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...
Transcription factor IIIC (TFIIIC) (or tau) is a large multisubunit and multifunctional factor required for transcription of all class III genes in Saccharomyces cerevisiae. It is responsible for promoter recognition and TFIIIB assembly. We report here the cloning and characterization of TFC6, an essential gene encoding the 91-kDa polypeptide, tau91, present in affinity-purified TFIIIC. Tau91 has a predicted molecular mass of 74 kDa. It harbors a central cluster of His and Cys residues and has basic and acidic amino acid regions, but it shows no specific similarity to known proteins or predicted open reading frames. The TFIIIC subunit status of tau91 was established by the following biochemical and genetic evidence. Antibodies to tau91 bound TFIIIC-DNA complexes in gel shift assays; in vivo, a B block-deficient U6 RNA gene (SNR6) harboring GAL4 binding sites was reactivated by fusing the GAL4 DNA binding domain to tau91; and a point mutation in TFC6 (tau91-E330K) was found to suppress the thermosensitive phenotype of a tfc3-G349E mutant affected in the B block binding subunit (tau138). The suppressor mutation alleviated the DNA binding and transcription defects of mutant TFIIIC in vitro. These results indicated that tau91 cooperates with tau138 for DNA binding. Recombinant tau91 by itself did not interact with a tRNA gene, although it showed a strong affinity for single-stranded DNA.
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