Interactions between the Tetratricopeptide Repeat-containing Transcription Factor TFIIIC131 and Its Ligand, TFIIIB70

Abstract

In the transcription of tRNA and 5 S genes by RNA polymerase III, recruitment of the transcription factor (TF)IIIB is mediated by the promoter-bound assembly factor TFIIIC. A critical limiting step in this process is the interaction between the tetratricopeptide repeat (TPR)-containing subunit of TFIIIC (TFIIIC131) and the TFIIB-related factor Brf1p/TFIIIB70. To facilitate biochemical studies of this interaction, we expressed a fragment of TFIIIC131, TFIIIC131-(1-580), that includes the minimal TFIIIB70 interaction domain defined by two-hybrid studies together with adjacent sequences, up to the end of TPR9, implicated in the assembly reaction. TFIIIC131-(1-580) interacts with TFIIIB70 in solution and inhibits the formation of TFIIIB70⅐TFIIIC⅐DNA complexes. In a coupled equilibrium binding assay, the formation of TFIIIC131-(1-580)⅐TFIIIB70 complexes was adequately described by a single-site binding model and yielded an apparent equilibrium dissociation constant of 334 ؎ 23 nM. CD spectroscopy and limited proteolysis experiments defined a well structured and largely protease-resistant core in TFIIIC131-(1-580) comprising part of the hydrophilic amino terminus, TPR1-5, the intervening non-TPR region, and TPR6 -8. CD spectra showed that trifluoroethanol induced significant ␣-helical structure in TFIIIC131-(1-580). A more modest monovalent ion-dependent CD difference was observed in mixtures of TFIIIC131-(1-580) and TFIIIB70, suggesting that formation of the binary complex may proceed with the acquisition of ␣-helicity. Transcription factor (TF)1 IIIC is a large multisubunit complex that is responsible for recruiting the RNA polymerase III (pol III) initiation factor, TFIIIB, to the DNA upstream of the transcription start site of 5 S rRNA, tRNA, and related genes (1, 2). On tRNA and other genes containing A and B block promoter elements, the assembly reaction begins with nucleosome repositioning or displacement to enable high affinity binding of TFIIIC to the promoter (3-5). Interactions with the A block are thermodynamically weaker than those with the B block, and yet simultaneous binding to both sites occurs over a wide range of distances without regard to helical phasing (2). The conformational flexibility of TFIIIC indicated by these studies is also seen during the recruitment of TFIIIB. This is reflected by changes in site-specific photocross-linking of the 131-kDa subunit (TFIIIC131) (6) and by the ability of TFIIIC to co-direct (with the TATA-binding protein (TBP)) the placement of TFIIIB at different locations on the DNA (7).In addition to TFIIIB recruitment, recent studies have shown that TFIIIC has other functions and activities in pol III transcription. Subunits of TFIIIC in yeast (8) and humans (9, 10) have been shown to interact either genetically or biochemically with subunits of the polymerase that have been highly conserved through evolution. These studies suggest possible roles for TFIIIC in polymerase recruitment; pol III holoenzyme stabilization (11); and/or the cycle of elongation, termination, an...

The Brf1 and Bdp1 Subunits of Transcription Factor TFIIIB Bind to Overlapping Sites in the Tetratricopeptide Repeats of Tfc4

“…6, second row). The known interactions between Tfc4 and Brf1 (Nt-TPR5, TPRs1-5, TPRs6 -9 and Nt-TPR9, (23,24) were confirmed in this assay (Fig. 6, second and fourth rows).…”

“…Purity (77-80%) was confirmed by SDS-PAGE analysis using direct staining with Coomassie Blue and comparison to known protein standards (23). The preparation of recombinant TBP, Brf1, and Bdp1, yeast polymerase III and conditions for assaying transcription using whole cell extracts or purified components have been previously reported (6,24). The details for the assembly of TFIIIC-dependent TFIIIB or Brf1 complexes, quantitation and analysis of complex formation are all provided in detail in an earlier publication (23).…”

“…24). Recombinant wildtype and L469K fragments that encompass the TPR6 -9 array were expressed and purified under denaturing conditions and refolded as described previously (23).…”

“…The ligand binding activity of TPRs10 and 11 is unclear since the C-terminal region is neither required for nor independently binds Brf1 in a two-hybrid assay (1) yet binds Brf1 in a pull-down assay (22). The N-terminal half of Tfc4 (Nt-TPR9) forms a structure that is largely stable to limited proteolysis and binds Brf1 in solution, in Far-Western and two-hybrid assays (1,24). Yet the apparent Brf1-binding affinity of the Nt-TPR9 region is lower than that of either smaller Brf1-binding region (Nt-TPR5 and TPR6 -9) (23).…”

Identification, Molecular Cloning, and Characterization of the Sixth Subunit of Human Transcription Factor TFIIIC

“…Nevertheless, it has recently been published that recombinant B is able to recruit TBP and to stimulate TFIIIB-directed transcription of a TATA box-containing tRNA gene, implying a combined contribution of A and B to TBP recruitment during preinitiation complex formation (31). Important roles for the A component Tfc4 (131) in recruitment of TFIIIB through interaction with its Brf1 and Bdp1 components (24,(32)(33)(34)(35)(36)(37)(38) and of RNA polymerase III via interactions with its C53 (39) and ABC10␣ subunits (40) have been published.…”