A Chimeric Subunit of Yeast Transcription Factor IIIC Forms a Subcomplex with τ95

Abstract

The multisubunit yeast transcription factor IIIC (TFIIIC) is a multifunctional protein required for promoter recognition, transcription factor IIIB recruitment, and chromatin antirepression. We report the isolation and characterization of TFC7, an essential gene encoding the 55-kDa polypeptide, 55, present in affinity-purified TFIIIC. 55 is a chimeric protein generated by an ancient chromosomal rearrangement. Its C-terminal half is essential for cell viability and sufficient to ensure TFIIIC function in DNA binding and transcription assays. The N-terminal half is nonessential and highly similar to a putative yeast protein encoded on another chromosome and to a cyanobacterial protein of unknown function. Partial deletions of the N-terminal domain impaired 55 function at a high temperature or in media containing glycerol or ethanol, suggesting a link between PolIII transcription and metabolic pathways. Interestingly, 55 was found, together with TFIIIC subunit 95, in a protein complex which was distinct from TFIIIC and which may play a role in the regulation of PolIII transcription, possibly in relation to cell metabolism.In eucaryotic cells, the transcription of a variety of small genes is conducted by RNA polymerase III (PolIII) and requires several auxiliary factors. For yeast tRNA gene (tDNA) activation, preinitiation complexes are assembled in a defined order within and upstream of the transcription unit (18, 27, 52). Transcription factor IIIC (TFIIIC) plays a primary role in this multistep complex assembly by binding to the intragenic promoter elements of tRNA genes (the A and B blocks). Yeast TFIIIC is a remarkably large multisubunit factor made of two protein subassemblies, named A and B, that have distinct DNA binding properties, that can be visualized by electron microscopy in a free or DNA-bound form (46), and that can be cleaved by limited proteolysis (37). The B domain binds tightly to the B block (37) and has been shown to display all the properties of enhancer binding proteins (11). Binding of the A domain to the A block is weaker and mostly B block dependent. Once bound, TFIIIC promotes the binding of transcription factor IIIB (TFIIIB) upstream of the transcription start site (6,26,28,31). The process is similar for yeast 5S RNA gene activation, except that TFIIIC assembly is dependent upon the binding of transcription factor IIIA (TFIIIA) to the internal promoter sequence. TFIIIB by itself does not bind detectably to TATA-less PolIII genes but, once assembled via TFIIIC, interacts intimately with DNA and is sufficient, at least in the yeast system, for directing accurate initiation by PolIII during multiple rounds of transcription in vitro (28, 31). Hence, TFIIIB is the initiation factor required for the activation of all PolIII genes, whereas TFIIIC and TFIIIA act as assembly factors. However, it has been shown that TFIIIC is a multifunctional protein, involved not only in promoter recognition and TFIIIB recruitment but also in the displacement of nucleosomes to relieve the repression of transcrip...

Structural and Functional Characterization of a Phosphatase Domain within Yeast General Transcription Factor IIIC

“…We used this control wild type strain to guarantee the comparison of strains with the most similar genetic background possible. Consistent with the literature, we could not observe any obvious growth phenotypes between 55-HPD⌬ huf⌬ and the used wild type strain (10,36). In a subsequent step we compared the phosphoproteomes of both strains by mass spectrometry using differential isotopic dimethyl labeling.…”

“…Because no enzymatic activity had been detected for 55 and because 55-HPD is nonessential and not required for TFIIIC activity, only an auxiliary structural role of 55-HPD in stabilizing TFIIIC had been suggested (10). However, partial deletions of 55-HPD result in a reduced growth rate at 30°C or in thermo-sensitive phenotypes in glycerol-or ethanol-containing media, suggesting a possible role for 55-HPD in directly linking Pol III transcription with metabolic pathways.…”

“…However, partial deletions of 55-HPD result in a reduced growth rate at 30°C or in thermo-sensitive phenotypes in glycerol-or ethanol-containing media, suggesting a possible role for 55-HPD in directly linking Pol III transcription with metabolic pathways. In addition, S. cerevisiae 55 and 95 form a complex separate from TFIIIC and not required for TFIIIC activity that has been also suggested to exert additional metabolic functions (10 also not been explored. There is increasing awareness about the cross-talk between the metabolic state of the cell and Pol I and Pol III transcription allowing cells to adapt to changing environmental conditions.…”

“…In Vivo Identification of Potential 55-HPD and Huf-regulated Phosphosites-Although 55-HPD and Huf are conserved in the Saccharomyces clade and 55-HPD is conserved in all hemiascomycetes, there are no obvious phenotypes connected to the deletion of the two described phosphatase domains under normal growth conditions in S. cerevisiae (10,36). Considering the above described highly specific in vitro peptide phosphatase activities, we investigated the effects of deleting these two potentially redundant phosphatase domains on the yeast phosphoproteome.…”

“…55-HPD Is a Functional Phosphatase-As the catalytic residues of 55-HPD and Huf are highly conserved and harbor all necessary active site residues, we wondered whether these proteins have catalytic activities, which were missed in previous studies (10). We tested purified 55, 55-HPD, and Huf proteins (Fig.…”