Proc. Nati. Acad. Sci. USA Vol. 88, pp. 7308-7312, August 1991 Biochemistry
Two essential components of the Saccharomyces cerevisiae transcription factor TFIIIH: Transcription and DNAbinding properties (RNA polymerase IMl/transcription factor TFUIIC/transcrlption complexes/photocrossinldng/DNase I protection)
GEORGE A. KASSAVETIS*, BLAINE BARTHOLOMEW, JAIME A. BLANCO, TERENCE E. JOHNSON, AND E. PETER GEIDUSCHEK Department of Biology and Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0634
Contributed by E. Peter Geiduschek, May 30, 1991
ABSTRACT RNA polymerase III transcription factor TFIB from Saccharomyces cerevisiae contains at least two polypeptides, with apparent masses of90 and 70 kDa, that were previously identified by photocrosslinking to DNA. It is shown here that TFUIB can be chromatographically separated into two components, each of which is required for efficient tRNA gene transcription. DNA-protein photocrosslinking experiments show these two components separately contain the 90and 70-kDa TFIB-specific polypeptides. The 70-kDa component forms a heparin-sensitive complex with transcription factor TFIUC and DNA, stabilizes TFHIC interaction with the tRNA gene promoter elements, and protects against DNase I digestion in the 3' portion of the upstream DNA sequence that is occupied by TFIB. The 90-kDa component of TFIB, which only detectably interacts with the TFIIIC-DNA complex when the 70-kDa component is also present, generates the complete DNase I protection pattern of TFIIB -and bestows heparin-insensitivit on the TFUIB-DNA complex. The resolution of TFMB into two functional components further defines the probable steps and interactions involved in the formation of stable transcription complexes.
tRNA gene. These two polypeptides displayed all the readily testable properties normally ascribed to TFIIIB. In this report, we show that TFIIIB can be separated into two components that contain the 90- and 70-kDa TFIIIBspecific polypeptides identified by the photocrosslinking analysis. Both components are essential for efficient transcription of tRNA genes. The 70-kDa protein-containing component alone is capable of assembling onto TFIIIC-DNA complexes but lacks the heparin-insensitive binding property of TFIIIB. The 90-kDa protein-containing component does not, by itself, detectably bind to the TFIIIC-DNA complex but does so when the 70-kDa polypeptide component is also present and confers heparin insensitivity to both the 70- and 90-kDa components of their joint protein-DNA complex.
MATERIALS AND METHODS Plasmids pTZ1, pTZ2, and p2A2B containing the SUP4 tRNAT rG62-+ C promoter-up mutant and DNA purification have been described (4, 9). DNA-affinity-purified TFIIIC,
Sequence-specific transcription by RNA polymerase III (pol III) requires the participation of multiple transcription factors (TFIII) that were initially defined as chromatographically separable fractions (e.g., refs. 1-3). Two functionally conserved factors, TFIIIB and -C, are required in all eukaryotes to transcribe genes coding for- tRwNA§ and a number of small viral and cellular RNAs. A third-factor, TFIIIA, is required for 5S rRNA gene transcription. In Saecharomyces cerevisiae, both TFIIIC and TFIIIA function as assembly factors that place TFIIIB within a 40-base-pair region directly upstream of the site of transcription initiation; TFIIIB, alone, functions to direct accurate transcription by pol III (4, 5). S. cerevisiae TFIIIC is a single large protein of =-:600 kDa (6) that contains at least four, and probably five, different subunits (7-9). The orientation of these subunits along the DNA helix has been examined by UV crosslinking (7, 9, 10). Recently, some of us (10) extended our photocrosslinking analysis of the pol III transcription factor complex on the SUP4 tRNATYr gene upstream of the transcriptional start to the DNA-binding region of TFIIIB. We found only the 135-kDa subunit of TFIJIC detectably protruding upstream of the transcriptional start, implicating this subunit in assembling TFIIIB into its DNA-binding site. Two polypeptides with apparent masses of 90 and 70 kDa that are present in our TFIIIB preparation were also found to photocrosslink specifically in the upstream TFIIIB-binding region of the SUP4
