INTRODUCTION

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The yeast Saccharomyces cerevisiae has served as a useful organism for detailed characterization of the factors that direct accurate initiation of transcription by RNA polymerase III and for investigation of the molecular interactions involved in the assembly of stable initiation complexes (reviewed in references 27 and 29). The three accessory transcription factors of S. cerevisiae that are minimally required to promote accurate initiation of transcription of the 5S RNA gene by RNA polymerase III are TFIIIA, TFIIIB, and TFIIIC. These factors assemble sequentially onto the 5S RNA gene in vitro to form a stable preinitiation complex that recruits RNA polymerase III to the start site of transcription (reviewed in references 29 and 86). TFIIIA, a sequence-specific DNA-binding protein that contains nine zinc fingers of the Cys₂-His₂ type, binds to the internal control region (ICR) of the 5S RNA gene as the first step in the in vitro assembly of this multifactor complex. This is followed by incorporation of the large, multisubunit TFIIIC (or ) into the TFIIIA-DNA complex. Formation of the TFIIIC-TFIIIA-DNA complex is necessary for recruitment of TFIIIB, a multisubunit factor that consists of TFIIIB70/Brf, TFIIIB90/Tfc5, and the TATA-binding protein, TBP (10, 46). In the TFIIIB-TFIIIC-TFIIIA-DNA complex, TFIIIB is stably bound upstream of the start site of transcription and recruits RNA polymerase III for multiple rounds of transcription (45).

TFIIIA is required only for transcription of the 5S RNA gene. On tRNA genes, TFIIIC binds directly to the intragenic A- and B-box promoter elements and acts to place TFIIIB upstream of the start site of transcription (47). Despite the requirement for TFIIIA in the assembly of a preinitiation complex on the 5S RNA gene, the relative placement of the individual subunits of TFIIIC and TFIIIB in preinitiation complexes formed on a 5S RNA gene and on a tRNA is similar (5, 6, 9).

The gene, or cDNA, coding for TFIIIA has been identified from S. cerevisiae, various amphibian species, and humans. Although the deduced sequences of these TFIIIAs indicate that they are structurally similar in that they contain nine zinc fingers of the Cys₂-His₂ type, the extent of sequence identity among the TFIIIAs from these organisms is low (2, 3, 23, 28, 32, 87). Moreover, the 81-amino-acid domain that interrupts the otherwise repeating nature of the zinc finger motifs between fingers 8 and 9 of yeast TFIIIA is not present in human TFIIIA or Xenopus TFIIIA. These differences among TFIIIAs are consistent with the observation that several components of the RNA polymerase III transcriptional machinery differ extensively between organisms. For example, both human and yeast TFIIIBs contain a subunit, referred to as TFIIIB90 in the human factor (82) and as TFIIIB70/Brf in the yeast factor (11, 18, 55), that is related to TFIIIB, yet it is only the TFIIB-related amino-terminal portions of the proteins that show significant identity. Another striking example is the complete absence of sequence similarity between the subunit of mammalian TFIIIC that interacts with the B-box region of tRNA genes (49, 51) and the functionally related B-box binding subunit of yeast TFIIIC (50).

Although Xenopus TFIIIB and TFIIIC are relatively uncharacterized, Xenopus TFIIIA and its interaction with the 50-bp ICR of the amphibian 5S RNA gene have been studied extensively (reviewed in reference 74). The ICR of the Xenopus 5S RNA gene contains three elements that contribute to efficient transcription of the gene: the A box, which spans nucleotides +50 to +64; the intermediate element, which spans nucleotides +67 to +72; and the C box, which spans nucleotides +80 to +97 (8, 66, 67). Xenopus TFIIIA binds to the ICR (25) such that its amino terminus is oriented towards the 3' end of the ICR and its carboxyl terminus is positioned towards the 5' end of the ICR (59, 80). The three amino-terminal and three carboxyl-terminal fingers of the molecule are proposed to wrap around the major groove of the DNA helix at each end of the ICR; the zinc fingers in the middle of the protein are thought to lie on one side of the helix, with finger 5 contacting the major groove and fingers 4 and 6 each crossing the minor groove (16, 26, 33, 35, 36, 58). The three amino-terminal zinc fingers interact with the C box with an affinity that is comparable to that of the intact protein (53). The interaction between the carboxyl-terminal zinc fingers and the A box (16, 35) appears to be necessary for transcription (72). Indeed, mutations that disrupt any one of the three carboxyl-terminal zinc fingers lead to reduced transcription (17, 21, 70). These carboxyl-terminal fingers may play a role in transcription by properly positioning the portion of Xenopus TFIIIA that extends beyond the ninth zinc finger. A 14-amino-acid segment that is present in this carboxyl-terminal extension is essential for the transcription factor activity of this TFIIIA (57, 76, 80). Human TFIIIA is thought to bind to the 5S RNA gene in a manner analogous to that of Xenopus TFIIIA, as both the sizes and the patterns of the DNase I footprints generated by these TFIIIAs on their respective templates are similar (62, 81).

