Answer Key: Making Protein

BIOL230W Week 4 – Making Protein (Translation) We are continuing our journey following the flow of information in a cell. Last week we discussed how information from DNA is transferred to a mRNA molecule and how expression is regulated resulting in differential gene expression. This week we will be discussing the next step in gene expression, transferring the information in the mRNA into a functional protein. This process is known as translation. Day 1 Learning Objectives 1. Explain how mRNA is translated into different locations (cytoplasm vs. ER) and how this impacts final protein localization (cytoplasm vs. export vs. membrane). 2. Describe the structure and the role of the translocon, Sec 61, in the localization of proteins. 3. Describe the role of a peptide bond. 4. Explain the importance of R-groups or side chains in protein function. 5. Compare and contrast a DNA polymer and a polypeptide. 6. Describe the structure of tRNA and how its structure contributes to function. Central Dogma Recap Last week we determined that there is a deletion in the DNA of a patient (Maggie) with cystic fibrosis. This week we want to continue our exploration of how this different genotype results in a different phenotype. Use the image on the screen to answer the following questions. 1. Where, in relation to the cell, do proteins function (Hint - think of three general areas)? Endoplasmic reticulum, mitochondria, chloroplast, cytoplasm, and plasma membrane. 2. Which organelle will read the information in the mRNA and convert it to a protein sequence? Ribosomes 3. Where in the cell is the ribosome located at the start of translation? Cytoplasm Mini-Lecture: Co-translation Translocation Look out for the answers to these questions during the mini-lecture! 4. The translocon allows passage between which two areas of the cell? The endoplasmic reticulum and the cytoplasm 5. If the signal peptide of a new protein directs the ribosome to the translocon would it need to be translated first or last? First 6. If translated through the translocon, where will be the protein’s ultimate destination? Endosome 7. What characteristic do amino acids that span the hydrophobic core of a membrane share? 8. What would cause a protein normally destined for the PM to get “stuck” in the ER? 1 BIOL230W Week 4 – Making Protein (Translation) Small Group Discussion: What is a protein? 9. What monomers make up a protein polymer? Polypeptides (amino acids) 10. What makes one amino acid different from another? Side chain structures 11. What is the name of the bond that connects monomers into the polymer (polypeptide)? Peptide bond Small Group Discussion: Compare and contrast polypeptide and DNA polymer. 12. How is a polypeptide analogous to a molecule of DNA or mRNA? The backbones repeat, both have an N-terminus and a C-terminus (or a 5’ and 3’ end in the case of DNA and mRNA) 13. How are they different? DNA is double-stranded, polypeptide is single-stranded. Additionally, they have different side chains Mini Lecture: What is a protein? Small group discussion: (Ready!) Player 1 - tRNA Now that we have all that information from the DNA transcribed to RNA, how is the information translated to the language of protein? We will be focusing on three main “players” or molecules that complete this process: tRNA, G-proteins, and the ribosome. The first player is another non-coding RNA molecule, tRNA, that acts as an adapter between the two languages! 14. Using the image on the screen, describe in your own words how the tRNA molecule can act as an adapter between RNA and protein. tRNA decodes mRNA and translates it into amino acids Day 2 Learning Objectives 1. Describe how G-proteins are activated and inactivated. 2. Explain the role of G-proteins in the process of translation. 3. Describe the structure of a ribosome and how its structure contributes to its function (e.g., ribozyme function, A-site, P-site, E site). 4. Compare and contrast the function of different non-coding RNAs we have discussed in class. 5. Outline the factors (proteins) and the sequence of events involved in initiation of translation. 6. Analyze the interaction of codons and anticodons and match them to the appropriate amino acid using the provided genetic code. 2 BIOL230W Week 4 – Making Protein (Translation) 7. Describe how the genetic code is redundant and how it affects codon: anticodon pairing (wobble). 