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

2003; Elsevier BV; Volume: 278; Issue: 45 Linguagem: Inglês

10.1074/jbc.m308354200

ISSN

1083-351X

Autores

Yanling Liao, Ian M. Willis, Robyn D. Moir,

Tópico(s)

DNA Repair Mechanisms

Resumo

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 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) 1The abbreviations used are: pol III, RNA polymerase III; TPR, tetratricopeptide repeat; NTA, nitrilotriacetic acid.1The abbreviations used are: pol III, RNA polymerase III; TPR, tetratricopeptide repeat; NTA, nitrilotriacetic acid. transcription. Although multiple subunits of both TFIIIB and TFIIIC interact (1Chaussivert N. Conesa C. Shaaban S. Sentenac A. J. Biol. Chem. 1995; 270: 15353-15358Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 2Deprez E. Arrebola R. Conesa C. Sentenac A. Mol. Cell. Biol. 1999; 19: 8042-8051Crossref PubMed Scopus (31) Google Scholar, 3Hsieh Y.J. Wang Z. Kovelman R. Roeder R.G. Mol. Cell. Biol. 1999; 19: 4944-4952Crossref PubMed Scopus (53) Google Scholar, 4Hsieh Y.J. Kundu T.K. Wang Z. Kovelman R. Roeder R.G. Mol. Cell. Biol. 1999; 19: 7697-7704Crossref PubMed Scopus (88) Google Scholar) 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 (5Lopez-de-Leon A. Librizzi M. Puglia K. Willis I.M. Cell. 1992; 71: 211-220Abstract Full Text PDF PubMed Scopus (108) Google Scholar, 6Moir R.D. Sethy-Coraci I. Puglia K. Librizzi M.D. Willis I.M. Mol. Cell. Biol. 1997; 17: 7119-7125Crossref PubMed Scopus (37) Google Scholar, 7Sethy-Coraci I. Moir R.D. Lopez-de-Leon A. Willis I.M. Nucleic Acids Res. 1998; 26: 2344-2352Crossref PubMed Scopus (37) Google Scholar). 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 (8Kassavetis G.A. Joazeiro C.A. Pisano M. Geiduschek E.P. Colbert T. Hahn S. Blanco J.A. Cell. 1992; 71: 1055-1064Abstract Full Text PDF PubMed Scopus (182) Google Scholar). 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 (9Kassavetis G.A. Braun B.R. Nguyen L.H. Geiduschek E.P. Cell. 1990; 60: 235-245Abstract Full Text PDF PubMed Scopus (359) Google Scholar, 10Kumar A. Grove A. Kassavetis G.A. Geiduschek E.P. Cold Spring Harb. Symp. Quant. Biol. 1998; 63: 121-129Crossref PubMed Scopus (23) Google Scholar, 11Grove A. Kassavetis G.A. Johnson T.E. Geiduschek E.P. J. Mol. Biol. 1999; 285: 1429-1440Crossref PubMed Scopus (30) Google Scholar, 12Cloutier T.E. Librizzi M.D. Mollah A.K. Brenowitz M. Willis I.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9581-9586Crossref PubMed Scopus (35) Google Scholar). Tfc4 contains eleven copies of a ubiquitous protein-protein interaction motif known as a tetratricopeptide repeat (13Marck C. Lefebvre O. Carles C. Riva M. Chaussivert N. Ruet A. Sentenac A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4027-4031Crossref PubMed Scopus (53) Google Scholar, 14Rameau G. Puglia K. Crowe A. Sethy I. Willis I. Mol. Cell. Biol. 1994; 14: 822-830Crossref PubMed Scopus (45) Google Scholar). TPR motifs, as the name implies, are typically 34 amino acids in length and fold into two antiparallel α-helices (designated A and B, Ref. 15Das A.K. Cohen P.W. Barford D. EMBO J. 1998; 17: 1192-1199Crossref PubMed Scopus (705) Google Scholar). 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 (15Das A.K. Cohen P.W. Barford D. EMBO J. 1998; 17: 1192-1199Crossref PubMed Scopus (705) Google Scholar, 16Lamb J.R. Tugendreich S. Hieter P. Trends Biochem. Sci. 1995; 20: 257-259Abstract Full Text PDF PubMed Scopus (545) Google Scholar, 17Main E.R. Xiong Y. Cocco M.J. D'Andrea L. Regan L. Structure (Camb.). 