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22 Proteins, the Essence of Life Proteins, the Essence of Life Simulating the Process of Protein Synthesis Proteins are the molecules that carry out the “business of living”. Humans can synthesize over 50,000 different proteins. How does a cell synthesize so many different proteins? The blueprint for the synthesis of these proteins is found in molecules of DNA. DNA contains the genetic information needed for protein synthesis. DNA is restricted to the nucleus in eukaryotic cells yet the actual synthesis of proteins occurs on ribosomes found in the cytoplasm of cell. This implies that there must be some sort of intermediate molecule that is responsible for moving the information from the nucleus out to the cytoplasm. That intermediate is RNA. There are two parts to protein synthesis, transcription and translation. Transcription is the process of converting the information encoded in the DNA molecule into a messenger RNA molecule. This occurs in the nucleus. Translation is the actual synthesis of a protein or polypeptide chain. This occurs in the cytoplasm and requires several components. The ribosome serves as the workbench for this process by bringing together the mRNA instructions with the tRNA carrier of the amino acid. PURPOSE In this activity you will simulate the process of transcription and translation. These processes will lead to the formation of a protein from a DNA gene. MATERIALS copy of a portion of a double helix DNA molecule scissors kits to simulate protein synthesis copy of mRNA codons clear tape PROCEDURE Transcription 1. The following should be cut prior to class: DNA strand and mRNA codons. Tape the two strands of the DNA molecule together to form one long DNA strand. Obtain a kit that contains the models needed for this simulation. Answer questions 1-3 on the student answer sheet. 2. To begin transcription, enzymes break the weak hydrogen bonds between the base pairs of the two DNA strands. To simulate this use scissors to cut the double stranded DNA into two single strands. This demonstrates how the DNA molecule unwinds. Answer questions 4-6 on the student answer page. 560 Laying the Foundation in Biology Proteins, the Essence of Life 22 3. Notice that many of the DNA nucleotides are in groups of three nucleotides. This grouping represents a codon. A DNA codon is a group of three nucleotides that will determine which amino acid will be placed in the polypeptide chain. Answer question 7 on the student answer page. 4. Transcription occurs when mRNA nucleotides base pair with DNA nucleotides. The enzyme, RNA polymerase, is responsible for base pairing the correct RNA nucleotides to the correct DNA nucleotides. Only one side of the DNA molecule is transcribed. Transcribe the top side of the DNA molecule by base pairing the appropriate mRNA base pairs using RNA polymerase. Tape the RNA nucleotides together to make one long mRNA strand. Answer questions 8 and 9. 5. Remove the mRNA from the DNA gene and “zip” the DNA gene back together with tape. At this point the mRNA is an immature mRNA molecule and must be processed. In eukaryotic genes, there are nucleotides that are not used or expressed. One might view these DNA nucleotides as “junk DNA”. They are called introns because they are intervening sequences. RNA polymerase can not tell the difference between these intron (intervening) and exon (expressed) regions. Therefore these introns are also found on the mRNA. Look at the mRNA. Notice a sequence that is not grouped in three nucleotides. This represents an intron. In reality, a eukaryotic gene has many introns and introns are much longer than four nucleotides. The intron must be removed, and a structure called a spliceosome is responsible for removing the intron. Place the mRNA on the paper spliceosome. Using scissors, remove the intron. The mRNA nucleotides that remain are called exons because they will be expressed. The spliceosome will splice the remaining exons together. Tape the exons together using tape. Answer questions 10-12. 6. The next thing that occurs is the addition of a 5’ cap, which is composed of a number of RNA nucleotides. The purpose of the 5’ cap is to protect the RNA molecule from being degraded in the cytoplasm and also aids in recognition between the ribosome and mRNA. Cut out the 5’ cap and add it to the mRNA. Answer question 13. 7. At the 3’ end, a series of adenine nucleotides, called a poly-A-tail, are added. The function of the poly-A-tail seems to also aid in protecting the mRNA from being degraded and plays a role in determining how many times the mRNA can be translated. The tail can average between 100 and 200 adenines long. Answer question 14. Laying the Foundation in Biology 561 22 Proteins, the Essence of Life 8. After splicing and adding the 5’ cap and 3’ tail, this mature piece of mRNA is ready for the actual synthesis of a protein. The process of synthesizing a protein is called translation. The sequence of the mRNA nucleotides determines the order of the amino acids in the protein. Notice that the RNA nucleotides are in groups of three nucleotides. This grouping represents a codon. The RNA codon is a group of three nucleotides that will determine what amino acid will be placed in the polypeptide chain. Remember the RNA codon has it sequence determined by the original DNA codon. Below is the genetic code based RNA codons. For example if an mRNA codon is UUU, this is the code for the amino acid, phenylalanine. Answer questions 15 and 16. 9. Determine sequence of amino acids encoded in your mRNA and record them on your student answer page under question 17. Translation 1. Translation is the actual synthesis of a polypeptide chain. Move your mRNA from the hypothetical nucleus to the hypothetical cytoplasm on your desk. There are three necessary components for translation to take place. They are: a. MRNA — This will determine the sequence of the amino acids b. tRNA charged with an appropriate amino acid — This molecule brings the appropriate amino acid to the correct codon on the mRNA c. Ribosome — This is a workbench where the actual synthesis of the polypeptide will occur. Answer question 19. 