Chapter 9. Coordination of Eukaryotic Polymerases

Introduction

In eukaryotes, the three RNA polymerases, Pol I, II, and III, each transcribe unique genes required for the synthesis of ribosomes: 25S and 18S rRNAs (Pol I), 5S rRNA (Pol III), and mRNAs for ribosomal proteins (Pol II). Researchers have long speculated that the activities of the three RNA polymerases are coordinately regulated according to the demand for ribosome synthesis: high demand in replicating cells in rich nutrient conditions and low demand when nutrients are scarce. To determine whether the activities of the three polymerases are coordinated, Arnaud Laferte and colleagues engineered a strain of yeast to be partially resistant to the inhibition of cell growth by the drug rapamycin (2006, Genes Dev. 20:2030–2040). As discussed in Chapter 10, rapamycin inhibits a protein kinase called TOR (for target of rapamycin) that regulates the overall rate of protein synthesis and ribosome synthesis. When TOR is inhibited by rapamycin, the transcription of rRNAs by Pol I and Pol III and of ribosomal protein mRNAs by RNA polymerase II are all rapidly repressed. Part of the inhibition of rRNA synthesis results from the dissociation of the Pol I transcription factor Rrn3 from Pol I. In the strain constructed by Laferte and colleagues, the wild-type Rrn3 gene and the wild-type A43 gene, encoding the Pol I subunit to which Rrn3 binds, were replaced with a gene encoding a fusion of the A43 Pol I subunit with Rrn3. The idea was that the covalent fusion of the two proteins would prevent the Rrn3 dissociation from Pol I otherwise caused by rapamycin treatment. The resulting CARA (constitutive association of Rrn3 and A43) yeast strain was found to be partially resistant to rapamycin. In the absence of rapamycin, the CARA strain grew at the same rate and had as many ribosomes as wild-type cells.

Question

a. To analyze rRNA transcription by Pol I, total RNA was isolated from rapidly growing wild-type (WT) cells and from CARA cells at various times following the addition of rapamycin. The concentration of the 35S rRNA precursor transcribed by Pol I (see Figure 10-41) was assayed by the primer-extension method. Since the 5′ end of the 35S rRNA precursor is degraded during the processing of 28S and 18S rRNA, this method measures the relatively shortlived pre-rRNA precursor. It is therefore an indirect measure of the rate of rRNA transcription by Pol I. The results of this primer-extension assay are shown below. How does the CARA strain’s Pol I–Rrn3 fusion affect the response of Pol I transcription to rapamycin?

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In untreated cells (t = 0 minutes), both wild-type and CARA cells transcribed equivalent amounts of ribosomal RNAs, suggesting that the mutant Pol I functions normally under control conditions. This finding is consistent with the observation that the growth rate of yeast harboring this mutation was indistinguishable from wild-type cells. However, when the drug rapamycin was added, Pol I transcription in wild-type cells was completely inhibited by 100 minutes after addition of the drug. In contrast, in the CARA cells, Pol I transcription was much less inhibited and continued at a significant level even at 100 minutes after addition of rapamycin. Consequently, Pol I transcription in the CARA cells was only partially inhibited by rapamycin.

Question

b. The concentrations of four mRNAs encoding ribosomal proteins, RPL30, RPS6a, RPL7a, and RPL5, and of the mRNA for actin (ACT1), a protein present in the cytoskeleton, were assessed in wild-type and CARA cells by Northern blotting at various times after addition of rapamycin to rapidly growing cells (upper autoradiograms). 5S rRNA transcription was assayed by pulse-labeling rapidly growing WT and CARA cells with ³H uracil (for 20 minutes) at various times after addition of rapamycin to the media. Total cellular RNA was isolated and subjected to gel electrophoresis and autoradiography. The lower autoradiogram shows the region of the gel containing 5S rRNA. Based on these data, what can be concluded about the influence of Pol I transcription on the transcription of ribosomal protein genes by Pol II and of 5S rRNA genes by Pol III?