Cibacron blue-Sepharose-purified TFIIIB, and Mono Q-purifled pol III were prepared as described (5). Quantities of TFIIIC and TFIIIB are specified in fmol of DNA-binding activity determined by double-reciprocal plots of DNA and DNA-TFIIIC complex saturation curves, respectively, densitometrically determined from gel retardation (TFIIIC) or DNase I-protection analysis (TFIIIB; ref. 4). Pol III is specified in terms of fmol of active molecules determined by a single round of transcription (4, 5). The impure TFIIIC/pol III fraction used to assay TFIIIB in some experiments is a 250 mM NaCl step fraction from the first DEAE-Sephadex A25 column used in the TFIIIB purification (4). This material was dialyzed and rechromatographed on DEAE-Sephadex to remove trace amounts of TFIIIB transcription factor activity. Gel-retardation, photocrosslinking, DNase I protection analysis, the preparation of 3'-end-labeled HindIII-EcoRI fiagments of pTZ1 and pTZ2, the preparation of photocrosslinking DNA probes -38/-37, -17/-12, and -9, and conditions for single- and multiple-round transcription assays have been described (4, 5, 9-11). In the assays involving the B" fraction from Mono S (see Figs. 4 and 5), a revised optimal assay buffer was used in which the acetate salts of Tris base and Mg2' replaced chlorides, and 160 mM KOAc replaced 80 mM NaCl. In these experiments, assays with Cibacron blue-Sepharose-purified TFIIIB contained 120 mM KOAc and 40 mM NaCI instead of the previously used 80 mM NaCl. Preparation of the Mono S B' (70 kDa) and B" (90 kDa) fractions was as follows. Trailing fractions from two preparations of TFIIIB purified to the 700 mM NaCl step elution
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: pol III, RNA polymerase III; UBR, upstream binding region. *To whom reprint requests should be addressed. 7308
Biochemistry: Kassavetis et al. from Cibacron blue-Sepharose (4, 5) were combined (11 ml), diluted to 150 mM NaCl and 20% (vol/vol) glycerol with buffer D [40 mM NaHepes, pH 7.8/7 mM MgCl2/10 mM 2-mercaptoethanol/0.01% Tween 20/0.5 mM phenylmethylsulfonyl fluoride/leupeptin (1 ,ug/ml)/pepstatin (1 ;Lg/ml)/ 20% glycerol], and loaded onto a Mono S (Pharmacia, HR 5/5) column. The column was washed with 5 ml of 150 mM NaCl in buffer D and a 20-ml 150-400 mM NaCl gradient in buffer D was applied, followed by 1 M NaCl in buffer D. Fractions were assayed for TFIIIC-dependent upstream footprinting activity on the nontranscribed strand of the SUP4 tRNA gene and for transcription factor activity with the impure TFIIIC/pol III DEAE-Sephadex fraction (see above). Both activities eluted at 275 mM NaCl. This material (1.5 ml) is referred to as the Mono S B' fraction in the text. Side fractions (1.5 ml) were also utilized after 10-fold concentration with a Centricon-30 unit (Amicon). This B' fraction was highly enriched in a 70-kDa protein, which represented -5% of the total silver-stainable material distributed among 10 polypeptides (data not shown). The activity that complements the B' fraction for specific transcription (termed B") was purified from the 250 mM NaCl step fraction off the first DEAE-Sephadex column of the TFIIIB purification scheme (4). The B" content in this fraction varied from trace to minor, relative to the TFIIIB activity in the 100 mM NaCl flow through ofthis column (data not shown). Further purification of the B" activity was conveniently achieved as a side product of our TFIIIC and pol III purification scheme where this