2003; 11: 497-508Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Multiple adjacent repeats form a right-handed superhelix in which the inner channel (formed by the A-helices) provides a ligand binding interface (18Gatto Jr., G.J. Geisbrecht B.V. Gould S.J. Berg J.M. Nat. Struct. Biol. 2000; 7: 1091-1095Crossref PubMed Scopus (290) Google Scholar, 19Lapouge K. Smith J.S. Walker A.P. Gamblin J.S. Smerdon J.S. Rittinger K. Mol. Cell. 2000; 6: 899-907Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 20Scheufler C. Brinker A. Bourenkov G. Pegoraro S. Moroder L. Bartunik H. Hartl F.U. Moarefi I. Cell. 2000; 101: 199-210Abstract Full Text Full Text PDF PubMed Scopus (991) Google Scholar). 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 minimal sequence conservation, and two solo repeats in the C terminus (depicted in Fig. 1 and Refs. 13Marck C. Lefebvre O. Carles C. Riva M. Chaussivert N. Ruet A. Sentenac A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4027-4031Crossref PubMed Scopus (53) Google Scholar, 14Rameau G. Puglia K. Crowe A. Sethy I. Willis I. Mol. Cell. Biol. 1994; 14: 822-830Crossref PubMed Scopus (45) Google Scholar, and 21Dumay-Odelot H. Acker J. Arrebola R. Sentenac A. Marck C. Mol. Cell. Biol. 2002; 22: 298-308Crossref PubMed Scopus (21) Google Scholar). Conservation of the number of TPRs and their organization in Tfc4 orthologues suggests that the function of the protein is based on the preservation of a common TPR-based tertiary structure (21Dumay-Odelot H. Acker J. Arrebola R. Sentenac A. Marck C. Mol. Cell. Biol. 2002; 22: 298-308Crossref PubMed Scopus (21) Google Scholar). The TPR motifs of Tfc4 contain multiple independent binding sites for Brf1 (3Hsieh Y.J. Wang Z. Kovelman R. Roeder R.G. Mol. Cell. Biol. 1999; 19: 4944-4952Crossref PubMed Scopus (53) Google Scholar, 22Khoo B. Brophy B. Jackson S.P. Genes Dev. 1994; 8: 2879-2890Crossref PubMed Scopus (108) Google Scholar, 23Moir R.D. Puglia K.V. Willis I.M. J. Biol. Chem. 2002; 277: 694-701Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) although the step(s) in complex assembly in which these sites are used is not known. The N-terminal region of Tfc4 in combination with the first repeat (Nt-TPR1) can support Brf1 binding, as detected by a two-hybrid interaction (1Chaussivert N. Conesa C. Shaaban S. Sentenac A. J. Biol. Chem. 1995; 270: 15353-15358Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), although the first array (TPRs1–5) is minimally sufficient in the absence of the Nt-region (23Moir R.D. Puglia K.V. Willis I.M. J. Biol. Chem. 2002; 277: 694-701Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The second TPR array (TPRs6 –9) contributes an additional binding site for Brf1 (3Hsieh Y.J. Wang Z. Kovelman R. Roeder R.G. Mol. Cell. Biol. 1999; 19: 4944-4952Crossref PubMed Scopus (53) Google Scholar, 23Moir R.D. Puglia K.V. Willis I.M. J. Biol. Chem. 2002; 277: 694-701Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). 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 (1Chaussivert N. Conesa C. Shaaban S. Sentenac A. J. Biol. Chem. 1995; 270: 15353-15358Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) yet binds Brf1 in a pull-down assay (22Khoo B. Brophy B. Jackson S.P. Genes Dev. 1994; 8: 2879-2890Crossref PubMed Scopus (108) Google Scholar). 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 (1Chaussivert N. Conesa C. Shaaban S. Sentenac A. J. Biol. Chem. 1995; 270: 15353-15358Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 24Moir R.D. Puglia K.V. Willis I.M. J. Biol. Chem. 2000; 275: 26591-26598Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). 