Yeast TFIIIA, like its amphibian counterpart, binds to the 5S RNA gene with its carboxyl terminus positioned towards the 5' end of the gene (61, 71). The ICR of the yeast 5S RNA gene, which is considerably smaller than that of the Xenopus 5S RNA gene, consists of only a C-box element between nucleotides +81 and +94 (13). The DNase I footprint obtained with yeast TFIIIA on the yeast 5S RNA gene is correspondingly smaller than that of Xenopus TFIIIA on the Xenopus 5S RNA gene; the region protected by yeast TFIIIA is 35 bp, extending from nucleotide +64 to nucleotide +99 of the 5S RNA gene (10, 71). The smaller DNase I footprint provided by yeast TFIIIA compared with the footprints generated by Xenopus TFIIIA and human TFIIIA can be accounted for by the absence of an intimate interaction of zinc fingers 6 through 9 of yeast TFIIIA with DNA (71). Site-specific DNA-protein photo-cross-linking suggests, however, that yeast TFIIIA may be positioned over a larger region of the gene than that detected by DNase I footprinting, particularly in the TFIIIB-TFIIIC-TFIIIA-DNA complex (9).

We previously analyzed a series of truncated forms of yeast TFIIIA for their ability to bind to the 5S RNA gene, incorporate TFIIIC into the TFIIIA-DNA complex, and support transcription of the 5S RNA gene (61). We found that a polypeptide containing the three amino-terminal zinc fingers binds to the ICR of the 5S RNA gene with an affinity comparable to that of intact TFIIIA and that this truncated form of TFIIIA can recruit TFIIIC (61, 71). The resultant TFIIIC-TFIIIA-DNA complex, however, is unable to support transcription of the 5S RNA gene. We found that the yeast-specific 81-amino-acid domain that is present between zinc fingers 8 and 9 is essential for the transcription factor activity of yeast TFIIIA (61). As a step towards understanding the role of this novel 81-amino-acid domain in establishing an active transcription complex, we carried out a mutational analysis to identify amino acids within this domain that are essential for its transcription factor activity. In addition, we assessed the potential role of zinc fingers 8 and 9 in the transcription factor activity of yeast TFIIIA.

	MATERIALS AND METHODS

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Plasmids. pXS-TFC2, which served as the parental plasmid for introduction of mutations into the coding sequence for TFIIIA, was constructed as follows. First, an XbaI- and SspI-less derivative of pBluescript II SK(+) was generated. The unique XbaI site of pBluescript II SK(+) was destroyed by digesting the plasmid with XbaI, filling in the overhanging ends with the Klenow form of DNA polymerase in the presence of deoxynucleoside triphosphates (dNTPs), and religating the DNA. The resultant plasmid was digested with SspI to yield a 2,831-bp fragment and a 130-bp fragment that contains the promoter for the -lactamase gene. The large fragment was gel purified and ligated with a 55-bp fragment that contains a weak bacterial promoter (kindly provided by D. E. Pulleyblank) to produce pXS, an XbaI- and SspI-less vector. pXS-TFC2 was generated by cloning a KpnI-BamHI fragment, which contains the coding region of TFIIIA obtained from pJA454 (3), between the corresponding sites in the polylinker of pXS. This places the coding sequence for TFIIIA downstream of a promoter for T7 RNA polymerase.

In vitro synthesis of TFIIIA. Wild-type and mutant versions of TFIIIA were synthesized in vitro by using the TnT coupled transcription-translation system (Promega), in which a rabbit reticulocyte lysate supports translation of transcripts synthesized by T7 RNA polymerase. The reactions were carried out according to the manufacturer's instructions and with the addition of ZnSO₄ to 0.1 mM. pXS-TFC2 or its variants were used as the template unless otherwise indicated. TFIIIA(1-397) and TFIIIA(1-365) were synthesized in vitro from pJA454 that had been linearized with SspI and from pJA454-1 that had been linearized with BamHI, respectively (61). Wild-type and mutant proteins that had been synthesized in the presence of [³⁵S]methionine were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis to confirm that protein of the appropriate size was synthesized (data not shown). In vitro-synthesized proteins used in gel mobility shift assays and transcription assays were not radiolabeled.

EMSAs and transcription assays. Electrophoretic mobility shift assays (EMSAs) were performed as described elsewhere (71) except that 0.25 µg of pBluescript II SK(+) was included as a competitor DNA in the reactions. A 20-µl reaction mixture contained 2 µl of an in vitro transcription-translation reaction mixture that had been programmed to produce the indicated version of TFIIIA, 2 µl of partially purified TFIIIC where indicated, and a 270-bp radioactively end-labeled DNA fragment, which was excised from p19-5S and contains the yeast 5S RNA gene (13). The partially purified TFIIIC-containing fraction derived from yeast was prepared as described for fraction j in reference 77. In vitro transcription assays were performed as described elsewhere (77) with the yeast 5S RNA gene (p19-5S) as a template. A 50-µl reaction mixture contained 4.5 µl of an in vitro transcription-translation reaction mixture that had been programmed to produce the indicated version of TFIIIA and 12 µl of a yeast-derived heparin-agarose fraction (fraction h) that contained TFIIIC, TFIIIB, and RNA polymerase III (77).

Yeast media, culture conditions, and transformations. Rich medium (yeast extract-peptone-dextrose [YPD]) and minimal medium (synthetic dextrose [SD]) were as previously described (38). All yeast cultures were grown at 30°C. Transformation of yeast cells was performed by the lithium acetate method of Geitz et al. (30).

