A differential response of wild type and mutant promoters to TFIIIB70 overexpression in vivo and in vitro

Sethy-Coraci, Indra; Moir, Robyn D.; López-De-León, Alfredo; Willis, Ian M.

doi:10.1093/nar/26.10.2344

Cited by 41 publications

(41 citation statements)

References 48 publications

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“…The same mutations have also been shown to increase transcription of wild-type and mutant templates in vitro by facilitating the recruitment of TFIIIB70 (25). Similar observations have been made in cells and in extracts of strains overexpressing TFIIIB70 (23,26). These findings are complementary and reveal that the interaction between TFIIIC131 and TFIIIB70 represents a limiting step in the assembly of a pol III transcription complex.…”

supporting

confidence: 78%

“…Although multiple subunits of both TFIIIB and TFIIIC participate in this assembly reaction (10,15,16,22), biochemical studies indicate that in yeast, the interaction between TFIIIB70 and TFIIIC⅐DNA is the major thermodynamically limited step (23,25,36). The binding of TFIIIB70 to the TFIIIC⅐DNA complex is mediated by the TPR-containing subunit, TFIIIC131 (15,16), and potentially by TFIIIC95 (based on an interaction between the homologous proteins in humans) (10).…”

Section: Discussionmentioning

confidence: 99%

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

Moir

Puglia

Willis

2000

Journal of Biological Chemistry

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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...

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supporting

confidence: 78%

Section: Discussionmentioning

confidence: 99%

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

Moir

Puglia

Willis

2000

Journal of Biological Chemistry

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“…Capillary transfer of the RNA to Nytran Plus membranes was performed using 20ϫ SSC (1ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and was followed by UV cross-linking as described above. [ 32 P]-labeled oligonucleotide probes were prepared as described previously (26) for detecting all tRNA precursors, TCM1 and CYH2 mRNAs, U4 snRNA, and U3 small nucleolar RNA (snRNA) (probe sequences are available upon request). KAR2 and ACT1 mRNA probes were prepared by random priming and runoff transcription, respectively (22).…”

Section: Methodsmentioning

confidence: 99%

Repression of Ribosome and tRNA Synthesis in Secretion-Defective Cells Is Signaled by a Novel Branch of the Cell Integrity Pathway

Moir²,

Sethy-Coraci

et al. 2000

Molecular and Cellular Biology

120

115

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The transcription of ribosomal DNA, ribosomal protein (RP) genes, and 5S and tRNA genes by RNA polymerases (Pols) I, II, and III, respectively, is rapidly and coordinately repressed upon interruption of the secretory pathway in Saccharomyces cerevisiae. We find that repression of ribosome and tRNA synthesis in secretion-defective cells involves activation of the cell integrity pathway. Transcriptional repression requires the upstream components of this pathway, including the Wsc family of putative plasma membrane sensors and protein kinase C (PKC), but not the downstream Bck1-Mkk1/2-Slt2 mitogen-activated protein kinase cascade. These findings reveal a novel PKC effector pathway that controls more than 85% of nuclear transcription. It is proposed that the coordination of ribosome and tRNA synthesis with cell growth may be achieved, in part, by monitoring the turgor pressure of the cell.The production of ribosomes and associated protein-synthetic machinery consumes substantial amounts of metabolic energy. It is estimated that yeast cells growing with a generation time of about 100 min devote at least 60% of their total nuclear transcription to the synthesis of the large rRNAs by RNA polymerase (Pol) I and as much as 50% of the initiation events by Pol II to the transcription of ribosomal protein (RP) genes (30). This level of transcription is needed to sustain the production of some 2,000 ribosomes per min and allow the doubling of the cell's ribosome content during each cell cycle. The large investment of high-energy phosphate in ribosome synthesis provides a biological rationale for the evolution of mechanisms that safeguard the cell's metabolic economy. These mechanisms couple the synthesis of ribosomes and ribosomal substrates, such as tRNAs, with the protein-synthetic needs of the cell and the availability of nutrients and/or growth factors (33,35).A potentially important mechanism in higher eukaryotic cells that may help to achieve metabolic economy and coordinate protein-synthetic capacity with cell growth involves transcriptional repression of Pols I and III by the tumor suppressor protein Rb (32). This activity of Rb results from its inhibition of preinitiation complex assembly and/or function through direct binding of the Pol I-and Pol III-specific transcription factors UBF and TFIIIB, respectively (2,3,15,29,34). In addition to Rb and the related pocket proteins, p107 and p130 (27), other mechanisms for controlling transcription of the protein-synthesizing machinery appear to exist. In yeast cells, for example, the production of ribosomal components and tRNAs in response to nutrient availability and growth rate is regulated predominantly at the transcriptional level in the absence of an Rb homolog (11,24,33,35). These observations suggest that yeast may be a good model system in which to examine the fundamental mechanisms coordinating ribosome synthesis with cell growth.In the past several years, studies with Saccharomyces cerevisiae have uncovered a regulatory circuit that connects the synthesis of th...