material [from 1.7 kg (wet weight) of cells] was fractionated by gradient elution from multiple heparin-agarose columns as described (5). B" activity was measured by transcription with purified TFIIIC, pol III, and the Mono S B' fraction and by formation of heparin-resistant B'- and TFIIIC-dependent DNA complexes that migrate on native gels identically with TFIIIB-DNA complexes (data not shown; ref. 5). Both activities coeluted on these columns at or near 350 mM (NH4)2SO4, whereas TFIIIC and pol III eluted at 475 and 560 mM (NH4)2SO4, respectively. The heparin-agarose B" fractions were pooled (61 ml), 54 ml of this material was dialyzed in 450 mM KOAc in buffer D, applied to Mono S, washed with 7 ml of the same buffer, followed by a 20-ml 450-1000 mM KOAc gradient. Most of the protein (>90%o) flowed through at this high ionic strength, but the B" activity peak was spread throughout the first half of the gradient. This material was pooled and approximately half of it was diluted 1:2 with buffer D, reapplied onto Mono S, and eluted with 700 mM KOAc in buffer D. RESULTS We have recently used a photocrosslinking method to show that S. cerevisiae TFIIIB contains at least two distinct polypeptides of 90 and 70 kDa (10). Although our current preparation of TFIIIB has undergone five column chromatographic steps of purification, polypeptides of the above sizes represent minor components in our final (Cibacron blue) fraction, as judged by SDS/PAGE (data not shown; ref. 4). Upon further purification of the Cibacron blue fraction on Mono S (cation exchanger), TFIIIC-dependent-upstreamsequence DNA-binding activity eluted at 275 mM NaCI (data not shown, but see below). In a multiple-round transcription assay utilizing a DEAE-Sephadex fraction as a convenient, but impure source of TFIIIC and pol III (but lacking any detectable TFIIIB activity), the Mono S fraction exhibiting the DNA-binding activity retained the TFIIIB transcription activity of the input Cibacron blue fraction (Fig. LA, compare lanes g and h with lanes a and b). A single-round transcription assay likewise showed that the Mono S fraction was 74% as active as the input Cibacron blue fraction (compare lanes i
Proc. Natl. Acad. Sci. USA 88 (1991) A
a b c de f g h i
7309
j kI
LlI Blue B (90+70): 2 4 4 2 4 4 2 4 4 2 4 4 ..1 MonoS B' (70): - crude C.Pol 111: + + - + + pureC PolI11: - - - + + + - - - + Tx Single Multiple: M M S M M S M M S M M S -
+
+
+
B
+
DNA probes -38/-37 and -17/-12 a b c d e f
135 -a(C-
135 kDa (C)-_ M
do
90 kDa 70 kDa- _.f
0-
_
+.
_
++
Heparin TEIIIC B' (70 kDa.) DEAE 0.25 M step
_
+0
+ +
FIG. 1. Characterization of the B' fraction. (A) Transcription analysis with crude and purified TFIIIC and pol III. Single-round transcription (Tx) (lanes c, f, i, and l) or 15 min of multiple-round transcription (lanes a, b, d, e, g, h, j, and k) was performed after formation of the 17-mer ternary complex on the SUP4 tRNA gene (4). A crude TFIIIC/pol III fraction (2 ,ul) or 30 fmol of purified TFIIIC and 2 fmol of purified pol III were incubated with Cibacron blueSepharose-purified TFIIIB (3.7 fmol/,ll) or the Mono S-purified B' fraction as indicated. Densitometric units of specific transcription were as follows. Lanes: a, 695; b, 990; c, 171; d, 259; e, 459; f, 128; g, 731; h, 831; i, 126; j, 7; k, 11; 1, 4. -, Not added; +, added; M, multiple rounds of transcription; S, single round of transcription. (B) Photocrosslinking analysis. Purified TFIIIC (35 fmol), 5 ,ul of the Mono S B' fraction, and 2 .l of the crude TFIIIC/pol III DEAESephadex fraction were incubated with the photocrosslinking DNA probes -38/-37 (1 fmol) and -17/-12 (1 fmol) as indicated. Complexes treated with heparin (300 ,ug/ml) prior to UV irradiation are also indicated. The 135-kDa subunit of TFIIIC and the 90- and 70-kDa polypeptide components of TFIIIB are identified at the left of the figure.