In vivo analysis of the mutant versions of TFIIIA. The haploid yeast strain YRW1 (MAT can1-100 his3-11 leu2-3,112 trp1-1 ura3-1 ade2-1 tfc2::LEU2, harboring pJA230) was constructed to test the ability of the variant forms of TFIIIA to support cell viability (60). As the first step in construction of YRW1, the plasmid pRKO (60) was digested with BssHII and BamHI to release a DNA fragment that contains the yeast LEU2 gene flanked by 213 and 300 bp of noncoding sequence from the regions upstream and downstream, respectively, of the TFC2 gene. This fragment was used to replace the entire coding region of the chromosomal TFC2 gene with the LEU2 gene by integrative transformation as follows. The diploid strain LP112 (64) was transformed with the gel-purified BssHII-BamHI fragment, and replacement of one chromosomal copy of the TFC2 gene by LEU2 was confirmed by Southern blot analysis of a Leu⁺ transformant. Plasmid pJA230, a CEN/ARS-based plasmid with a URA3 selectable marker and a 10-kbp insert of yeast DNA containing RPO26 and TFC2 (4), was then introduced into the TFC2/tfc2::LEU2 strain. Sporulation of a Ura⁺ transformant generated the haploid strain YRW1. Since TFC2 is an essential gene (3), viability of YRW1 depends on the presence of pJA230.

Preparation of anti-TFIIIA and Western blot analysis. We confirmed that the various forms of pG3-encoded TFIIIA were expressed in vivo by standard Western blot analysis. Polyclonal antibodies were generated by injection of rabbits with 500 µg of bacterially expressed and purified full-length yeast TFIIIA emulsified with an equal volume of complete Freund's adjuvant. Purification of yeast TFIIIA was carried out as described previously (71) except that as a final step the protein band corresponding to TFIIIA was excised from an SDS-polyacrylamide gel to achieve further purification. Rabbits were boosted every 4 weeks with 100 µg of yeast TFIIIA emulsified with an equal volume of incomplete Freund's adjuvant. Blood was collected from the rabbits 2 weeks after each boost.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

The nine zinc fingers of yeast TFIIIA occur in succession except for zinc fingers 8 and 9, which are separated by an 81-amino-acid domain (Fig. 1A). We have previously shown that TFIIIA lacking the 81-amino-acid domain recruits TFIIIC to a TFIIIA-5S RNA gene complex; the resultant complex, however, is unable to promote transcription (61). We also found that the ninth zinc finger of TFIIIA, although not essential, contributes to efficient transcription (61). In this study we have defined the region of the 81-amino-acid domain that is essential for its function, which we refer to as its transcription factor activity, and we have further assessed the requirement for the adjacent zinc fingers in supporting efficient transcription.

View larger version (47K): [in this window] [in a new window]   FIG. 1.   Effects of alanine replacement of charged residues within the 81-amino-acid domain on activity of TFIIIA. (A) Schematic representation of yeast TFIIIA. The numbered boxes represent the nine zinc fingers of yeast TFIIIA, the stippled region between fingers 8 and 9 represents the 81-amino-acid domain, and the diagonally striped boxes represent the 48-amino-acid amino-terminal region and the 35-amino-acid carboxyl-terminal region. (B) Amino acid sequence of yeast TFIIIA from residues 253 to 397. The sequences for zinc fingers 8 and 9, with the zinc-coordinating cysteines and histidines in boldface, and for the intervening 81-amino-acid domain are given. The residue number, relative to the initiator methionine, of the last amino acid on each line is given on the right. Minus and plus symbols above residues in the 81-amino-acid domain indicate negatively and positively charged amino acids, respectively. Double arrowheads indicate pairs of charged residues that were mutated to alanine; the positions of the residues are given below the lines connecting the arrowheads. (C) Abilities of mutant versions of TFIIIA to bind to the 5S RNA gene and to recruit TFIIIC to the TFIIIA-DNA complex, as assessed by EMSA. A radioactively labeled DNA fragment containing the yeast 5S RNA gene was incubated with in vitro-synthesized versions of TFIIIA in the absence (odd-numbered lanes) or presence (even-numbered lanes) of partially purified yeast TFIIIC prior to electrophoresis on a nondenaturing polyacrylamide gel. Lanes 1 and 2, in vitro transcription-translation reactions that were not programmed to synthesize TFIIIA; lanes 3 and 4, wild-type TFIIIA; lanes 5 to 22, TFIIIA with the following mutations: K287A/K289A (lanes 5 and 6), D299A/H300A (lanes 7 and 8), K308A/H309A (lanes 9 and 10), D314A/E315A (lanes 11 and 12), K324A/R325A (lanes 13 and 14), D342A/E344A (lanes 15 and 16), K345A/R346A (lanes 17 and 18), E348A/E351A (lanes 19 and 20), and R363A/K364A (lanes 21 and 22). The positions of free DNA (minus sign), TFIIIA-DNA complexes (open arrowhead), and TFIIIC-TFIIIA-DNA complexes (solid arrowhead) are indicated on the right. (D) Abilities of mutant versions of TFIIIA to support in vitro transcription of the 5S RNA gene. In vitro transcription reaction mixtures contained the yeast 5S RNA gene as a template; partially purified yeast TFIIIC, TFIIIB, and RNA polymerase III; and the version of in vitro-synthesized TFIIIA indicated above the lane. RL, reticulocyte lysate (in vitro transcription-translation reaction not programmed to synthesize TFIIIA); (WT, wild type. The RNAs synthesized in vitro were analyzed on a 7 M urea-10% polyacrylamide gel. The autoradiogram shows the portion of the gel containing 5S RNA.

