In eukaryotes, the three RNA polymerases, Pol I, II, and III, each transcribes unique genes required for the synthesis of ribosomes: 25S and 18S rRNAs (Pol 1), 55 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 in replicating cells in rich nutrient conditions and low when nutrients are scarce. To determine whether the activities of the three polymerases are coordinated, 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 8, 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 ribosomal protein mRNAs by RNA polymerase II are all rapidly repressed. Part of the inhibition of Pol I rRNA synthesi'> results from the dissociation of the Pol I transcription factor Rrn3 from Pol I. In thestrain constructed by Lafertc and colleagues, the wild-typeRnr3 gene and the wild-type A43 gene, encoding the Pol Isubunit to which Rrn3 binds, were replaced with a gene encoding a fusion protein of the A43 Poll subunit with Rrn3.The idea was that the covalent fusion of the two proteins would prevent the Rrn3 dissociarion from Pol I otherwise caused by rapamycin treatment. The resulting CARA (constitutiveassociation of Rrn3 and A43) strain was found tobe partially resistant to rapamycin. In the absence of rapamycin, the CARA strain grew at the same rate and had equal numbers of ribosomes as wild-type cells.

a. To analyze rRNA transcription by Pol I, total RNA was isolated from rapidly growing wild type (WT) and CARA cells at various times following the addition of rapamycin. The concentration of the 355 rRNA precursor transcribed by Pol I (sec Figure 8-38) was assayed by the primer-extension method. Since the 5' end of the 35S rRNA precursor is degraded during the processing of 255 and 18S rRNA, this method measures the relatively short-lived pre-rRNA precursor. This is 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 Pol I-Rrn3 fusion affect the response of Poll transcription to rapamycin?

b. The concentrations of four mRNAs encoding ribosomal proteins, RPL30, RPS6a, RPL7a, and RPL5, and the mRNA for actin (ACTl ), 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 auto radiograms). 5S rRNA transcription was assayed by pulse labeling rapidly growing WT

and CARA cells with 1H uracil (for 20 minutes) at various rimes 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 5S rRNA by Pol Ill?

c. To determine whether the difference in behavior of wild-type and CARA cells can be observed under normal physiological conditions (i.e., without drug treatment), cells were subjected to a shift in their food source, from nutrientrich media to nutnent-poor media. Under these condition<>, 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 rapamycm, 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 this ratio, indicated by the x axis. The results of hybndization to open reading frames encoding mRNAs for ribosomal proreins 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?

Transcribed Image Text:

Minutes after rapamycin WT CARA O 20 40 60 80 100 O 20 40 60 80 100 35S FRNA WT, O 20 40 60 80 100 CARA Minutes after rapamycin O 20 40 60 80 100 RPL30 RPS6A RPL7A RPL5 ACT1 Minutes after WT CARA rapamycin O 20 40 60 80 100 O 20 40 60 80 100