and c). In contrast, the same Mono S fraction was transcriptionally deficient when the crude TFIIIC/pol III fraction was replaced with highly purified TFIIIC and pol III (lanes j-l): whereas the Cibacron blue fraction formed 75% as many active transcription complexes with purified TFIIIC and pol III as it did with impure TFIIIC and pol III (compare lanes f and c), the comparable proportion for the Mono S fraction was
Two essential components of the Saccharomyces cerevisiae transcription factor TFIIIH: Transcription and DNAbinding properties (RNA polymerase IMl/transcription factor TFUIIC/transcrlption complexes/photocrossinldng/DNase I protection)
GEORGE A. KASSAVETIS*, BLAINE BARTHOLOMEW, JAIME A. BLANCO, TERENCE E. JOHNSON, AND E. PETER GEIDUSCHEK Department of Biology and Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0634
Contributed by E. Peter Geiduschek, May 30, 1991
ABSTRACT RNA polymerase III transcription factor TFIB from Saccharomyces cerevisiae contains at least two polypeptides, with apparent masses of90 and 70 kDa, that were previously identified by photocrosslinking to DNA. It is shown here that TFUIB can be chromatographically separated into two components, each of which is required for efficient tRNA gene transcription. DNA-protein photocrosslinking experiments show these two components separately contain the 90and 70-kDa TFIB-specific polypeptides. The 70-kDa component forms a heparin-sensitive complex with transcription factor TFIUC and DNA, stabilizes TFHIC interaction with the tRNA gene promoter elements, and protects against DNase I digestion in the 3' portion of the upstream DNA sequence that is occupied by TFIB. The 90-kDa component of TFIB, which only detectably interacts with the TFIIIC-DNA complex when the 70-kDa component is also present, generates the complete DNase I protection pattern of TFIIB -and bestows heparin-insensitivit on the TFUIB-DNA complex. The resolution of TFMB into two functional components further defines the probable steps and interactions involved in the formation of stable transcription complexes.
tRNA gene. These two polypeptides displayed all the readily testable properties normally ascribed to TFIIIB. In this report, we show that TFIIIB can be separated into two components that contain the 90- and 70-kDa TFIIIBspecific polypeptides identified by the photocrosslinking analysis. Both components are essential for efficient transcription of tRNA genes. The 70-kDa protein-containing component alone is capable of assembling onto TFIIIC-DNA complexes but lacks the heparin-insensitive binding property of TFIIIB. The 90-kDa protein-containing component does not, by itself, detectably bind to the TFIIIC-DNA complex but does so when the 70-kDa polypeptide component is also present and confers heparin insensitivity to both the 70- and 90-kDa components of their joint protein-DNA complex.
MATERIALS AND METHODS Plasmids pTZ1, pTZ2, and p2A2B containing the SUP4 tRNAT rG62-+ C promoter-up mutant and DNA purification have been described (4, 9). DNA-affinity-purified TFIIIC,
Sequence-specific transcription by RNA polymerase III (pol III) requires the participation of multiple transcription factors (TFIII) that were initially defined as chromatographically separable fractions (e.g., refs. 1-3). Two functionally conserved factors, TFIIIB and -C, are required in all eukaryotes to transcribe genes coding for- tRwNA§ and a number of small viral and cellular RNAs. A third-factor, TFIIIA, is required for 5S rRNA gene transcription. In Saecharomyces cerevisiae, both TFIIIC and TFIIIA function as assembly factors that place TFIIIB within a 40-base-pair region directly upstream of the site of transcription initiation; TFIIIB, alone, functions to direct accurate transcription by pol III (4, 5). S. cerevisiae TFIIIC is a single large protein of =-:600 kDa (6) that contains at least four, and probably five, different subunits (7-9). The orientation of these subunits along the DNA helix has been examined by UV crosslinking (7, 9, 10). Recently, some of us (10) extended our photocrosslinking analysis of the pol III transcription factor complex on the SUP4 tRNATYr gene upstream of the transcriptional start to the DNA-binding region of TFIIIB. We found only the 135-kDa subunit of TFIJIC detectably protruding upstream of the transcriptional start, implicating this subunit in assembling TFIIIB into its DNA-binding site. Two polypeptides with apparent masses of 90 and 70 kDa that are present in our TFIIIB preparation were also found to photocrosslink specifically in the upstream TFIIIB-binding region of the SUP4