Alanine-scanning mutagenesis through charged regions of the 81-amino-acid domain. As a first approach towards identification of amino acids within the 81-amino-acid domain that are involved in the transcription factor activity of TFIIIA, we performed alanine-scanning mutagenesis of charged residues. A charged region of a protein is likely to be solvent exposed, and a charged amino acid(s) on this surface may be involved in interprotein contacts in a multiprotein complex. The choice of alanine for substitutions within a target region allows for a consistent series of mutations with a small amino acid that is unable to provide a side-chain interaction involving atoms beyond the  carbon. This approach has been used extensively for identification of amino acids that are critical for protein-protein interactions (15, 84).

Effects of carboxyl-terminal truncations on the transcription factor activity of TFIIIA. Because site-directed mutagenesis of charged amino acids did not identify any residues as critical for the transcription factor activity of TFIIIA, we next analyzed a series of TFIIIAs that contained carboxyl-terminal truncations that extended into the 81-amino-acid domain. We showed previously that a form of TFIIIA that is truncated at the end of the eighth zinc finger, and therefore lacks the entire 81-amino-acid domain, is unable to support transcription of the 5S RNA gene, whereas a form of TFIIIA that is truncated at the beginning of the ninth zinc finger, and therefore contains the 81-amino-acid domain, supports transcription of the 5S RNA gene, albeit less efficiently than does wild-type TFIIIA (61).

View larger version (32K): [in this window] [in a new window]   FIG. 2.   Effects of carboxyl-terminal deletions on activity of TFIIIA. (A) Amino acid sequences of zinc finger 8, the 81-amino-acid domain, and zinc finger 9 of yeast TFIIIA as shown in Fig. 1B. Arrowheads indicate the positions of the carboxyl termini of truncated forms of TFIIIA; the number of the last residue of each truncated protein is given below the bent tail. (B) Abilities of carboxyl-terminal-truncated versions of TFIIIA to bind to the 5S RNA gene and to recruit TFIIIC to the TFIIIA-DNA complex, as outlined in the legend for Fig. 1C. Lanes 1 and 2, no transcription-translation mixture; lanes 2 and 3, in vitro transcription-translation reactions that were not programmed to synthesize TFIIIA; lanes 5 and 6, wild-type TFIIIA synthesized in vitro; lanes 7 to 22, in vitro-synthesized, truncated versions of TFIIIA extending from the initiator methionine to residue 397 (lanes 7 and 8), 378 (lanes 9 and 10), 365 (lanes 11 and 12), 359 (lanes 13 and 14), 353 (lanes 15 and 16), 347 (lanes 17 and 18), 339 (lanes 19 and 20), or 311 (lanes 21 and 22). (C) Abilities of carboxyl-terminal-truncated versions of TFIIIA to support in vitro transcription of the 5S RNA gene. For details, see the legend for Fig. 1D. (D) Abilities of carboxyl-terminal-truncated versions of TFIIIA to support cell viability. A plasmid shuffle system was used to test the abilities of mutant versions of TFIIIA to replace wild-type TFIIIA in vivo. pG3-derived plasmids that expressed truncated versions of TFIIIA were transformed into the yeast strain YRW1 (see the text for details), and the abilities of these cells to grow on medium containing 5-FOA were monitored. vector, pG3 not expressing TFIIIA; WT, pG3 expressing wild-type TFIIIA; aa1-n, pG3 ex- pressing a version of TFIIIA that extends from the initiator methionine to residue n. (E) Assessment by Western blot analysis of in vivo expression of truncated versions of TFIIIA. Protein extracted from YRW1 yeast cells containing pG3-derived plasmids was separated on an SDS-10% polyacrylamide gel and electrotransferred to a nitrocellulose filter, and the filter was probed with anti-TFIIIA antibody. Lane 1, TFIIIA partially purified from yeast; lane 2, yeast TFIIIA purified from bacteria; lane 3, protein extract of YRW1 cells; lanes 4 to 9, protein extract of YRW1 cells containing pG3 expressing no TFIIIA (lane 4), wild-type TFIIIA (lane 5), TFIIIA(1-365) (lane 6), TFIIIA(1-359) (lane 7), TFIIIA(1-353) (lane 8), and TFIIIA(1-347) (lane 9). The asterisk on the left indicates a cross-reacting molecule not related to TFIIIA. The positions and sizes (in kilodaltons) of molecular mass markers are shown on the right.

Effects of internal deletions within the 81-amino-acid domain of TFIIIA. As described above, analysis of the effects of carboxyl-terminal deletions suggested that a residue(s) within the leucine-rich sequence adjacent to amino acid 359 of TFIIIA was essential for the activity of the protein. Because the hydrophobic, nonpolar nature of this segment suggested the possibility that it might play a role in folding of the 81-amino-acid domain, we assessed the effects of a series of internal deletions within the 81-amino-acid domain (Fig. 3A) on the activity of TFIIIA. We anticipated that if the leucine-rich segment did contribute to folding of the 81-amino-acid domain rather than being directly involved in its function, this approach might identify another region(s) that contributed to the function of TFIIIA.