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) (23Moir R.D. Puglia K.V. Willis I.M. J. Biol. Chem. 2002; 277: 694-701Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). These data suggest that the Brf1 binding sites are masked in the context of the Nt-TPR9 region (23Moir R.D. Puglia K.V. Willis I.M. J. Biol. Chem. 2002; 277: 694-701Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) and indeed the entire Tfc4 protein (1Chaussivert N. Conesa C. Shaaban S. Sentenac A. J. Biol. Chem. 1995; 270: 15353-15358Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). A study of the human homolog of Tfc4 (hT-FIIIC102) (3Hsieh Y.J. Wang Z. Kovelman R. Roeder R.G. Mol. Cell. Biol. 1999; 19: 4944-4952Crossref PubMed Scopus (53) Google Scholar) showed that repeats in each TPR array bind to both HsBrf1 and hTFIIIC63 (the homolog of S. cerevisiae Tfc1 that together with Tfc4 and Tfc7 form the τA domain of TFIIIC) (25Schultz P. Marzouki N. Marck C. Ruet A. Oudet P. Sentenac A. EMBO J. 1989; 8: 3815-3824Crossref PubMed Scopus (49) Google Scholar). The TPRs also contribute to the interaction between Tfc4 and the TFIIIB subunit Bdp1. Although the Bdp1-binding site(s) in Tfc4 has not yet been defined, analysis of deletion mutants of Tfc4 (21Dumay-Odelot H. Acker J. Arrebola R. Sentenac A. Marck C. Mol. Cell. Biol. 2002; 22: 298-308Crossref PubMed Scopus (21) Google Scholar, 26Ruth J. Conesa C. Dieci G. Lefebvre O. Dusterhoft A. Ottonello S. Sentenac A. EMBO J. 1996; 15: 1941-1949Crossref PubMed Scopus (77) Google Scholar) and the effect of amino acid substitutions in both TPR2 (27Ishiguro A Kassavetis G.A. Geiduschek E.P. Mol. Cell. Biol. 2002; 22: 3264-3275Crossref PubMed Scopus (42) Google Scholar) and TPR7 (28Rozenfeld S. Thuriaux P. Mol. Genet. Genomics. 2001; : 705-710Crossref PubMed Scopus (14) Google Scholar) suggest that the TPR arrays are involved in the association between Tfc4 and Bdp1. The TPRs in Tfc4 may therefore provide binding sites for multiple ligands. A phylogenetic analysis of the first array in Tfc4, TPRs1–5, found that the conserved non-structural TPR residues are distributed between the A and B helices and loop regions of the repeats (23Moir R.D. Puglia K.V. Willis I.M. J. Biol. Chem. 2002; 277: 694-701Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Dominant gain of function mutations in Tfc4 map in and around TPR2 and increase the recruitment of Brf1 to TFIIIC-DNA (6Moir R.D. Sethy-Coraci I. Puglia K. Librizzi M.D. Willis I.M. Mol. Cell. Biol. 1997; 17: 7119-7125Crossref PubMed Scopus (37) Google Scholar). A structural model of TPRs1–3 showed that the conserved residues and sites of the gain of function mutations cluster into two potential binding sites: one that traverses the ligand binding channel and another that lies across the back side of the array (29Moir R.D. Willis I.M. Conaway J.W. Conaway R.C. The Assembly of the Initiation Factor TFIIIB. Academic Press, New York2004Google Scholar). Biochemical studies of the T167I (PCF1–2) gain of function mutation show that the channel formed by TPRs1–3 binds Brf1 directly. 2R. Moir, K. Puglia, Y. Liao, and I. Willis, manuscript in preparation.2R. Moir, K. Puglia, Y. Liao, and I. Willis, manuscript in preparation. In contrast, characterization of the H190Y(PCF1-1) mutation shows that Brf1 does not interact on the back side of the TPR1–5 array. This latter mutation is proposed to affect an intramolecular interaction that indirectly influences Brf1 binding, leading to relief of autoinhibition (23Moir R.D. Puglia K.V. Willis I.M. J. Biol. Chem. 2002; 277: 694-701Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Unlike the first TPR array, the conserved non-structural residues in TPRs6 –9 map predominantly to the A-helices (23Moir R.D. Puglia K.V. Willis I.M. J. Biol. Chem. 2002; 277: 694-701Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Thus, the ability of TPRs6 –9 to bind Brf1 in vitro suggests that these conserved residues form a binding site for Brf1. This was confirmed in the current study by in vivo and in vitro analysis of mutations at specific sites in the second TPR array. We show that conserved residues in TPRs6 –9 cluster into two groups; one in TPR9 that affects Brf1 binding and the other in TPRs7 and 8 that generates a more complex effect on ligand binding. Biochemical studies on one mutant, L469K in TPR7, reveals effects on two steps in complex assembly; Brf1 and Bdp1 incorporation into TFIIIB-TFIIIC-DNA complexes. The minimal region of Tfc4 required for detectable Bdp1 binding in our assays was mapped to TPRs1–9, a region previously defined as containing two binding sites for Brf1. We discuss the implications of two ligands, Brf1 and Bdp1, apparently competing for binding to Tfc4, in TFIIIC-directed TFIIIB complex assembly. Mutagenesis of Tfc4 —The Transformer™ Mutagenesis kit (Clonetech) was used to introduce amino acid substitutions at selected positions in TPR7–9 in Tfc4. The following mutagenic primers were used to introduce mutations in the plasmid pRS313/PCF1+ 16E (the underlined nucleotides mark the mutation site(s): L469K, 5′-GACGTTGCGGATAAATATTTTGAGGCTGC-3′; E472K, 5′GTTGCGGATTTATATTTTAAGGCTGCAAC-3′; E498K, 5′GCCGTTGTTATCCCTTAAGGAATGGC-3′; V504K, 5′-GTACCACTGACAAGTTCAAACCACTAGC-3′; C511A, 5′-CCACTAGCAAGAGCCTACAAGGAAATCG-3′; I515K, 5′-GCTACAAGGAAAAGGAAAGTTATGAAACG-3′; D537K, 5′-CCAGATGATTTAAAGATTCGTGTATC-3′; S541I, 5′-GATATTCGTGTAATTTTGGCAGAAGTTTAC-3′; L542G, 5′-GATATTCGTGTATCTGGGGCAGAAGTTTAC-3′ and E564K, 5′-GTTGCGTTGTAAAGATGAGGGAAACACC-3′. The selection primer 5′-GATACCGTCGACGGATCCGGGGGGCCCGGTAC was used to identify successfully mutated plasmids through loss of XhoI and gain of BamH1 (underlined) restriction sites in vector sequence. Candidate clones were retransformed into DH5α and sequenced to confirm the identity of the amino acid substitution. Mutant PCF1 alleles on pRS313 were transformed into the yeast strain supAC1+ that contained a wild-type PCF1+ gene and the pol III reporter gene sup9-e A19-supS1 (14Rameau G. Puglia K. Crowe A. Sethy I. Willis I. Mol. Cell. Biol. 1994; 14: 822-830Crossref PubMed Scopus (45) Google Scholar) and the rescuing wild-type plasmid (pRS316PCF1+) was evicted on 5-FOA-containing media. Single colonies were obtained and assayed for growth and suppression phenotypes at 16, 30 or 37 °C. Strains were grown to early log phase (OD 1.0) in synthetic complete medium before dilution and spotting onto minimally complete or selective media. Each suppressor defective strain was transformed with plasmids that overexpressed either Brf1 (YEp24TDS4, Ref. 30Buratowski S. Zhou H. Cell. 1992; 71: 221-230Abstract Full Text PDF PubMed Scopus (109) Google Scholar), TBP (pDE31–7, a gift from Greg Prelich) or Bdp1 (pRS426B90, recloned from pRS313, Ref. 7Sethy-Coraci I. Moir R.D. Lopez-de-Leon A. Willis I.M. Nucleic Acids Res. 1998; 26: 2344-2352Crossref PubMed Scopus (37) Google Scholar). Transcription and Complex Assembly—Extracts from wild-type and L469K mutant strains were fractionated on Biorex 70 and DEAE-A25 resins before sequential gradient purification on heparin-agarose and mono Q resins (as previously described in Ref. 24Moir R.D. Puglia K.V. Willis I.M. J. Biol. Chem. 2000; 275: 26591-26598Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Recombinant wild-type and L469K fragments that encompass the TPR6 –9 array were expressed and purified under denaturing conditions and refolded as described previously (23Moir R.D. Puglia K.V. Willis I.M. J. Biol. Chem. 2002; 277: 694-701Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The calculated extinction coefficients were used to determine protein concentration at 280 nm. Purity (77–80%) was confirmed by SDS-PAGE analysis using direct staining with Coomassie Blue and comparison to known protein standards (23Moir R.D. Puglia K.V. Willis I.M. J. Biol. Chem. 2002; 277: 694-701Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). 