8. Outline the major steps of translation (initiation, elongation, termination) using appropriate key words. Small Group Discussion: Player 2 - G-Proteins Using the image on the screen answer the following questions: 1. G-proteins bind to a variety of target proteins in addition to one common molecule. What molecule do all G-proteins bind to and hydrolyze? GTPase, guanine nucleotide binding protein 2. Describe the mechanism of action of the GEF and its effect on G-protein function. It helps turn GDP into GTP 3. Describe the mechanism of action of the GAP and its effect on G-protein function. GAP turns GTP into GDP via hydrolyzation Mini-Lecture: Player 3 – Ribosome (rRNA, proteins and ribozyme) Mini- Lecture: Initiation of Translation 4. What is similar between the function of rRNAs of the small ribosomal subunit and the tRNA methionine in the initiation of transcription (Hint – this is similar to the function of non-coding RNA’s we have discussed)? Both rRNAs and tRNA help with sequence specificity Small Group Activity: Elongation of Translation Using this Figure 1 and Figure 2 answer the following questions concerning the three main events required for elongation (building a polypeptide from the information in an mRNA molecule). 3 BIOL230W Week 4 – Making Protein (Translation) Figure 1. Steps of elongation. Figure 2. The universal genetic code. Use this table to match codons to their respective amino acids. Note the codons are organized such that the first nucleotide of the codon in each row is listed to the left of the table and the second on the top. The third is listed to the right. The sequence listed is the sequence of the CODON from 5’ to 3’. Step 1: Binding of aminoacyl tRNA. 5. What is the name and function of the G-protein in this step of elongation? Elongation Factor (EF-TU) 4 BIOL230W Week 4 – Making Protein (Translation) 6. If the mRNA codon 5’ ACU 3’ is in the A-site of the ribosome, what is the sequence of the complementary tRNA anticodon 5’_______ 3’ and which amino acid would be added? 3’ UCA 5’, which would add Ser 7. If the anticodon 5’ ACU 3’ is found on the incoming aminoacyl tRNA, which amino acid would it be carrying? Thr Step 2: Peptide bond formation – Ribozyme! 8. Describe the covalent bond that is formed (give the name of the bond and what molecules it connects) and the bond that is broken (what molecules was the bond connecting) by the peptidyl transferase (i.e., the ribozyme) center of the ribosome? Peptide bonds with amino acids are made at P and A sites. The amino acid is broken when a covalent bond with tRNA is formed 9. Compare and contrast the ribozyme with the other non-coding RNAs we have talked about so far. It is not as specific as others (represents enzyme activity) Step 3: Translocation 10. How many nucleotides does the ribosome move during translocation (not shown in image)? 3 11. What is the name of the G-protein that functions in this event? EF - G 12. What is functioning as the GAP in this part of elongation (Hint: what does a GAP do)? Ribosome facilitates hydrosis Small Group Activity: Elongation Practice 13. Now think back to Maggie’s DNA. Using the provided genetic code, translate Maggie’s mRNA and compare it to the protein sequence of the individual without CF. Primary Transcript (pre-mRNA): 5’ GCGUAUGAAAAACAGUUGCAG......AUCAUCUUUGGU...CGGUAAAGC...3’ Mature mRNA: 5’ GCGUAUGAAAAACCAG......AUCAUCUUUGGU...CGGUAAAGC...3’ Amino Acid Sequence: Ala-Tyr-GluLys-Pro…Ile-Ile-Ser-Gly…Arg-STOP 5 BIOL230W Week 4 – Making Protein (Translation) Primary Transcript (pre-mRNA): 5’ GCGUAUGAAAAACAGUUGCAG......AUCAUCGGU...CGGUAAAGC...3’ Mature mRNA: 5’ GCGUAUGAAAAACCAG......AUCAUCGGU...CGGUAAAGC...3’ Amino Acid Sequence: Ala-Tyr-Glu-Lys-Pro…Ile-Ile-Gly…Arg-STOP Figure 2. Sequence of the CFTR gene from an individual without CF (top) and the patient’s DNA (bottom). 14. What is different between Maggie’s Protein and the individual without CF? Maggie is missing the Ser protein 15. What is the characteristic of the phenylalanine side-group? Nonpolar and hydrophobic Mini-Lecture: Elongation, Wobble and Translation Termination Day 3 Learning Objectives: 1. Describe the different levels of protein structure (primary, secondary etc…). 2. Explain which interactions dictate the levels of protein structure. 3. Compare and contrast structural motifs (super-secondary structure) and domains. 