View larger version (58K): [in this window] [in a new window]   FIG. 3.   Effects of deletions within the 81-amino-acid domain on activity of TFIIIA. (A) Amino acid sequences of zinc finger 8, the 81-amino-acid domain, and zinc finger 9 of yeast TFIIIA, as shown in Fig. 1B. Rectangles and numbers above the amino acid sequence represent regions deleted from the 81-amino-acid domain. The deletions in TFIIIA(284-364), which lacks the entire 81-amino-acid domain, and in TFIIIA(282-353) are not represented. (B) Abilities of versions of TFIIIA with deletions within the 81-amino acid-domain to bind the 5S RNA gene and to recruit TFIIIC to the TFIIIA-DNA complex, as outlined in the legend for Fig. 1C. Lanes 1 to 4 were as described for Fig. 1C. Lanes 5 to 20, in vitro-synthesized versions of TFIIIA lacking residues 284 to 364 (lanes 5 and 6), 282 to 287 (lanes 7 and 8), 282 to 315 (lanes 9 and 10), 282 to 353 (lanes 11 and 12), 316 to 334 (lanes 13 and 14), 328 to 341 (lanes 15 and 16), 342 to 351 (lanes 17 and 18), or 352 to 359 (lanes 19 and 20). (C) Abilities of versions of TFIIIA with deletions in the 81-amino-acid domain to support in vitro transcription of the 5S RNA gene. For details, see the legend to Fig. 1D. (D) Abilities of versions of TFIIIA with deletions in the 81-amino-acid domain to support cell viability. A plasmid shuffle system was used to test the abilities of mutant versions of TFIIIA to support cell viability as described in the legend to Fig. 2D. Growth of cells containing pG3-derived plasmids on medium containing 5-FOA is shown. vector, pG3 not expressing TFIIIA; WT, pG3 expressing wild-type TFIIIA; n-n, pG3 expressing a version of TFIIIA that lacks amino acids n to n. (E) Assessment by Western blot analysis of in vivo expression of TFIIIA containing deletions in the 81-amino-acid domain. The analysis was carried out as described in the legend to Fig. 2E. Protein in lanes 1 to 9 was extracted from YRW1 cells containing pG3-derived plasmids expressing no TFIIIA, wild-type TFIIIA, TFIIIA(284-364), TFIIIA(282-315), TFIIIA(282-353), TFIIIA(316-334), TFIIIA(328-341), TFIIIA(342-351), and TFIIIA(352-359), respectively. The asterisk on the left indicates a cross-reacting molecule not related to TFIIIA. Only the portion of the blot that contains TFIIIA is shown.

Effects of double alanine-scanning mutations of hydrophobic and nonpolar amino acids in the carboxyl-terminal portion of the 81-amino-acid domain. We next subjected the region from residue 343 to 359, which spans the leucine-rich segment at the carboxyl-terminal end of the 81-amino-acid domain, to alanine-scanning mutagenesis (Fig. 4A). For ease of reference, forms of TFIIIA with double mutations were assigned code names beginning with the letter D. We found that TFIIIA(N352A/N355A) (D3), TFIIIA(L354A), TFIIIA(L356A), and TFIIIA(N359A) supported efficient in vitro transcription of the 5S RNA gene (Fig. 4C, lanes 6, 8, 9, and 11). TFIIIA(L343A/L347A) (D2), TFIIIA(L354A/L356A) (D4), and TFIIIA(L357A/L358A) (D5) had reduced, but readily detectable, activity (Fig. 4C, lanes 5, 7, and 10). Of these mutants, we consistently found that TFIIIA(L343A/L347A) (D2) was more active than TFIIIA(L357A/L358A) (D5) and that TFIIIA(L354A/L356A) (D4) was the most compromised for activity (Fig. 4C, lanes 5, 7, and 10). For comparison, we also monitored the effect of replacement by alanine of the hydrophobic residues at positions 296 and 297 and at positions 336 and 337. We found that both TFIIIA(L296A/V297A) and TFIIIA(F336A/L337A) (D1) were as active as wild-type TFIIIA (Fig. 4C, lanes 3 and 4).