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 (6Moir R.D. Sethy-Coraci I. Puglia K. Librizzi M.D. Willis I.M. Mol. Cell. Biol. 1997; 17: 7119-7125Crossref PubMed Scopus (37) Google Scholar, 24Moir R.D. Puglia K.V. Willis I.M. J. Biol. Chem. 2000; 275: 26591-26598Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). 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 (23Moir R.D. Puglia K.V. Willis I.M. J. Biol. Chem. 2002; 277: 694-701Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). TFIIIC-DNA complexes were assembled, with Brf1, TBP and Bdp1 as required in specific experiments, at 20 °C for 60 min before native gel electrophoresis. TPR6 –9 fragments were added to preformed TFIIIC-DNA complexes prior to the addition of Brf1. Protein-Protein Interactions—Recombinant His-tagged Brf1 or Bdp1 was immobilized on NiNTA-resin (Qiagen) in buffer containing 50 mm sodium phosphate, pH 7.5, 5 mm magnesium acetate, 150 mm potassium acetate, 1 mm dithiothreitol, and 10% glycerol with protease inhibitors (protein refolding buffer supplemented with 150 mm NaCl, Ref. 31Librizzi M.D. Moir R.D. Brenowitz M. Willis I.M. J. Biol. Chem. 1996; 271: 32695-32701Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Subclones of Tfc4 that encoded untagged versions of the following regions; Nt-TPR9, TPR1–9, TPR6 –9, and TPR1–5, were individually transcribed and translated in vitro (SMLnT®-coupled reticulocyte lysate system, Promega) to generate 35S-labeled proteins. In each 40-μl pull-down reaction, 5 μl of Brf1-(50 pmol) or Bdp1 (12.5 pmol)-bound resin was pre-equilibrated in binding buffer that contained 10 mm imidazole, 3 μg/μl bovine serum albumin, and 0.1% Triton X-100 for 30–60 min, before incubation with 5 μl of labeled Tfc4 fragment at 4 °C for 2 h. Resins were then washed three times in binding buffer that contained 15–20 mm imidazole. Control resins, that lacked Brf1 or Bdp1, were processed in parallel. Samples were boiled, analyzed by SDS-PAGE, and Tfc4 protein detected by direct autoradiography. Two-hybrid interactions were assayed using Brf1 or Bdp1 in pASCYH2 and wild-type or mutant Tfc4, or the respective subfragments, in pACTII. The resulting β-galactosidase activity in strain Y190, was measured and normalized to protein concentration and expressed as Miller units (units of β-galactosidase activity per mg of protein, Ref. 32Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley and Sons, New York1995Google Scholar). Amino Acid Substitutions at Phylogenetically Conserved Positions in TPRs6 –9 of Tfc4 Generate Defects in pol III Transcription—A phylogenetic analysis of TPRs6 –9 from four yeast and three metazoan species identified four absolutely conserved amino acids, one located in each A-helix of the array at positions that were not predicted to contribute to the TPR-fold (15Das A.K. Cohen P.W. Barford D. EMBO J. 1998; 17: 1192-1199Crossref PubMed Scopus (705) Google Scholar, 33Moir R.D. Puglia K.V. Willis I.M. Mol. Cell. Biol. 2002; 22: 6131-6141Crossref PubMed Scopus (12) Google Scholar). In addition, a number of highly conserved acidic, basic, and hydrophobic residues