4. Compare and contrast and identify the 3-dimensional structures of different proteins using domain box diagrams, ribbon diagrams and ball and stick diagrams Small Group Discussion: CFTR Protein Structure Now that the protein has been translated it needs to fold into a structure that will allow it to function correctly. This will involve the interaction of atoms at several levels within the polypeptide and sometimes with other polypeptides, as well. Use the image of Figure 3 on the screen to answer the following questions. Figure 3. Ribbon diagram of the CFTR protein. The image on the left shows a normal CFTR protein and the image on the right indicates a protein harboring the ∆508 mutation. Tubular structures represent alpha helices and flat arrow indicate beta strands. Indicated in yellow is the phenylalanine that is missing in the ∆ 508 mutant as well as the two amino acids upstream of the phenylalanine for purposes of comparison in the mutant. 6 BIOL230W Week 4 – Making Protein (Translation) 1. From Day 2, is there a change in primary structure in the mutant gene? Addition (circled) 2. What secondary structures do you see in the images? Alpha helicase and beta strands (sheets) 3. Is there a change in secondary structure? Yes, there is an additional helix 4. Is there a change in tertiary structure? Yes, 3’ extra alpha helix changes the shape 5. Is there a change in quaternary structure? No Now use the image of figure 4 on the screen to answer questions 35-39 Figure 4. Stick and ball representation of the normal and ∆508 mutant CFTR proteins. (Left) Shown in yellow are the phenylalanine amino acid that is missing the ∆508 mutant as well as the tryptophan that it interacts with on the interior of the protein. (Right) Shown in yellow are the amino acids proximal to the missing. phenylalanine (above) and the tryptophan (below) that is now been positioned toward the exterior of the protein. 6. What is the characteristic of the phenylalanine side-group? Nonpolar and hydrophobic 7. Where would you expect phenylalanine to be in a globular protein? On the inside of the protein 8. What other amino acids would you expect to interact with phenylalanine in the center of the protein? Alanine, Leucine, Tryptophan, Tyrosine 9. What is the characteristic of the tryptophan side-group? Nonpolar and hydrophobic 10. What does the change in position of the tryptophan in the mutant suggest about the folding of the protein? 7 BIOL230W Week 4 – Making Protein (Translation) Mini lecture: Hierarchy of Protein Structure Small Group Discussion: Protein Domains Figure 6. (A) Domain diagram of RecQ helicases from several organisms. The Zn-binding motif (brown), unique to the RecQ family, is depicted in the C-terminus of the ATPase domain. Most RecQs also share the HRDC (helicase-andribonuclease D/C-terminal) domain at the C-terminus, although it is not essential for helicase activity. (B) Structure of WRN RQC domain (blue, ribbon model) bound to the 14-base-pair DNA molecule (orange, tubular model). The secondary and supersecondary structures are labeled. Side chains of the key interacting amino acids are shown as stick models. (C) A view following 90° rotation along the y- axis. 11. In Fig 6(A), what do the “boxed” structures represent? What do the thinner lines represent? Thin structural proteins (thinner) and 3’ domains (boxed) 12. Does the E. coli protein have more or less domains compared to the WRN human? Why is this evolutionarily important? Fewer domains, less functionality 13. What hierarchy of structure is represented in Fig 6 (B) and (C)? 8 BIOL230W Week 4 – Making Protein (Translation) Secondary, super secondary 14. Does helicase activity require energy (*hint, look at domain functions)? Why or why not? Yes, it has the ATPase domain which requires ATP for activity 15. Are there any super secondary structures represented in the figure? How do these generally differ from domains? Yes, there are alpha helix super secondary structures 45. What secondary structures (sheets, helices or both) appear to lead to the association of WRN with one of the grooves of DNA? (*hint – what part of the domain appears to be interacting with the nucleobases?) End of Week 3 Summary Drawing - Optional Summarize what you have learned this week with a drawing on a blank sheet of paper. Draw and label a ribosome translating an mRNA. Include the ribosome (both subunits), the mRNA (5’ and 3’ ends labelled), an uncharged tRNA, and aminoacyl tRNA, and active and an inactive EF-Tu, a start and a stop codon. Add anything else you can think of! Note: Drawing is a research-supported strategy to retain information, so we encourage you to complete this for practice. 9