View larger version (63K): [in this window] [in a new window]   FIG. 4.   Effects of double and quadruple alanine-scanning mutations within the carboxyl-terminal portion of the 81-amino-acid domain on activity of TFIIIA. (A) Schematic representation of TFIIIA, showing the positions of mutated residues within the amino acid sequence from residue 335 to 365. The series of mutations that were introduced into the sequence from amino acid 335 to amino acid 365 are listed on the left, preceded by code names for the double (D1 to D5) and the quadruple (Q1 to Q8) alanine substitutions, and are represented by A's aligned below the amino acid sequence. Dashes represent wild-type amino acids. For ease of alignment, Phe336 and Leu residues are shaded on all lines. A circled or boxed A indicates that alanine has replaced a negatively or positively charged residue, respectively. The two columns at the right summarize the transcription factor activity of each mutant TFIIIA with respect to its ability to support in vitro transcription of the 5S RNA gene (as shown in panels C and E) and cell viability (as shown in panels F and G). Left column: +++, the ability to support in vitro transcription was similar to that of wild-type TFIIIA; , only a background level of in vitro transcription was observed. Right column: +, the mutant was able to support cell viability; , the mutant was not able to support cell viability. (B and D) Abilities of versions of TFIIIA with single or double alanine-scanning mutations (B) or with quadruple alanine-scanning mutations (D) within the 81-amino-acid domain to bind to the 5S RNA gene and to recruit TFIIIC to the TFIIIA-DNA complex, as outlined in the legend to Fig. 1C. Lanes 1 to 4 were as described for Fig. 1C. (B) Lanes 5 to 22, TFIIIA with the following mutation(s): L296A/V297A (lanes 5 and 6), F336A/L337A (lanes 7 and 8), L343A/L347A (lanes 9 and 10), N352A/N355A (lanes 11 and 12), L354A/L356A (lanes 13 and 14), L354A (lanes 15 and 16), L356A (lanes 17 and 18), L357A/L358A (lanes 19 and 20), or N359A (lanes 21 and 22). (D) Lanes 5 to 20, TFIIIA with the following mutations: F336A/L337A/L343A/L347A (lanes 5 and 6), D342A/E344A/E348A/E351A (lanes 7 and 8), L343A/L347A/L354A/L356A (lanes 9 and 10), L343A/L347A/L357A/L358A (lanes 11 and 12), K345A/R346A/L357A/L358A (lanes 13 and 14), K345A/R346A/R363A/K364A (lanes 15 and 16), L354A/L356A/L357A/L358A (lanes 17 and 18), or L357A/L358A/R363A/K364A (lanes 19 and 20). (C and E) Abilities of versions of TFIIIA with single or double (C) or quadruple (E) alanine-scanning mutations in the carboxyl-terminal portion of the 81-amino-acid domain to support in vitro transcription of the 5S RNA gene. For details, see the legend to Fig. 1D. Where appropriate, the D and Q codes from panel A are given above the lanes. (F and G) Abilities of versions of TFIIIA with double and quadruple alanine-scanning mutations to support cell viability. A plasmid shuffle system was used to test the abilities of mutant versions of TFIIIA to support cell viability, as described in the legend to Fig. 2D. Cells containing pG3-derived plasmids that did not express TFIIIA (vector) or that expressed wild-type TFIIIA (WT) or versions of TFIIIA with the double or quadruple alanine-scanning mutations (D and Q codes, respectively) diagrammed in panel A were streaked onto 5-FOA-containing medium. (H) Assessment by Western blot analysis of in vivo expression of mutant forms of TFIIIA. Protein extracted from YRW1 yeast cells containing pG3-derived plasmids was analyzed as described in the legend to Fig. 2E. Protein in lanes 1 to 12 was extracted from cells containing pG3 expressing no TFIIIA (lane 1), wild-type TFIIIA (lane 2), TFIIIA(L343A/L347A) (lane 3), TFIIIA(L354A/L356A) (lane 4), TFIIIA(L357A/L358A) (lane 5), TFIIIA(L343A/L347A/L354A/L356A) (lane 6), TFIIIA(L343A/L347A/L357A/L358A) (lane 7), TFIIIA(F336A/L337A/L343A/L347A) (lane 8), TFIIIA(K345A/R346A/L357A/L358A) (lane 9), TFIIIA(K345A/R346A/R363A/K364A) (lane 10), TFIIIA(L354A/L356A/L357A/L358A) (lane 11), and TFIIIA(L357A/L358A/R363A/K364A) (lane 12). Asterisk, a cross-reacting molecule not related to TFIIIA; arrow, full-length TFIIIA.

Effects of quadruple alanine-scanning mutations within the carboxyl-terminal portion of the 81-amino-acid domain. Although we found that TFIIIA(352-359) was inactive both in vitro and in vivo, double substitutions in this region (N352A/N355A, L354A/L356A, and L357A/L358A) did not abolish the transcription factor activity of TFIIIA (see above). We therefore next assessed the effects of quadruple mutations in this region, anticipating that replacement of four leucines by alanine would be sufficiently deleterious to abolish function. For ease of reference, forms of TFIIIA with quadruple mutations were assigned code names beginning with the letter Q. Indeed, combining the double mutations L354A/L356A and L357A/L358A, both of which compromised the activity of TFIIIA (Fig. 4C, lanes 7 and 10, respectively), with each other or with the double mutation L343A/L347A, which on its own led to a modest decrease in activity of TFIIIA (Fig. 4C, lane 5), completely inactivated TFIIIA. We found that TFIIIA(L354A/L356A/L357A/L358A) (Q7), TFIIIA(L343A/L347A/L354A/L356A) (Q3), and TFIIIA(L343A/L347A/L357A/L358A) (Q4) failed to support in vitro transcription of the 5S RNA gene (Fig. 4E, lanes 5, 6, and 9) and failed to support cell viability (Fig. 4F and G). For comparison, we combined the double mutation L343A/L347A, which led to a modest decrease in the activity of TFIIIA (Fig. 4C, lane 5), with mutation of the hydrophobic residues F336 and L337, which as a double mutation had no effect on the activity of TFIIIA (Fig. 4C, lane 4). The resultant TFIIIA(F336A/L337A/L343A/L347A) (Q1) supported a very low level of 5S RNA gene transcription in vitro (Fig. 4E, lane 3) and supported cell viability (Fig. 4G). We also monitored the effect of mutation of four negatively charged residues (D342, E344, E348, and E351) in this region. TFIIIA(D342A/E344A/E348A/E351A) (Q2) was as active as wild-type TFIIIA in supporting in vitro transcription of the 5S RNA gene (Fig. 4E, lane 4).

