7.06_2004_exam3 - Name: 7.06 Cell Biology EXAM #3 April 22,...

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Name: 7.06 Spring 2004 Exam 3 1 of 8 7.06 Cell Biology EXAM #3 April 22, 2004 This is an open book exam, and you are allowed access to books and notes. Please write your answers in pen to the questions in the space allotted; if you use the back of a sheet make it clear which answer is where. And be sure to put your name on each page in case they become separated! Good luck! Name: Student ID #: Question 1. ___________________________ Question 2. ___________________________ Question 3. ___________________________ Question 4. ___________________________ Question 5. _____________________ Question 6.
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Name: 7.06 Spring 2004 Exam 3 2 of 8 1. 25 pts) Chloroplasts contain the enzyme ribulose –5 phosphate kinase (RPK), an essential enzyme in the Calvin cycle of photosynthetic carbon fixation. The enzyme is found in the chloroplast stroma, the space inside the chloroplast inner membrane and outside of the thylakoid vesicles. Using RPK DNA and RNA polymerase you have made pure RPK mRNA. You have developed a cell- free system , containing spinach chloroplasts and a wheat germ cytosol capable of translating the RPK mRNA, in which the RPK protein is synthesized and incorporated into chloroplasts. a. 4 pts.) Using the cell free system, how can you measure uptake of newly- synthesized RPK protein into the chloroplast? b. 4 pts.) How would you determine whether incorporation occurs cotranslationally or post-translationally? c. 4 pts.) You have identified an unusual sequence of amino acids at the C-terminus of the RPK protein, containing six glutamic acids in a row that you think might be the chloroplast signal- uptake sequence. How would you show that this sequence is necessary for uptake of RPK into the chloroplast? d. 4 pts.) How would you show that this sequence is sufficient for uptake of RPK into the chloroplast? 1e. 9 pts.) For various reasons you want to localize RPK to the intermembrane space of the mitochondrion rather than to the chloroplast stroma. How would you alter the RPK cDNA (i.e. delete or change some sequences, add others, etc.) such that the RPK
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Name: 7.06 Spring 2004 Exam 3 3 of 8 protein would become resident in the intermembrane space? In answering, it is all right if the relocalized RPK protein has extra amino acids on one end or the other. You can
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7.06_2004_exam3 - Name: 7.06 Cell Biology EXAM #3 April 22,...

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