Cibacron blue-Sepharose-purified TFIIIB, and Mono Q-purifled pol III were prepared as described (5). Quantities of TFIIIC and TFIIIB are specified in fmol of DNA-binding activity determined by double-reciprocal plots of DNA and DNA-TFIIIC complex saturation curves, respectively, densitometrically determined from gel retardation (TFIIIC) or DNase I-protection analysis (TFIIIB; ref. 4). Pol III is specified in terms of fmol of active molecules determined by a single round of transcription (4, 5). The impure TFIIIC/pol III fraction used to assay TFIIIB in some experiments is a 250 mM NaCl step fraction from the first DEAE-Sephadex A25 column used in the TFIIIB purification (4). This material was dialyzed and rechromatographed on DEAE-Sephadex to remove trace amounts of TFIIIB transcription factor activity. Gel-retardation, photocrosslinking, DNase I protection analysis, the preparation of 3'-end-labeled HindIII-EcoRI fiagments of pTZ1 and pTZ2, the preparation of photocrosslinking DNA probes -38/-37, -17/-12, and -9, and conditions for single- and multiple-round transcription assays have been described (4, 5, 9-11). In the assays involving the B" fraction from Mono S (see Figs. 4 and 5), a revised optimal assay buffer was used in which the acetate salts of Tris base and Mg2' replaced chlorides, and 160 mM KOAc replaced 80 mM NaCl. In these experiments, assays with Cibacron blue-Sepharose-purified TFIIIB contained 120 mM KOAc and 40 mM NaCI instead of the previously used 80 mM NaCl. Preparation of the Mono S B' (70 kDa) and B" (90 kDa) fractions was as follows. Trailing fractions from two preparations of TFIIIB purified to the 700 mM NaCl step elution
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: pol III, RNA polymerase III; UBR, upstream binding region. *To whom reprint requests should be addressed. 7308
Biochemistry: Kassavetis et al. from Cibacron blue-Sepharose (4, 5) were combined (11 ml), diluted to 150 mM NaCl and 20% (vol/vol) glycerol with buffer D [40 mM NaHepes, pH 7.8/7 mM MgCl2/10 mM 2-mercaptoethanol/0.01% Tween 20/0.5 mM phenylmethylsulfonyl fluoride/leupeptin (1 ,ug/ml)/pepstatin (1 ;Lg/ml)/ 20% glycerol], and loaded onto a Mono S (Pharmacia, HR 5/5) column. The column was washed with 5 ml of 150 mM NaCl in buffer D and a 20-ml 150-400 mM NaCl gradient in buffer D was applied, followed by 1 M NaCl in buffer D. Fractions were assayed for TFIIIC-dependent upstream footprinting activity on the nontranscribed strand of the SUP4 tRNA gene and for transcription factor activity with the impure TFIIIC/pol III DEAE-Sephadex fraction (see above). Both activities eluted at 275 mM NaCl. This material (1.5 ml) is referred to as the Mono S B' fraction in the text. Side fractions (1.5 ml) were also utilized after 10-fold concentration with a Centricon-30 unit (Amicon). This B' fraction was highly enriched in a 70-kDa protein, which represented -5% of the total silver-stainable material distributed among 10 polypeptides (data not shown). The activity that complements the B' fraction for specific transcription (termed B") was purified from the 250 mM NaCl step fraction off the first DEAE-Sephadex column of the TFIIIB purification scheme (4). The B" content in this fraction varied from trace to minor, relative to the TFIIIB activity in the 100 mM NaCl flow through ofthis column (data not shown). Further purification of the B" activity was conveniently achieved as a side product of our TFIIIC and pol III purification scheme where this material [from 1.7 kg (wet weight) of cells] was fractionated by gradient elution from multiple heparin-agarose columns as described (5). B" activity was measured by