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_feedback: As for rRNA precursor synthesis by Pol I analyzed in (a), both ribosomal protein mRNAs transcribed by Pol II and 5S rRNA synthesis by Pol III were rapidly inhibited by the addition of rapamycin in wild-type cells. Also, as observed for Pol I in (a), transcription of ribosomal protein and 5S rRNA genes was partially resistant to rapamycin and continued for a longer period after the addition of rapamycin in CARA cells compared to wild-type cells. Since the genetic modification of CARA cells directly affects Pol I, but not Pol II or Pol III, these differences from wild-type cells are probably a consequence of the continued Pol I transcription of the large rRNA precursor in CARA cells. Thus, these results indicate that transcription of ribosomal protein mRNAs by Pol II and of 5S rRNA by Pol III is coordinated with the sup-ply of the large rRNA precursor. However, the molecular mechanisms that coordinate the transcription of these genes by Pol II and Pol III with the sup-ply of the large rRNA precursor transcribed by Pol I remain to be discovered. This strategy for regulating the transcription of ribosomal protein genes and 5S rRNA genes seems logical. Since the ribosomal proteins and 5S rRNA are assembled onto the large rRNAs transcribed by Pol I during the synthesis of the ribosomal subunits, it makes sense for the cell to increase synthesis of these proteins and 5S rRNA in response to the supply of the large rRNAs.

Question

c. To determine whether the difference in the behavior of wild-type and CARA cells could be observed under normal physiological conditions (i.e., without drug treatment), cells were subjected to a shift from nutrient-rich media to nutrient-poor media. Under these conditions, in wild-type cells, the TOR protein kinase becomes inactive. Consequently, shifting cells from nutrient-rich media to nutrient-poor media should result in a normal physiological response that is equivalent to treating cells with rapamycin, which inhibits TOR. To determine how the CARA fusion protein affected the response to this media shift, RNA was extracted from wild-type and CARA cells and used to probe microarrays containing all yeast open reading frames. The extent of RNA hybridization with the arrays was quantified and is expressed in the graphs as log₂ of the ratio of CARA-cell RNA concentration to wild-type-cell RNA concentration for each open reading frame. A value of zero indicates that the two strains of yeast exhibit the same level of expression for those specific RNAs. A value of 1 indicates that the CARA cells contain twice as much of that particular RNA as do wild- type cells. The graphs below show the number of open reading frames (y axis) that have values for log₂ of each ratio, indicated by the x axis. The results of hybridization to open reading frames encoding mRNAs for ribosomal proteins are shown by black bars, those for all other mRNAs by white bars. The graph on the left gives results for cells grown in nutrient-rich medium, the graph on the right for cells shifted to nutrient-poor medium for 90 minutes. What do these data suggest about the regulation of ribosomal-protein gene transcription by Pol II?

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_feedback: In rich media, the CARA cells express the majority of genes, including the ribosomal protein genes, at a level similar to that of wild-type cells. Only a very small number of genes were expressed at more than twofold or less than one-half the level in wild-type cells. However, when the cells were shifted to poor media, the ribosomal mRNAs, but not other mRNAs, were expressed at a higher (twofold) level in the CARA cells than in the wild-type cells. Thus, these mRNAs were overrepresented in CARA cells under conditions that do not involve the use of drugs, but simply a decrease in nutrient supply. This overrepresentation in the mutant cells may be explained as follows: In wild-type cells, the shift to the poor media results in repression of Pol I synthesis of the large ribosomal RNAs and, coordinately, repression of Pol II synthesis of mRNAs encoding ribosomal proteins. In CARA cells, where the genetic manipulation affects Pol I and not Pol II, the continued expression of Pol I in low nutrient medium resulted in increased transcription of the ribosomal protein genes, confirming that transcription of ribosomal protein genes is stimulated in response to the production of the large rRNAs by Pol I.