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“…Previous work has established that transcription of the sup9-e A19-supS1 reporter gene in vivo is sensitive to the level of Brf1 as well as to the Brf1 binding activity of Tfc4 (5)(6)(7)33). Thus, it was interesting to find that only two of the mutations (S541I and L542G in TPR9, Fig.…”

Section: Amino Acid Substitutions At Phylogenetically Conserved Positmentioning

confidence: 99%

“…Although multiple subunits of both TFIIIB and TFIIIC interact (1-4) TFIIIB assembly is mediated initially by protein-protein interactions between the TFIIIC subunit, Tfc4, and the TFIIIB subunit, Brf1. Biochemical and genetic studies in yeast indicate that the recruitment of Brf1 by TFIIIC is a major limiting step in preinitiation complex assembly (5)(6)(7). Subsequent binding of the TBP and Bdp1 subunits of TFIIIB generates a series of structural changes in Tfc4, Brf1 and DNA that leads to progressively increased accessibility of Brf1 to DNA (8).…”

mentioning

confidence: 99%

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

Liao

Willis

Moir

2003

Journal of Biological Chemistry

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The RNA polymerase III initiation factor TFIIIB is assembled onto DNA through interactions involving the Tfc4 subunit of the assembly factor TFIIIC and two subunits of TFIIIB, Brf1 and Bdp1. Tfc4 contains two arrays of tetratricopeptide repeats (TPRs), each of which provides a binding site for Brf1. Dominant mutations in the ligand binding channel of the first TPR array, TPRs1-5, and on the back side of this array, increase Brf1 binding by Tfc4. Here we examine the biological importance of the second TPR array, TPRs6 -9. Radical mutations at phylogenetically conserved residues in the ligand binding channel of TPRs6 -9 impair pol III reporter gene transcription. Biochemical studies on one such mutation, L469K in TPR7, revealed a defect in the recruitment of Brf1 into TFIIIB-TFIIIC-DNA complexes and diminished the direct interaction between Tfc4 and Brf1. Multicopy suppression analysis implicates TPR9 in Brf1 binding and TPRs7 and 8 in binding to more than one ligand. Indeed, the L469K mutation also decreased the binding affinity for Bdp1 incorporation into TFIIIB-TFIIIC-DNA complexes and inhibited binary interactions between Bdp1 and Tfc4. The Bdp1 binding domain in Tfc4 was mapped to TPRs1-9, a domain that contains both TPR arrays and thus overlaps two of the known binding sites for Brf1. The properties of the L469K mutation identify both Brf1 and Bdp1 as ligands for the second TPR array.The assembly of the initiation factor TFIIIB by TFIIIC is a limiting step in RNA polymerase III (pol III) 1 transcription. Although multiple subunits of both TFIIIB and TFIIIC interact (1-4) TFIIIB assembly is mediated initially by protein-protein interactions between the TFIIIC subunit, Tfc4, and the TFIIIB subunit, Brf1. Biochemical and genetic studies in yeast indicate that the recruitment of Brf1 by TFIIIC is a major limiting step in preinitiation complex assembly (5-7). Subsequent binding of the TBP and Bdp1 subunits of TFIIIB generates a series of structural changes in Tfc4, Brf1 and DNA that leads to progressively increased accessibility of Brf1 to DNA (8). The protein-protein interactions between TFIIIB subunits, together with DNA bending, generate a highly stable TFIIIB complex, a structure that surrounds and kinetically traps the DNA (9 -12).Tfc4 contains eleven copies of a ubiquitous protein-protein interaction motif known as a tetratricopeptide repeat (13,14).TPR motifs, as the name implies, are typically 34 amino acids in length and fold into two antiparallel ␣-helices (designated A and B, Ref. 15). Although no position within the motif is invariant, a pattern of small and large hydrophobic residues defines the loose TPR consensus and provides stacking interactions between adjacent repeats (15-17). Multiple adjacent repeats form a right-handed superhelix in which the inner channel (formed by the A-helices) provides a ligand binding interface (18 -20). The TPRs in Tfc4 are organized into two arrays in the N terminus, TPR1-5 and TPR6 -9 (with five and four repeats, respectively) that are separated by a region of mini...

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A differential response of wild type and mutant promoters to TFIIIB70 overexpression in vivo and in vitro

Cited by 41 publications

References 48 publications

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

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

Repression of Ribosome and tRNA Synthesis in Secretion-Defective Cells Is Signaled by a Novel Branch of the Cell Integrity Pathway

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

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