562 Laying the Foundation in Biology Proteins, the Essence of Life 22 2. Another important RNA molecule used in the process of translation is transfer RNA or tRNA. For each mRNA codon in this model, there is a specific tRNA. tRNA is a single stranded molecule that folds and base pairs with itself to form a clover-leaf shaped structure. At the bottom of the tRNA is series of 3 nucleotides. These nucleotides are complementary to the mRNA codon (A-U and G-C) and are called the anti-codons. For example if an mRNA codon is UUU, then the tRNA anticodon found on the bottom of the tRNA is AAA. Look at the tRNA models in the kit and notice the anticodon found at the bottom. For each mRNA codon you listed in question 17, list the anticodon in question 18. 3. The function of the tRNA is to bring the appropriate amino acid to the appropriate mRNA codon. This is works because there is a charging enzyme responsible for bonding the correct tRNA with the correct amino acid. This bonding requires ATP. Find the charging enzyme. Notice that the active site fits a tRNA and an amino acid. 4. Find an amino acid. The Styrofoam ball represents the central carbon. Notice that hydrogen is attached as are an amine group, and a carboxyl group. Also notice there is a jumbo craft stick representing the variable R group. On the jumbo stick is a drawing of the variable R group. Each amino acid has a different colored stick and a different variable R group. Answer question 20. Laying the Foundation in Biology 563 22 Proteins, the Essence of Life 5. Now, using the charging enzyme, combine each tRNA with its correct amino acid. This is done by using a paper clip to attach the amino acid (on the jumbo stick) to the tRNA (at the top end of the pipe cleaner). The correct amino acid is bonded to the correct tRNA by a sequence of nucleotides on the anticodon and the codon of mRNA. The charging enzyme will release the charged tRNA once the amino acid is bonded to it. Look at the answers for questions 17 and 18 to make sure that the correct amino acid is bonded to the correct tRNA. At this point every tRNA should have an amino acid attached to it. 564 Laying the Foundation in Biology Proteins, the Essence of Life 22 6. Find the ribosome. Notice that it is composed of two subunits, one that is large and one that is small. There are three places for the tRNA’s and one place for the mRNA. Now simulate the process of making the polypeptide chain, using your mRNA and your tRNA that have been “charged” with amino acids. Put the start codon (AUG) on the mRNA under the P site. Now find the tRNA with the complementary anticodon and place it on the P site. Once this is done, notice that a second mRNA codon is positioned under the A site. Find the tRNA with the complementary anticodon and place it on the A site. This should bring two amino acids (found at the top of the tRNA) next to one another. 7. Join the two together amino acids together by joining the carboxyl group on the first amino acid to the amine group on the second amino acid. Fasten the two together with a paper clip at their amine and carboxyl sites. The paperclip simulates the covalent bond that will form between these two functional groups. It is called a peptide bond. Notice which atoms are no longer visible. This means that a water molecule has been removed from the chemical reaction. Laying the Foundation in Biology 565 22 Proteins, the Essence of Life 8. Remove the tRNA on the P site over to the E site leaving the amino acid behind. This tRNA then will leave the ribosome altogether. Move the mRNA down one codon and begin the process again. The tRNA at the A site moves over to the P site. The A site is now open to receive the next tRNA that is complementary to the mRNA below. This is called translocation. Continue this process until the stop codon is reached. 566 Laying the Foundation in Biology Proteins, the Essence of Life 22 9. When the stop codon is reached, a release factor binds with the mRNA stop codon. This causes the ribosome to disengage, releasing the polypeptide chain and the mRNA. This process is called termination. At this point you should have synthesized a model of a polypeptide chain. 10. Answer the remaining questions on your student answer page. Laying the Foundation in Biology 567 22 Proteins, the Essence of Life Name______________________________________ Period _____________________________________ Proteins, the Essence of Life Simulating the Process of Protein Synthesis SIMULATION QUESTIONS 1. Where is DNA located in the cell? Describe the structure of the DNA molecule. 2. DNA is a long polymer made of many subunits. What are those subunits called and what parts do they have? 3. Write out both the top and bottom portion of the DNA molecule in the space below. 4. Define the term gene and explain exactly what a gene does. 5. How many genes are found on a chromosome? 568 Laying the Foundation in Biology Proteins, the Essence of Life 22 6. What biological molecule do the scissors represent? 7. What is a DNA codon? 8. What enzyme is responsible for base pairing the RNA nucleotides to the DNA gene? 9. Fill in the chart below for the base pairing of DNA to RNA nucleotides DNA RNA Adenine ___________ __________ Guanine Thymine ____________ __________ Cytosine 10. What are introns and exons? 11. What molecule is responsible for removing introns and rejoining exons? 12. Write out the RNA codons that remain after the intron has been removed. Laying the Foundation in Biology 569 22 Proteins, the Essence of Life 13. What are two purposes of a 5’ cap? 14. What are two purposes of poly-A-tail? 15. How is mature mRNA different from immature mRNA? How does mRNA determine the sequence of a polypeptide chain? 16. What is an mRNA codon? 17. Determine the amino acid sequence using the mRNA codon chart below. 570 Laying the Foundation in Biology Proteins, the Essence of Life 22 18. List the complementary anticodons that will be found on the tRNA for the mRNA in question 17. 19. What is translation and where does it occur? What four items are needed for translation to occur? 20. What is the structure of an amino acid? 21. What type of bond is made when amino acids are joined? 22. How many water molecules were removed from the polypeptide chain? 23. Write in the space provided the amino acids were used to make this protein and what molecule was removed. 24. Describe the process of termination. Laying the Foundation in Biology 571