Weaver eoc answers 10

Weaver eoc answers 10 - Answers to Weaver end of chapter...

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Answers to Weaver end of chapter qeustions Chapter 10 Eukaryotic RNA Polymerases and Their Promoters 1. Diagram the elution pattern of the eukaryotic nuclear RNA polymerases from DEAE- Sephadex chromatography. Show what you would expect if you assayed the same fractions in the presence of 1 μ g/ml of α -amanitin. Figure 10.1 illustrates the results of the elution pattern and separation of three polymerases using DEAE-Sephadex chromatography. The order of elution is indicative of the binding affinity of the three enzymes to the DEAE resin, with Pol I displaying the weakest interaction and Pol III the strongest under the binding conditions given. Fig. 10.4 shows the sensitivity of the polymerases to α -amanitin. If the fractions in Fig 10.1 were assayed in the presence of 1 μ g/ml of α -amanitin, there would be no change to peak 1, peak 2 would disappear, and peak 3 would be reduced by ~10%. 2. Describe and give the results of an experiment that shows that polymerase I is located primarily in the nucleolus of the cell. Refer to figures 10.1 and 10.2. Extracts derived from purified nucleoli and nucleoplasm were subjected to DEAE chromatography. The nucleoplasmic fraction was enriched for Pol II activity (10.2a) whereas the nucleolar fraction was enriched for Pol I activity (10.2b) 3. Describe and give the results of an experiment that shows that polymerase III makes tRNA and 5s rRNA. Refer to figures 10.4 and 10.5. Figure 10.5 shows the results of an experiment where small labeled RNAs were synthesized in the presence of increasing amounts of α -amanitin and were then collected. The RNAs were analyzed by polyacrylamide gel electrophoresis (PAGE), after which the gel was sliced into fractions, and the amount of radioactivity in each slice determined. Control samples contained markers for 5S rRNA and 4S tRNA in adjacent lanes of the same gel. The positions of the markers are shown in the figures as blue lines, where inhibition of 5S rRNA and 4S tRNA synthesis can be seen. These results match the sensitivity of Pol III to α - amanitin (Fig. 10.4), therefore Pol III is the likely candidate for the synthesis of tRNAs and 5S rRNA. 4. How many subunits does yeast RNA polymerase II have? Which of these are core subunits? How many subunits are common to all three nuclear RNA polymerases? Yeast Pol II has 12 subunits. There are three core subunits, Rpb1, Rpb2, and Rpb3. and there are five common subunits to all RNA polymerases (Rpb5,6,8,10, and 12). 5. Describe how epitope tagging can be used to purify Pol II from yeast in one step. The principle of epitope tagging is illustrated in figure 10.6. In this case, a number of extra amino acids (epitope tag) have been genetically added to a known subunit of Pol II. The yeast strain can then be grown in the presence of labeled amino acids that will be incorporated into all proteins made by the organism. After cell growth and lysis, an antibody that recognizes the epitope is used to precipitate the tagged protein as well as all the other proteins with which it forms a complex stable under the conditions of the experiment. Contaminating proteins remain soluble and Pol II can be purified in a single step.
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