the polypeptide. When you and your partner reach this point, both partners should make their own full page, detailed sketch of the model. You may use the blank back page of this lab handout for the drawing. This sketch should use color and show the relationship of components of protein synthesis including the following: a. The small ribosomal subunit, clearly labeled b. The large ribosomal subunit, clearly labeled, showing the P site and A site c. mRNA, clearly labeled and with nucleotide sequence clearly represented d. tRNA clearly labeled, showing the nucleotide sequence of its anticodon and with the polypeptide attached. e. Another tRNA, clearly labeled, showing the nucleotide sequence of its anticodon and its amino acid ready to be added to the polypeptide f. A labeled arrow showing the direction of mRNA / tRNA / polypeptide movement through the ribosome during translocation g. An arrow showing how and where the fifth amino acid and the growing polypeptide will be joined together 10. Continue the process of protein synthesis until a stop codon is reached. When the mRNA stop codon enters the A site of the ribosome the amino acid chain is released from the tRNA in the P site. The amino acid chain is now called a polypeptide. The small and large ribosomal subunits separate from each other and translation is complete. 11. Remove all tape from laminated pieces and use a paper towel to erase the dry erase marker from the laminated pieces. Post Lab Questions Answer the following questions in complete sentences. 1. Explain the overall function and process of transcription including where it happens in a cell and the purpose of any enzymes that are required. 2. Before mRNA can leave the nucleus it is chemically modified. We did not model this today. What happens to the mRNA when it is chemically modified in the nucleus? 3. Transcription and DNA replication are different processes with different outcomes, but they also have some similarities. Describe three similarities and three differences between DNA replication and transcription? 4. Explain the overall function and process of translation including where it happens in a cell and the purpose of any enzymes that are required. 5. tRNA is sometimes called the interpreter molecule of protein synthesis. Explain.