Maf1p, a negative effector of RNA polymerase III in Saccharomyces cerevisiae

The yeast RNA polymerase III (Pol III) transcription system is well characterized. Small untranslated RNAs with essential housekeeping functions, such as tRNAs, 5S rRNA, or the U6 small nuclear RNA (snRNA) that is required for mRNA splicing, are synthesized by Pol III with the help of two general auxiliary factors, TFIIIC and TFIIIB. The large TFIIIC factor (six subunits) binds to the DNA promoter elements and assembles the initiation factor TFIIIB (three components) upstream of the start site. Once TFIIIB is in position, it recruits the Pol III enzyme (17 subunits) and directs accurate and multiple rounds of transcription. All of the polypeptide components of the Pol III apparatus (∼1,500 kDa) have been characterized and found to be essential for cell viability (8, 23). The identification of the components of the Pol III system has facilitated the description of a cascade of protein-protein interactions that leads to the recruitment of the Pol III enzyme (reviewed in reference 55). Detailed knowledge of the yeast Pol III transcription system contrasts with the limited information available on the control of class III gene expression in yeast. Cellular tRNA levels respond to cell growth rate (48, 49), to a nutritional upshift (27, 48) or to nitrogen starvation (36) but only modestly to amino acid starvation (41). Finally, Pol III transcription is repressed in secretion-defective cells (30). Although the mechanism of repression is not clear, it does involve activation of the cell integrity pathway (30). The effect of growth conditions on Pol III transcription is well mimicked in vitro with whole-cell extracts (11, 39). tRNA synthesis is downregulated in dense cell cultures approaching stationary phase, a result due essentially to reduced TFIIIB activity. The TFIIIB component Brf/TFIIIB70 was found to be the limiting factor in extracts from such cells (39). However, the occupancy of the TFIIIB binding site on the SUP53 gene encoding tRNALeu does not decrease in stationary-phase cells. Rather, in vivo footprinting data suggest reduced promoter occupancy by Pol III (25). In higher eukaryotic cells, Pol III transcription responds to growth rate, developmental phase, cell cycle position, and a number of pathological conditions (reviewed in reference 55). The regulation operates principally at the level of TFIIIB and TFIIIC (17, 20, 42, 46, 52). The tumor suppressors Rb and p53 inhibit TFIIIB (9, 10, 28, 53). Therefore, it is likely that the control of Pol III transcription rate is important in restraining tumor cell proliferation (54). No equivalent negative regulator of Pol III transcription has been found in yeast. Genes controlling tRNA synthesis in yeast can be identified by nonsense suppression approaches (22). One candidate for such a gene is Saccharomyces cerevisiae MAF1. It was originally identified by the isolation of maf1-1 as a temperature-sensitive mutation that decreases the efficiency of SUP11 (tRNA Tyr/UAA) suppression (34). A search for multicopy suppressors of maf1-1 revealed an intriguing genetic interaction between MAF1 and RPC160, the gene encoding the largest subunit of the RNA Pol III, C160. RPC160 genes with 3′ deletions in their open reading frame suppress the maf1-1 phenotypes when overexpressed (6). In the present work, we show that tRNA levels are elevated in maf1-1 cells and that spontaneous mutations in RPC160 which reduce tRNA levels also suppress the growth phenotype associated with maf1-1. maf1-1 cell extracts support increased levels of Pol III transcription in vitro compared to wild-type cells. Further, we show that Maf1p is a nuclear protein that physically interacts with RNA Pol III. Therefore, Maf1p appears to be a negative effector of Pol III activity, potentially regulating the level of cellular tRNA in response to external signals. A database search revealed that a variety of organisms have sequences similar to Maf1p, suggesting that this type of Pol III regulation may not be limited to yeast.