Structure of the TFIIIC subcomplex τA provides insights into RNA polymerase III pre-initiation complex formation.

Transcription factor (TF) IIIC is a conserved eukaryotic six-subunit protein complex with dual function. It serves as a general TF for most RNA polymerase (Pol) III genes by recruiting TFIIIB, but it is also involved in chromatin organization and regulation of Pol II genes through interaction with CTCF and condensin II. Here, we report the structure of the S. cerevisiae TFIIIC subcomplex τA, which contains the most conserved subunits of TFIIIC and is responsible for recruitment of TFIIIB and transcription start site (TSS) selection at Pol III genes. We show that τA binding to its promoter is auto-inhibited by a disordered acidic tail of subunit τ95. We further provide a negative-stain reconstruction of τA bound to the TFIIIB subunits Brf1 and TBP. This shows that a ruler element in τA achieves positioning of TFIIIB upstream of the TSS, and suggests remodeling of the complex during assembly of TFIIIB by TFIIIC.

Positive modulation of RNA polymerase III transcription by ribosomal proteins.

A yeast nuclear fraction of unknown composition, named TFIIIE, was reported previously to enhance transcription of tRNA and 5S rRNA genes in vitro. We show that TFIIIE activity co-purifies with a specific subset of ribosomal proteins (RPs) which, as revealed by chromatin immunoprecipitation analysis, generally interact with tRNA and 5S rRNA genes, but not with a Pol II-specific promoter. Only Rpl6Ap and Rpl6Bp, among the tested RPs, were found associated to a TATA-containing tRNA(Ile)(TAT) gene. The RPL6A gene also emerged as a strong multicopy suppressor of a conditional mutation in the basal transcription factor TFIIIC, while RPL26A and RPL14A behaved as weak suppressors. The data delineate a novel extra-ribosomal role for one or a few RPs which, by influencing 5S rRNA and tRNA synthesis, could play a key role in the coordinate regulation of the different sub-pathways required for ribosome biogenesis and functionality.

Reconstitution of the yeast RNA polymerase III transcription system with all recombinant factors.

Transcription factor TFIIIC is a multisubunit complex required for promoter recognition and transcriptional activation of class III genes. We describe here the reconstitution of complete recombinant yeast TFIIIC and the molecular characterization of its two DNA-binding domains, tauA and tauB, using the baculovirus expression system. The B block-binding module, rtauB, was reconstituted with rtau138, rtau91, and rtau60 subunits. rtau131, rtau95, and rtau55 formed also a stable complex, rtauA, that displayed nonspecific DNA binding activity. Recombinant rTFIIIC was functionally equivalent to purified yeast TFIIIC, suggesting that the six recombinant subunits are necessary and sufficient to reconstitute a transcriptionally active TFIIIC complex. The formation and the properties of rTFIIIC-DNA complexes were affected by dephosphorylation treatments. The combination of complete recombinant rTFIIIC and rTFIIIB directed a low level of basal transcription, much weaker than with the crude B'' fraction, suggesting the existence of auxiliary factors that could modulate the yeast RNA polymerase III transcription system.

Transcription of individual tRNA1Gly genes from within a multigene family is regulated by transcription factor TFIIIB.

Members of a multigene family from the silkworm Bombyx mori have been classified based on their transcriptions in homologous nuclear extracts, into three groups of highly, moderately and poorly transcribed genes. Because all these gene copies have identical coding sequences and consequently identical promoter elements (the A and B boxes), the flanking sequences modulate their expression levels. Here we demonstrate the interaction of transcription factor TFIIIB with these genes and its role in regulating differential transcriptions. The binding of TFIIIB to the poorly transcribed gene -6,7 was less stable compared with binding of TFIIIB to the highly expressed copy, -1. The presence of a 5' upstream TATA sequence closer to the coding region in -6,7 suggested that the initial binding of TFIIIC to the A and B boxes sterically hindered anchoring of TFIIIB via direct interactions, leading to lower stability of TFIIIC-B-DNA complexes. Also, the multiple TATATAA sequences present in the flanking regions of this poorly transcribed gene successfully competed for TFIIIB reducing transcription. The transcription level could be enhanced to some extent by supplementation of TFIIIB but not by TATA box binding protein. The poor transcription of -6,7 was thus attributed both to the formation of a less stable transcription complex and the sequestration of TFIIIB. Availability of the transcription factor TFIIIB in excess could serve as a general mechanism to initiate transcription from all the individual members of the gene family as per the developmental needs within the tissue.

