Zinc fingers 1 and 7 of yeast TFIIIA are essential for assembly of a functional transcription complex on the 5 S RNA gene.

The binding of transcription factor (TF) IIIA to the internal control region of the 5 S RNA gene is the first step in the assembly of a DNA-TFIIIA-TFIIIC- TFIIIB transcription complex, which promotes accurate transcription by RNA polymerase III. With the use of mutations that are predicted to disrupt the folding of a zinc finger, we have examined the roles of zinc fingers 1 through 7 of yeast TFIIIA in the establishment of a functional transcription complex both in vitro and in vivo. Our data indicate that, in addition to their role in DNA binding, the first and seventh zinc fingers contribute other essential roles in the assembly of an active transcription complex. Alanine-scanning mutagenesis identified residues within zinc finger 1 that are not required for DNA binding but are required for incorporation of TFIIIC into the TFIIIA-DNA complex. Although disruption of zinc finger 2 or 3 had a deleterious effect on the activity of TFIIIA both in vitro and in vivo, we found that increasing the level of their in vivo expression allowed these mutant proteins to support cell viability. Disruption of zinc fingers 4, 5 or 6 had minimal effect on the DNA binding and TF activities of TFIIIA.

Components of the ESCRT pathway, DFG16, and YGR122w are required for Rim101 to act as a corepressor with Nrg1 at the negative regulatory element of the DIT1 gene of Saccharomyces cerevisiae.

The divergently transcribed DIT1 and DIT2 genes of Saccharomyces cerevisiae, which belong to the mid-late class of sporulation-specific genes, are subject to Ssn6-Tup1-mediated repression in mitotic cells. The Ssn6-Tup1 complex, which is required for repression of diverse sets of coordinately regulated genes, is known to be recruited to target genes by promoter-specific DNA-binding proteins. In this study, we show that a 42-bp negative regulatory element (NRE) present in the DIT1-DIT2 intergenic region consists of two distinct subsites and that a multimer of each subsite supports efficient Ssn6-Tup1-dependent repression of a CYC1-lacZ reporter gene. By genetic screening procedures, we identified DFG16, YGR122w, VPS36, and the DNA-binding proteins Rim101 and Nrg1 as potential mediators of NRE-directed repression. We show that Nrg1 and Rim101 bind simultaneously to adjacent target sites within the NRE in vitro and act as corepressors in vivo. We have found that the ability of Rim101 to be proteolytically processed to its active form and mediate NRE-directed repression not only depends on the previously characterized RIM signaling pathway but also requires Dfg16, Ygr122w, and components of the ESCRT trafficking pathway. Interestingly, Rim101 was processed in bro1 and doa4 strains but was unable to mediate efficient repression.

Regulation of the premiddle and middle phases of expression of the NDT80 gene during sporulation of Saccharomyces cerevisiae.

The NDT80 gene of Saccharomyces cerevisiae, which encodes a global activator of transcription of middle sporulation-specific genes, is first expressed after the activation of early meiotic genes but prior to activation of middle sporulation-specific genes. Both upstream repression sequence 1 (URS1) and mid-sporulation element (MSE) sites are present in the promoter region of the NDT80 gene; these elements have been shown previously to contribute to the regulation of expression of early and middle sporulation-specific genes, respectively, by mediating repression in growing cells and activation at specific times during sporulation. In this study, we have shown that the overlapping windows of URS1- and MSE-mediated repression and activation are responsible for the distinctive premiddle expression pattern of the NDT80 gene. Our data suggest that a Sum1-associated repression complex bound at the NDT80 MSE sites prevents Ime1 tethered at the NDT80 URS1 sites from activating transcription of the NDT80 gene at the time that Ime1-dependent activation of early URS1-regulated meiotic genes is occurring. We propose that a decrease in the efficiency of Sum1-mediated repression as cells progress through the early events of the sporulation program allows the previously inactive Ime1 tethered at the URS1(NDT80) sites to promote a low level of expression of the NDT80 gene. This initial phase of URS1-dependent NDT80 expression is followed by Ndt80-dependent upregulation of its own expression, which requires the MSE(NDT80) sites and occurs concomitantly with Ndt80-dependent activation of a set of middle MSE-regulated sporulation-specific genes. Mutation of IME2 prevents expression of NDT80 in sporulating cells. We show in this study that NDT80 is expressed and that middle genes are activated in cells of an Deltaime2/Deltaime2 Deltasum1/Deltasum1 strain in sporulation medium. This suggests that Ime2 activates expression of NDT80 by eliminating Sum1-mediated repression.

Role of Ndt80, Sum1, and Swe1 as targets of the meiotic recombination checkpoint that control exit from pachytene and spore formation in Saccharomyces cerevisiae.

The meiotic recombination checkpoint, which is triggered by defects in recombination or chromosome synapsis, arrests sporulating cells of Saccharomyces cerevisiae at pachytene by preventing accumulation of active Clb-Cdc28. We compared the effects of manipulating the three known targets of the meiotic recombination checkpoint, NDT80, SWE1, and SUM1, in dmc1-arrested cells. Ndt80 is an activator of a set of middle sporulation-specific genes (MSGs), which includes CLB genes and genes involved in spore wall formation; Swe1 inhibits Clb-Cdc28 activity; and Sum1 is a repressor of NDT80 and some MSGs. Activation of the checkpoint leads to inhibition of Ndt80 activity and to stabilization of Swe1 and Sum1. Thus, dmc1-arrested cells fail to express MSGs, arrest at pachytene, and do not form spores. Our study shows that dmc1/dmc1 sum1/sum1 cells expressed MSGs prematurely and at high levels, entered the meiotic divisions efficiently, and in some cases formed asci containing mature spores. In contrast, dmc1/dmc1 swe1/swe1 cells expressed MSGs at a very low level, were inefficient and delayed in entry into the meiotic divisions, and never formed mature spores. We found that cells of dmc1/dmc1 sum1/sum1 ndt80/ndt80 and dmc1/dmc1 swe1/swe1 ndt80/ndt80 strains arrested at pachytene and that dmc1/dmc1 or dmc1/dmc1 swe1/swe1 cells overexpressing NDT80 were less efficient in bypassing checkpoint-mediated arrest than dmc1/dmc1 sum1/sum1 cells. Our results are consistent with previous suggestions that increased Clb-Cdc28 activity, caused by mutation of SWE1 or by an NDT80-dependent increase in CLB expression, allows dmc1/dmc1 cells to exit pachytene and that subsequent upregulation of Ndt80 activity by a feedback mechanism promotes entry into the meiotic divisions. Spore morphogenesis, however, requires efficient and timely activation of MSGs, which we speculate was achieved in dmc1/dmc1 sum1/sum1 cells by premature expression of NDT80.