transcription with purified TFIIIC, pol III, and the Mono S B' fraction and by formation of heparin-resistant B'- and TFIIIC-dependent DNA complexes that migrate on native gels identically with TFIIIB-DNA complexes (data not shown; ref. 5). Both activities coeluted on these columns at or near 350 mM (NH4)2SO4, whereas TFIIIC and pol III eluted at 475 and 560 mM (NH4)2SO4, respectively. The heparin-agarose B" fractions were pooled (61 ml), 54 ml of this material was dialyzed in 450 mM KOAc in buffer D, applied to Mono S, washed with 7 ml of the same buffer, followed by a 20-ml 450-1000 mM KOAc gradient. Most of the protein (>90%o) flowed through at this high ionic strength, but the B" activity peak was spread throughout the first half of the gradient. This material was pooled and approximately half of it was diluted 1:2 with buffer D, reapplied onto Mono S, and eluted with 700 mM KOAc in buffer D. RESULTS We have recently used a photocrosslinking method to show that S. cerevisiae TFIIIB contains at least two distinct polypeptides of 90 and 70 kDa (10). Although our current preparation of TFIIIB has undergone five column chromatographic steps of purification, polypeptides of the above sizes represent minor components in our final (Cibacron blue) fraction, as judged by SDS/PAGE (data not shown; ref. 4). Upon further purification of the Cibacron blue fraction on Mono S (cation exchanger), TFIIIC-dependent-upstreamsequence DNA-binding activity eluted at 275 mM NaCI (data not shown, but see below). In a multiple-round transcription assay utilizing a DEAE-Sephadex fraction as a convenient, but impure source of TFIIIC and pol III (but lacking any detectable TFIIIB activity), the Mono S fraction exhibiting the DNA-binding activity retained the TFIIIB transcription activity of the input Cibacron blue fraction (Fig. LA, compare lanes g and h with lanes a and b). A single-round transcription assay likewise showed that the Mono S fraction was 74% as active as the input Cibacron blue fraction (compare lanes i
Proc. Natl. Acad. Sci. USA 88 (1991) A
a b c de f g h i
7309
j kI
LlI Blue B (90+70): 2 4 4 2 4 4 2 4 4 2 4 4 ..1 MonoS B' (70): - crude C.Pol 111: + + - + + pureC PolI11: - - - + + + - - - + Tx Single Multiple: M M S M M S M M S M M S -
+
+
+
B
+
DNA probes -38/-37 and -17/-12 a b c d e f
135 -a(C-
135 kDa (C)-_ M
do
90 kDa 70 kDa- _.f
0-
_
+.
_
++
Heparin TEIIIC B' (70 kDa.) DEAE 0.25 M step
_
+0
+ +
FIG. 1. Characterization of the B' fraction. (A) Transcription analysis with crude and purified TFIIIC and pol III. Single-round transcription (Tx) (lanes c, f, i, and l) or 15 min of multiple-round transcription (lanes a, b, d, e, g, h, j, and k) was performed after formation of the 17-mer ternary complex on the SUP4 tRNA gene (4). A crude TFIIIC/pol III fraction (2 ,ul) or 30 fmol of purified TFIIIC and 2 fmol of purified pol III were incubated with Cibacron blueSepharose-purified TFIIIB (3.7 fmol/,ll) or the Mono S-purified B' fraction as indicated. Densitometric units of specific transcription were as follows. Lanes: a, 695; b, 990; c, 171; d, 259; e, 459; f, 128; g, 731; h, 831; i, 126; j, 7; k, 11; 1, 4. -, Not added; +, added; M, multiple rounds of transcription; S, single round of transcription. (B) Photocrosslinking analysis. Purified TFIIIC (35 fmol), 5 ,ul of the Mono S B' fraction, and 2 .l of the crude TFIIIC/pol III DEAESephadex fraction were incubated with the photocrosslinking DNA probes -38/-37 (1 fmol) and -17/-12 (1 fmol) as indicated. Complexes treated with heparin (300 ,ug/ml) prior to UV irradiation are also indicated. The 135-kDa subunit of TFIIIC and the 90- and 70-kDa polypeptide components of TFIIIB are identified at the left of the figure.
and c). In contrast, the same Mono S fraction was transcriptionally deficient when the crude TFIIIC/pol III fraction was replaced with highly purified TFIIIC and pol III (lanes j-l): whereas the Cibacron blue fraction formed 75% as many active transcription complexes with purified TFIIIC and pol III as it did with impure TFIIIC and pol III (compare lanes f and c), the comparable proportion for the Mono S fraction was