were identified. The majority of these residues also mapped to the A-helices in this array (summarized in Fig. 1, adapted from Ref. 23Moir R.D. Puglia K.V. Willis I.M. J. Biol. Chem. 2002; 277: 694-701Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). To examine the functional importance of TPRs6 –9, a subset of ten conserved A- and B-helix residues in the array were chosen for radical site-directed mutagenesis. The resulting mutant genes were plasmid shuffled into S. cerevisiae (strain supAC1+, Ref. 14Rameau G. Puglia K. Crowe A. Sethy I. Willis I. Mol. Cell. Biol. 1994; 14: 822-830Crossref PubMed Scopus (45) Google Scholar) and evaluated for growth at several temperatures on synthetic complete medium and for their ability to express the pol III reporter gene, sup9-e A19-supS1 (34Willis I. Schmidt P. Soll D. EMBO J. 1989; 8: 4281-4288Crossref PubMed Scopus (22) Google Scholar). None of the mutations were lethal at 30 °C since the URA3-marked wild-type rescuing plasmid could be successfully evicted on 5-FOA-containing medium. Moreover, the mutants exhibited growth phenotypes that were indistinguishable from wild-type at 30 °C and 37 °C on nutritionally complete medium (Fig. 2A and data not shown). Several mutants (L469K, E472K, V504K, S541I, and L542G) exhibited reduced growth at 16 °C relative to wild type (Fig. 2A, right panel, labeled in bold). This cold-sensitive slow growth phenotype was most pronounced for the mutation, L469K at position 3 in TPR7. Expression of the sup9-e A19-supS1 reporter gene was monitored at 30 °C by the suppression of auxotrophies in tryptophan and methionine biosynthesis. Since growth differences were not apparent under non-selective conditions at this temperature, differences observed on Trp-Met-medium reflect changes in sup9-e A19-supS1 transcription (34Willis I. Schmidt P. Soll D. EMBO J. 1989; 8: 4281-4288Crossref PubMed Scopus (22) Google Scholar). One mutation, I515K at position 14 in TPR8, conferred a modest increase in suppressor activity implying either the loss of an inhibitory interaction or the acquisition of a positive interaction. However, 5 of 10 mutations (including the L469K mutation) exhibited significantly lower suppressor activity than wild-type (Fig. 2A, middle panel). Whereas wild-type growth was apparent on selective medium after 5 days, the five defective strains did not show significant growth even after 14 days. Consistent with the idea that ligand binding in the second TPR array is important for Tfc4 function in vivo, all of the defective mutations mapped to residues in the A-helices of the repeat (Fig. 1). 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 (5Lopez-de-Leon A. Librizzi M. Puglia K. Willis I.M. Cell. 1992; 71: 211-220Abstract Full Text PDF PubMed Scopus (108) Google Scholar, 6Moir R.D. Sethy-Coraci I. Puglia K. Librizzi M.D. Willis I.M. Mol. Cell. Biol. 1997; 17: 7119-7125Crossref PubMed Scopus (37) Google Scholar, 7Sethy-Coraci I. Moir R.D. Lopez-de-Leon A. Willis I.M. Nucleic Acids Res. 1998; 26: 2344-2352Crossref PubMed Scopus (37) Google Scholar, 33Moir R.D. Puglia K.V. Willis I.M. Mol. Cell. Biol. 2002; 22: 6131-6141Crossref PubMed Scopus (12) Google Scholar). Thus, it was interesting to find that only two of the mutations (S541I and L542G in TPR9, Fig. 2B) showed increased sup9-e A19-supS1 activity in the presence of a multicopy plasmid containing Brf1. The interpretation of multicopy suppression data for presumed loss of function mutations in TFIIIC subunits is not unambiguous in the absence of supporting biochemical studies. The increase in reporter gene expression could indicate that these two mutations