7.06_2004_exam3key - 7 .06 S pring 2 004 Exam 3 KEY 1 of 6...

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7.06 Spring 2004 Exam 3 KEY 1 of 6 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 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! 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? ANSWER: Radio-label protein with 35-S and separate out chloroplasts from cell to see if they are radioactive. Better is to treat with a protease and look for intact protease- resistant radioactive protein. b. 4 pts.) How would you determine whether incorporation occurs cotranslationally or post-translationally? ANSWER: In vitro translate the protein, then once translation is complete add chloroplasts and see where the protein ends up. If in chloroplast then incorporation occurs 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? ANSWER: Delete the sequence from cDNA and see if translated protein still localizes to chloroplasts d. 4 pts.) How would you show that this sequence is sufficient for uptake of RPK into the chloroplast?
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7.06 Spring 2004 Exam 3 KEY 2 of 6 ANSWER: Get cDNA from a non-chloroplast protein (preferably a cytosolic protein), add the sequence to it and see if this protein gets incorporated into chloroplasts. e. 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 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 also assume that the sequence noted in part c is indeed the sole chloroplast signal- uptake sequence in the RPK protein.
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This note was uploaded on 01/23/2012 for the course LSM lsm1301 taught by Professor Seow during the Spring '11 term at National University of Singapore.

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7.06_2004_exam3key - 7 .06 S pring 2 004 Exam 3 KEY 1 of 6...

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