BIOL 200 (921): Final Exam Study Guide and Review questions

1. BIOL 200 (921):Final Exam Study Guide and Review questions FINAL EXAM (IN CLASS) FROM 1 PM TO 3:30 PM ON JULY 7, 2006 The final exam will cover material covered up to and including the Lecture on July 7 and will also include the pre-midterm material. Be prepared to integrate material covered in these lectures. Emphasis will be placed on understanding of general concepts, experimental approaches, and ability to interpret new information and data in light of what you know. You should supplement your class notes with details and questions from the textbook and the BIOL 200 web site material. You are responsible for the reading material listed in the outlines for each lecture, and the material provided in the lecture notes, tutorials and powerpoint slides. The Study Questions from the textbook given in the lecture outlines are a good way to review most of the topics covered. Reading in the text is designed to expand upon and support this material. You may be asked to consider information in such figures, as it relates to material we have covered in lecture.

2. BIOL 200 (921): Final Exam Study Guide and Review questions As a guide, here are some specific comments about questions in the relevant chapters: • Understand the relationship between structure, cellular composition and function of cell organelles and macromolecules. • Understand basic terminology and concepts in cell biology e.g. membrane transport systems, membrane transport processes, protein sorting and transport, photophosphorylation, oxidative phosphorylation, microtubules and associated proteins, cyclins and CDKs, DNA replication, cell division phases etc. • Understand how the biochemical and cell biological approaches/techniques we have discussed can be used to answer questions in cell biology. Be prepared to propose the use of systems and tools to approach a specific problem. • Concentrate on understanding cell biological processes e.g. membrane transport processes, protein sorting and transport, photophosphorylation, oxidative phosphorylation, microtubules/actin filaments and associated proteins, cyclins and CDKs, DNA replication, cell division, membrane structure and transport etc.

3. BIOL 200 (921): Final Exam Study Guide and Review questions Final exam preparation material: • For the Final exam, one sheet of 8.5 x 11 paper, double-sided, "study sheet" will be allowed as a memory aid. • Memorizing facts are not the goal of this course, you must be able to use information to solve problems and defend a point of view. Specific type of questions to consider: • There will be an essay question on a major cell biology topic in the final exam. • Short answer, Multiple-choice, definitions, true/false, fill in the blanks, small essay-type questions. Test objectives: Familiarity with terms, concepts, and basic principles covered in lectures. Ability to make connections between different topics covered. • Problem (e.g. explain experimental results which are presented, explain how to approach a particular problem, predict results from an experiment, etc). Test objectives: Ability to use information in new situations and to solve problems, depth of knowledge concerning basic concepts, understanding of approaches used to investigate cell biology. Ability to integrate information. Please study the problems and their solutions given in your textbook. • Structure-function relationships of cell, organelles, macromolecules etc. • Structural and functional differences between different cell types (e.g. animal, plant, bacterium) • Use of appropriate experimental methods/techniques to support cell biological hypotheses/theories/results • Ability to analyze a given set of data in the form of a Table or Figure pertaining to topics covered in lectures. • See the Study Questions from the textbook given in lecture outlines. • Some examples of exam questions are given below.

4. BIOL 200 (921): Final Exam Study Guide and Review questions Final Exam Review Questions Question 1. Briefly explain the structure-function relationship of the following: • Myosin-II filament: • trans Golgi network:

5. 1. The Myosin head tightly locked onto an actin filament. 2. ATP binds to the myosin head. The Myosin head released from actin. 3. The myosin head displaced by 5 nm. ATP hydrolysis. 4. The myosin head attaches to a new site on actin filament. Pi released. Myosin head regains its original conformation (power stroke). ADP released. 5. The myosin head is again locked tightly to the actin filament. 17_45_myosin_walks.jpg

6. one Golgi stack [Fig. 15-24]

7. Protein modifications in Golgi [Fig. 12-6 from Becker] Predict the location of enzymes, galactosyl transferase and sialic acid transferase

8. Question 2. Short Answer Explain in one or 2 sentences why plants need to have mitochondria in every cell, even when the sun is shining. There are no motor proteins that move on intermediate filaments. Can you suggest a reason based on the structure of the intermediate filaments? What does this tell you about motor proteins?

9. Question 3. Fill in the blanks: • A small GTP-binding protein called ___________ assembles around the neck of each coated vesicle and helps in pinching off the vesicle from the membrane. • __________ is the motor protein that moves along cytoplasmic microtubules and towards the plus end of the microtubule.