Zinc fingers 8 and 9 contribute to the transcription factor activity of TFIIIA. We fortuitously obtained by error-prone PCR mutagenesis a gene encoding TFIIIA with a mutation in a zinc-coordinating residue of finger 8 (H272R) and in a zinc-coordinating residue of finger 9 (C367Y) (see Materials and Methods) (Fig. 5A). This combination of mutations (H272R/C367Y), which would be expected to disrupt the structure of zinc fingers 8 and 9, abolished the ability of TFIIIA to support in vitro transcription of the 5S RNA gene (Fig. 5C, lane 3) and to support cell viability (Fig. 5D). We found, however, that TFIIIA that contained H272R or C367Y as a single mutation (see Materials and Methods) was only modestly compromised in its ability to support in vitro transcription of the 5S RNA gene (Fig. 5C, lanes 4 and 5) and was able to support cell viability (Fig. 5D). These data suggest that zinc fingers 8 and 9 make redundant contributions to the transcription factor activity of TFIIIA; the activity of TFIIIA is maintained on disruption of finger 8 or finger 9, but its activity is abolished on disruption of both fingers. We note that the assessment of protein-DNA complex formation by the EMSA shown in Fig. 5B suggests that TFIIIA(H272R/C367Y) was less active than wild-type TFIIIA in the formation of TFIIIA-DNA and TFIIIC-TFIIIA-DNA complexes. Because of the qualitative nature of this assay, however, we do not know if this apparent difference is significant.

View larger version (52K): [in this window] [in a new window]   FIG. 5.   Effects of mutations that disrupt the structure of zinc fingers 8 and 9 on activity of TFIIIA. (A) Schematic representation of yeast TFIIIA as described for Fig. 1A. The approximate positions of mutations H272R and C367Y within zinc fingers 8 and 9, respectively, are indicated by arrows. (B) Abilities of versions of TFIIIA with disruptions of zinc fingers 8 and 9 to bind to the 5S RNA gene and to recruit TFIIIC to the TFIIIA-DNA complex, as outlined in the legend to Fig. 1C. Lanes 1 to 4 were as described for Fig. 1C. Lanes 5 to 10, in vitro-synthesized versions of TFIIIA containing the following mutation(s): H272R/C367Y (lanes 5 and 6), H272R (lanes 7 and 8), or C367Y (lanes 9 and 10). (C) Abilities of versions of TFIIIA with disruptions of zinc fingers 8 and 9 to support in vitro transcription of the 5S RNA gene. For details, see the legend to Fig. 1D. (D) Abilities of versions of TFIIIA with disruptions of zinc fingers 8 and 9 to support cell viability. A plasmid shuffle system was used to test the abilities of mutant versions of TFIIIA to support cell viability, as described in the legend to Fig. 2D. (E) Assessment by Western blot analysis of in vivo expression of versions of TFIIIA with disruptions in zinc fingers 8 and 9. The analysis was carried out as described in the legend to Fig. 2E. Protein was extracted from YRW1 cells containing pG3-derived plasmids expressing no TFIIIA (lane 1), wild-type TFIIIA (lane 2), TFIIIA(H272R/C367Y) (lane 3), TFIIIA(H272R) (lane 4), or TFIIIA(C367Y) (lane 5).

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

We previously found that an 81-amino-acid domain that is present between zinc fingers 8 and 9 of yeast TFIIIA is essential for the transcription factor activity of the protein. TFIIIA lacking this domain binds efficiently to the 5S RNA gene and recruits TFIIIC, but the resultant TFIIIC-TFIIIA-DNA complex is unable to promote transcription (61). As a step towards understanding the role of this region in assembly of a functional preinitiation complex, we have defined specific residues within the 81-amino-acid domain that are critical for TFIIIA-mediated transcription. We tested versions of TFIIIA containing carboxyl-terminal deletions, internal deletions, and substitutions within the 81-amino-acid domain for their ability to support transcription of the 5S RNA gene in vitro and in vivo. We note that the only essential function of TFIIIA in vivo is as a transcription factor for the 5S RNA gene (12). In this study, we found that a short hydrophobic segment within the 81-amino-acid domain, from residue 352 to 359, was crucial for TFIIIA-mediated transcription.

Essential role of a hydrophobic patch within the 81-amino-acid domain. We found that the 81-amino-acid domain of yeast TFIIIA was surprisingly tolerant to mutation. We were unable to identify any charged residue within this domain that was essential for its function (Fig. 1). Moreover, forms of TFIIIA with extensive deletions within the 81-amino-acid domain retained the ability to support transcription of the 5S RNA gene (Fig. 3). We found that TFIIIA that lacked the sequence from residue 282 to 315, from residue 316 to 334, from residue 328 to 341, or from residue 342 to 351 supported in vitro transcription of the 5S RNA gene, although in some instances to a reduced level, and supported cell viability (Fig. 3). However, deletion of an asparagine- and leucine-rich segment (NGLNLLLN; residue 352 to 359) within the carboxyl-terminal portion of the 81-amino-acid domain destroyed the activity of TFIIIA. Our data suggest that no other segment within the 81-amino-acid domain can substitute for this asparagine- and leucine-rich segment: TFIIIA lacking the carboxyl-terminal portion of the protein beyond residue 359 supported cell viability, whereas TFIIIA lacking the carboxyl-terminal portion beyond residue 353 did not support viability (Fig. 2).