Assembly and isolation of intermediate steps of transcription complexes formed on the human 5S rRNA gene.

By employing purified transcription factors and RNA polymerase III (pol III), we generated active pol III transcription complexes on the human 5S rRNA gene. These large complexes were separated by size exclusion chromatography from non- incorporated proteins. In addition, we succeeded in isolating specific intermediate stages of complex formation. Such isolated partial complexes require complementation with the missing activities for full transcription activity. One central finding is that a 5S DNA-TFIIIA-TFIIIC2-TFIIIBbeta complex could be isolated which had been assembled in the absence of the general pol III transcription factor IIIC1. Thus TFIIIC1 is not an assembly factor for other transcription factors. Although pol III has the potential to bind unspecifically to DNA, such polymerase molecules cannot be rendered initiation competent by direct recruitment to a 5S DNA-TFIIIA-TFIIIC2- TFIIIBbeta complex, but this process strictly requires additional TFIIIC1 activity. This clearly demonstrates that in contrast to yeast cells, hTFIIIB(beta), although required, does not suffice for the functional recruitment of polymerase III. These data document that TFIIIC1 is the second transcription factor required for the recruitment of pol III in mammalian cells.

Characterization of RNA polymerase III transcription factor TFIIIC from the mulberry silkworm, Bombyx mori.

Fractionation of nuclear extracts from posterior silk glands of mulberry silkworm Bombyx mori, resolved the transcription factor TFIIIC into two components (designated here as TFIIIC and TFIIIC1) as in HeLa cell nuclear extracts. The reconstituted transcription of tRNA genes required the presence of both components. The affinity purified TFIIIC is a heteromeric complex comprising of five subunits ranging from 44 to 240 kDa. Of these, the 51-kDa subunit could be specifically crosslinked to the B box of tRNA1Gly. Purified swTFIIIC binds to the B box sequences with an affinity in the same range as of yTFIIIC or hTFIIIC2. Although an histone acetyl transferase (HAT) activity was associated with the TFIIIC fractions during the initial stages of purification, the HAT activity, unlike the human TFIIIC preparations, was separated at the final DNA affinity step. The tRNA transcription from DNA template was independent of HAT activity but the repressed transcription from chromatin template could be partially restored by external supplementation of the dissociated HAT activity. This is the first report on the purification and characterization of TFIIIC from insect systems.

Isolation and cloning of four subunits of a fission yeast TFIIIC complex that includes an ortholog of the human regulatory protein TFIIICbeta.

Eukaryotic tRNA genes are controlled by proximal and downstream elements that direct transcription by RNA polymerase (pol) III. Transcription factors (TFs) that reside near the initiation site are related in Saccharomyces cerevisiae and humans, while those that reside at or downstream of the B box share no recognizable sequence relatedness. Human TFIIICbeta is a transcriptional regulator that exhibits no homology to S. cerevisiae sequences on its own. We cloned an essential Schizosaccharomyces pombe gene that encodes a protein, Sfc6p, with homology to the S. cerevisiae TFIIIC subunit, TFC6p, that extends to human TFIIICbeta. We also isolated and cloned S. pombe homologs of three other TFIIIC subunits, Sfc3p, Sfc4p, and Sfc1p, the latter two of which are conserved from S. cerevisiae to humans, while the former shares homology with the S. cerevisiae B box-binding homolog only. Sfc6p is a component of a sequence-specific DNA-binding complex that also contains the B box-binding homolog, Sfc3p. Immunoprecipitation of Sfc3p further revealed that Sfc1p, Sfc3p, Sfc4p, and Sfc6p are associated in vivo and that the isolated Sfc3p complex is active for pol III-mediated transcription of a S. pombe tRNA gene in vitro. These results establish a link between the downstream pol III TFs in yeast and humans.