10. Question 4. Design an experimentusing appropriateexperimental technique(s) to study the kinesin-assisted transport of macromolecules. • Immunofluorescence microscopy: Primary antibodies bind to cytoskeletal proteins. Secondary antibodies labeled with a fluorescent tag bind to the primary antibody. Cytoskeletal proteins glow in the fluorescence microscope. • Fluorescence techniques: Fluorescent versions of cytoskeletal proteins are made and introduced into living cells. Fluorescence microscopy and video cameras are used to view the proteins as they function in the cell. • Computer-enhanced digital videomicroscopy: High resolution images from a video camera attached to a microscope are computer processed to increase contrast and remove background features that obscure the image. • In vitro and in vivo studies [Figs. 17-19 to 17-22, pp 585-88]

11. GFP protein of interest 1. GFP gene fused to gene coding for protein of interest 2. Transform cell with GFP-protein gene fusion. 3.Gene is expressed, targeted, protein functions. 4. Localize Green fluorescence with fluorescent. light microscopy (or confocal) GFP=green fluorescent protein Control-cytoplasmic GFP, i.e. no protein of interest fused onto GFP.

12. Question 5. Three phospholipids X, Y and Z are distributed in the plasma membrane as shown. For which of these phospholipids does a flippase probably exist? a. X onlyb. Z onlyc. X and Yd. Y and Ze. X and ZExplain your answer briefly.

13. ANSWER: Three phospholipids X, Y and Z are distributed in the plasma membrane as shown. For which of these phospholipids does a flippase probably exist? C = X and Y Explain your answer briefly. Lipids are inserted into the membrane on the lumen face of the SER (which corresponds to cytosolic side of the plasma membrane). Therefore both X and Y would need flippases to move to the external leaflet. Must know that flipping can’t occur spontaneously and that all lipids are inserted into the same leaflet. Must mention SER.

14. Question 6. Based on your understanding of cell structure and function, please state if the following statements are TRUE or FALSE and provide an explanation for your choice. • a) Membrane-bound and free ribosomes, are structurally and functionally identical and differ only in the proteins that they happen to be making at a particular time. • b) All of the glycoproteins in the intracellular membranes have their oligosaccharide chains facing the lumen, whereas those in the plasma membrane have their oligosaccharide chains facing the outside of the cell • c) The pH of the chloroplast thylakoid space (or lumen) increases in the light.

15. a) Membrane-bound and free ribosomes, are structurally and functionally identical and differ only in the proteins that they happen to be making at a particular time. TRUE. Cytosolic ribosomes are translating proteins with no sorting signals/ proteins destined to remain in the cytoplasm. Ribosomes of the RER are translating proteins with ER signal sequence, i.e. the subsequent events leading to attachment of ribosomes to the ER membrane are a result of the interaction of the signal peptide amino acid sequence with the signal recognition particle (SRP) and the SRP receptor in the ER membrane.

16. b) All of the glycoproteins in the intracellular membranes have their oligosaccharide chains facing the lumen, whereas those in the plasma membrane have their oligosaccharide chains facing the outside of the cell TRUE. For glycoproteins as the oligosaccharide chains are transferred onto the protein in the ER lumen, then further modified in the Golgi. When those proteins are secreted via exocytosis, the oligosaccharide chains are facing the extracellular space or comment on extracellular space being topologically equivalent to endomembrane lumen.

17. c) The pH of the chloroplast thylakoid space (or lumen) increases in the light. FALSE. The pH of the thylakoid space decreases due to accumulation of protons driven into the space by the flow of electrons along the photosynthetic electron transport chain.

18. QUESTION 7: Tubulin polymerizationA typical time course for polymerization of purified tubulin to form microtubules is shown below. Explain the different parts of the curve labeled A, B and C. How would the curve change if centrosomes were added at the onset?

19. ANSWER: Tubulin polymerization A typical time course for polymerization of purified tubulin to form microtubules is shown below. A – lag phase. Nucleation of MTs B – rapid polymerization C - equilibrium How would the curve change if centrosomes were added at the onset? Lag phase eliminated

20. Question 8. Circle the correct answer on the exam. A. What are the molecular components of ATP? • adenine, thymine, and phosphates • adenine plus three phosphates • adenine, ribose and three phosphates • alanine, ribose and three phosphates • alanine, threonine and phosphate B. A yeast strain that has a mutation that prevents vesicle fusion with the plasma membrane may have a mutation in the gene that codes for: • Clathrin • COP protein • Dynamin • Adaptin • V SNARE C. What keeps the Golgi apparatus in the middle of the cell, and away from the periphery? • Intermediate filaments • Kinesin • Dynein • Myosin • Actin

21. Question 9. Essay Question. Sorting of proteins to the correct intracellular compartment is essential to cells. I-cell disease is a rare human disorder in which enzymes normally found in lysosomes are actually secreted from the cell. Describe the process of synthesis of lysosomal enzymes in normal individuals in comparison with individuals affected by I-cell disease. Your description should begin with the mature mRNA in the cytoplasm that encodes a lysosomal enzyme and describe how the protein produced by translation of this mRNA is sorted through each successive organelle. Do not describe the details of the translation process.