Requirement for hydrophobic residues within the carboxyl-terminal portion of the 81-amino-acid domain. Alanine-scanning mutagenesis indicated that no single amino acid in the region from residue 354 to 359 (LNLLLN) was essential for the activity of TFIIIA (Fig. 4). Analysis of the effect of double mutations in the segment ³⁵²NGLNLLLN³⁵⁹ indicated that the leucine residues were more important for function than the asparagine residues. TFIIIA containing the double mutation N352A/N355A appeared to be as active as wild-type TFIIIA, whereas TFIIIA containing the double mutation L354A/L356A or L357A/L358A had reduced activity. TFIIIA in which L354, L356, L357, and L358 were all mutated was unable to support cell viability. The double mutation L343A/L347A, which consists of residues amino-terminal to the hydrophobic ³⁵²NGLNLLLN³⁵⁹ segment, had only a very minor effect on the in vitro activity of TFIIIA. Interestingly, combining this double mutation with either the double mutation L354A/L356A or the double mutation L357A/L358A led to forms of TFIIIA that were unable to support cell viability. This observation suggests that the overall hydrophobicity of the region that encompasses ³⁵²NGLNLLLN³⁵⁹ is more important for its function than is any single residue. In support of this notion, we found that combining the double mutations F336A/L337A and L343A/L347A, which are in residues upstream of the ³⁵²NGLNLLLN³⁵⁹ hydrophobic segment and which by themselves had little effect on the activity of TFIIIA, led to a dramatic decrease in the activity of TFIIIA. It is interesting that TFIIIA with an internal deletion that eliminated residues 342 to 351 was just as active in vitro as wild-type TFIIIA. In this case, however, L337 occupied the same position, at least at the primary sequence level, as L347 normally does relative to residue 352, and consequently L337 may have fulfilled the role of L347.

Role of the leucine-rich segment of the 81-amino-acid domain in TFIIIA-mediated transcription. We previously presented a model for the role of yeast TFIIIA in the assembly of a transcription complex on the yeast 5S RNA gene (61) in which we took into consideration the fact that TFIIIC (or ) consists of two domains, _A and _B, which interact with the A box and B box, respectively, of tRNA genes (27). It is the interaction of the _A domain with the A box of a tRNA gene that is responsible for appropriate positioning of TFIIIB upstream of the start site of transcription (43; also reviewed in reference 86). In our model, we proposed that the amino-terminal zinc fingers of yeast TFIIIA interact with the _B domain of TFIIIC and we speculated that once TFIIIC had been recruited to a TFIIIA-DNA complex by this interaction, the 81-amino-acid domain of TFIIIA interacted with the _A domain of TFIIIC (61). The latter interaction was predicted to be responsible for docking the multisubunit TFIIIC on the TFIIIA-DNA complex with the appropriate topography so that TFIIIB could be properly positioned to fulfill its role as an initiation factor. The present study implicates a hydrophobic segment in the carboxyl-terminal portion of the 81-amino-acid domain as essential for its function and, according to our model, leads to the suggestion that this segment interacts with a region of TFIIIC.

Comparison of yeast and Xenopus TFIIIAs. A 14-amino-acid segment (KRSLASRLTGYIPP) that is present in the portion of Xenopus TFIIIA that extends beyond the ninth zinc finger is essential for the transcription factor activity of this TFIIIA (57). Zinc fingers 8 and 9 also contribute to the transcription factor activity of Xenopus TFIIIA (21, 70); these fingers may have a direct role in assembly of a functional transcription complex or they may act indirectly, through their interaction with the A box of the ICR, to appropriately position the 14-amino-acid segment to fulfill its role in promoting transcription. We find no sequence similarity between this 14-amino-acid segment of Xenopus TFIIIA and the leucine-rich region of the 81-amino-acid domain of yeast TFIIIA that we have defined as being important for its transcription factor activity. This lack of sequence similarity is not surprising, as components of the RNA polymerase III transcriptional machinery appear to be quite divergent among species (see the introduction and reference 83). It is also possible that the molecular details of assembly of a functional transcription complex differ between yeast and Xenopus. For example, the activity of the transcription-activating region of Xenopus TFIIIA appears to be more position dependent (57) than the activity of the transcription-activating region of yeast TFIIIA (this study), as assessed by monitoring the effects of deletion of adjacent sequences. Zinc fingers 8 and 9 of both Xenopus TFIIIA (21, 70) and yeast TFIIIA (this study) appear to contribute to the transcription factor activity of TFIIIA. However, we found that yeast TFIIIA remained active if either finger 8 or finger 9 was disrupted by mutation; disruption of both these fingers was required in order to abolish the ability of TFIIIA to promote transcription of the 5S RNA gene. This suggests that these zinc fingers play a redundant role in promoting transcription. These fingers may help establish the overall topography of the preinitiation complex either by extending the interaction of TFIIIA with DNA (9), by interacting with another component of the complex, or by contributing to the function of the 81-amino-acid domain.