have a defect in Brf1 binding that is rescued by elevated Brf1 protein levels. Alternatively, these mutations may cause a defect in a step that precedes Brf1-binding (e.g. TFIIIC-DNA binding). In contrast, sup9-e A19-supS1 expression conferred by the mutations L469K, E472K, and V504K was not increased by overexpression of Brf1 (Fig. 2B) or either of the other two TFIIIB components (see Fig. 2C for L469K and data not shown). The different pattern of multicopy suppression seen for the S541I and L542G mutations in TPR9 versus the L469K, E472K, and V504K mutations, suggests that TPR9 is functionally distinct from TPRs7 and 8 in its ligand interactions. The latter group is most likely affected at more than a single step in transcription complex assembly or function. The presence of non-conserved proline residues in the B-helix of TPR7 and in the A-helix of TPR8 in S. cerevisiae Tfc4 (23Moir R.D. Puglia K.V. Willis I.M. J. Biol. Chem. 2002; 277: 694-701Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) complicates the construction of a molecular model for the entire TPR6 –9 array. However, the structures of TPRs6 and 7A and TPRs8B and 9 have been successfully modeled and show that residues Leu469 and Glu472 in TPR7, Val504 in TPR8 and Ser541 in TPR9 project into the ligand-binding groove formed by the repeats (29Moir R.D. Willis I.M. Conaway J.W. Conaway R.C. The Assembly of the Initiation Factor TFIIIB. Academic Press, New York2004Google Scholar). The predicted surface accessibility of Leu469 in the TPR channel together with the cold-sensitive growth phenotype and the pol III transcription defect of the L469K mutation led us to choose this mutant for biochemical analysis. The L469K Mutation in Tfc4 Affects Brf1 Binding in Vitro— The specific activity of TFIIIC fractions prepared from wild-type and L469K extracts were analyzed in parallel for DNA binding and transcription activity (see “Experimental Procedures”). TFIIIC preparations from L469K and wild-type strains were indistinguishable in protein composition (as determined by the abundance of TFIIIC subunits; Tfc4, Tfc1, and Tfc3, data not shown) and yielded comparable TFIIIC-DNA binding activity (data not shown). TFIIIC-DNA complexes were assembled based on the empirically determined DNA binding activity and assayed for the ability to support transcription in a reconstituted system. These transcription reactions contain TFIIIC, RNA polymerase III, and the TFIIIB factors (recombinant TBP and Brf1 and yeast-purified Bdp1). Transcription of the tRNALeu template (shown in Fig. 3) is dependent on TFIIIC (Fig. 3, lane 1) and generates highly reproducible amounts of transcript (duplicate reactions generate standard deviations of less than 10%, data not shown). Quantitation of the transcription products showed a linear dependence on Brf1 levels as reported previously (6Moir R.D. Sethy-Coraci I. Puglia K. Librizzi M.D. Willis I.M. Mol. Cell. Biol. 1997; 17: 7119-7125Crossref PubMed Scopus (37) Google Scholar) and regression analysis of the data in Fig. 3 yielded r 2 values of >0.99 (data not shown). Under the conditions employed, the L469K mutant TFIIIC supported a 2-fold lower level of transcription than did wild-type TFIIIC (Fig. 3, compare lanes 2–4 to 5–7). The previously documented interaction between the TPR6 –9 array and Brf1 (23Moir R.D. Puglia K.V. Willis I.M. J. Biol. Chem. 2002; 277: 694-701Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) suggested that the L469K mutation likely affected the interaction between Tfc4 and Brf1 and generated a defect in the assembly of TFIIIB complexes.

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