22. Normal cotranslational insertion steps up to the occurrence of the defect. Protein synthesis begins on a free ribosome in the cytosol The first part of the protein synthesized (N terminus) contains a signal sequence. This sequence binds to a signal recognition particle (SRP) which binds to the ribosome. It causes translation to pause The SRP binds to a SRP receptor in the ER membrane. This leads to the formation of a translocation channel that is connected to the ribosome. The SRP is released. Translation resumes. The hydrophobic signal sequence binds to the hydrophobic core of the membrane that is exposed in the interior of the translocation channel. The rest of the protein proceeds into the lumen of the ER. The signal sequence is cleaved off of the protein by the signal peptidase enzyme that is in the ER membrane, and is degraded. The protein is now free in the lumen of the ER. Glycosylation. In the ER an enzyme transfers an oligosaccharide tree containing the sugar mannose from a membane glycolipid/dolichol to the lysosomal protein. Vesicle transport from ER to Golgi, through Golgi The protein is then transferred to the cis Golgi via COP-coated transport vesicles. In the cis golgi an enzyme normally phosphorylates mannose residues in the oligosaccharide attached to the lysosome- destined protein. This enzyme is missing or defective in individuals with I cell disease.

23. Normal processing of lysosomal proteins: The lysosome destined proteins proceed through the Golgi stack, presumably through a vesicle transport process. In the trans Golgi network normal lysosome-destined proteins are bound to mannose-6-phosphate receptors in the membrane, and are incorporated into clathrin- coated vesicles that are targeted to late endosomes. In the late endosome the acidic environment causes the lysosome-destined protein to separate from the mannose-6-phosphate receptor. The receptors are recycled to the trans Golgi network by vesicle transport. The lysosome-destined proteins proceed to lysosomes Alternate processing of proteins normally destined to lysosomes in individuals with I cell disease. - Since the normal targeting signal for lysomal proteins (mannose-6-phosphate) is not present, the proteins will enter the constitutive secretory pathway and be delivered to the cell surface. - As the vesicles fuse with the plasma membrane the proteins normally destined for lysosomes will be deposited in the extracellular space.

24. Question 10. Essay question. Beginning with the electron donor molecule, describe in detail, using essay format (NO DIAGRAMS) the cellular events that result in electron transport, proton pumping and ATP synthesis during oxidative phosphorylation or photophosphorylation • Discuss the initial electron donor, multiprotein complexes, mobile electron carriers/proteins, final electron acceptor in ETC in mitochondria and chloroplast • Mention the site(s) and direction of proton pumping in mitochondria and chloroplast • Discuss the subcellular site, structure, function and mechanism of ATP synthesis in mitochondria and chloroplast • Discuss the chemiosmotic theory • Role of light energy in electron flow in chloroplast • Energy transformation in oxidative- and photo-phosphorylation

25. Electron transport and H+ pumping in mitochondria 14_10_resp_enzy_comp.jpg 4 4 2

26. Electrons flow from –ve to +ve redox potential carriers 14_21_Redox_potential .jpg

27. Electron transport and H+ pumping in chloroplast 14_36_thylakoid_memb.jpg

28. Photophosphorylation and NADP reduction [Fig. 14-37]

29. FO-F1 ATP synthase complex: Uses proton electrochemical gradient to make ATP [Fig. 14-14]

30. Binding-change mechanism of ATP synthesis Lehninger Principles of Biochemistry

31. An overview of cytoskeleton • Three major cytoskeletal systems and their general properties • a) Intermediate Filaments - a system of elastic fibers - used to strengthen cells and to transmit mechanical strain across cells in a tissue. These filaments are purely skeletal in nature. • b) Microtubules - rigid protein tubules. These are involved in generation of cell shape and provide substrate for two different types of motor systems, dyneins and kinesins. The mitotic spindle is a variant form of the microtubular cytoskeleton. • c) Microfilaments (actin filaments) - this is the most complex system. Occurs in many forms, bundles of fibers or networks. Interacts with many types of molecules including its own class of motor proteins, the myosins. Its most elaborate form occurs in striated muscle. Microfilaments are responsible for cytoplasmic streaming and amoeboid motion.

32. Cyclin-CDK complex [Fig. 18-5] Cyclin-amount varies during cell cycle Cyclin-dependent kinase (Cdk)

33. Factors affecting the activity of Cdks • Cyclin degradation by ubiquitination • Phosphorylation and dephosphorylation • Positive feedback • Cdk inhibitor proteins

34. 18_11_M_Cdk_active.jpg Selective phosphorylation and dephosphorylationactivate M-Cdk [Fig. 18-11] Thr 14, Tyr 15 Thr 161

35. II. DNA Replication • DNA synthesis starts at replication origins • New DNA is synthesized by DNA polymerase at replication forks (Y-shaped junctions in DNA) • The DNA replication forks are asymmetric in nature • DNA polymerase has self-correcting (proofreading) activity

36. DNA replication forks are asymmetrical [Fig. 6-12] 06_12_asymmetrical.jpg

37. 06_17_group proteins.jpg A group of proteins act together at a replication fork