| Nucleic Acids Research | Pages |
The activity binding to the termination region of several pol III genes represents a separate entity and is distinct from a novel component enhancing U6 snRNA transcription
Introduction
Materials And Methods
Plasmids
Preparation of nuclear extract and purification of transcription factors
In vitro transcription
DNAse I footprint analysis
Results
Purification of the termination site binding activity TBA from TFIIIC0 fractions
The TFIIIC0 fraction additionally contains a TFIIIC1 related activity (TFIIIC1-like)
TBA stimulates VAI transcription only in a system containing TFIIIC1
Identification of an activity necessary for U6 snRNA transcription
TFIIIU is distinct from TBA
Discussion
The termination site binding activity (TBA) is a distinct entity and can be completely separated from TFIIIC1 and related activities
TBA stimulates VAI transcription only in the presence of TFIIIC1 but not TFIIIC1-like
A novel activity, TFIIIU, stimulates U6 snRNA transcription
Acknowledgements
References
The activity binding to the termination region of several pol III genes represents a separate entity and is distinct from a novel component enhancing U6 snRNA transcription
ABSTRACT
INTRODUCTION
Genes transcribed by RNA polymerase III (pol III) can be classified by the promoter structures which control their expression (1). While type 1 genes (5S rRNA) contain internally located A- and C-boxes as well as an intermediate element, type 2 genes (e.g. tRNA and VAI) are controlled by A- and B-boxes within the gene. Expression of type 2 pol III genes requires transcription factors TFIIIC and TFIIIB, whereas synthesis of 5S rRNA additionally requires the primarily DNA-binding protein TFIIIA. Type 3 pol III promoters in higher eukaryotic cells (e.g. U6, 7SK) are governed exclusively by promoter sequences located upstream of the initiation site which include a TATA-box, as well as a proximal sequence element (PSE) and a distal sequence element (DSE). Transcription of these latter genes minimally requires the TATA-binding protein TBP, the PSE-binding protein PBP (2) also designated as PTF (3-5) or SNAPc (6-8), TFIIIB and pol III.
Accurate initiation of transcription of pol III genes is best understood in the yeast system and in the case of the tRNA genes this process depends on the sequence specific interaction of TFIIIC with the B-box promoter element and the subsequent incorporation of the initiation factor TFIIIB and the polymerase itself by protein-protein interactions (1,9-11).
In mammalian cells the situation is more complex. First, it has been shown that human transcription factor IIIB can be separated into functionally different forms TFIIIB-[alpha] and TFIIIB-[beta]. The latter is a TBP/TAF complex required for expression of genes with intragenic promoters. In contrast, TFIIIB-[alpha] is devoid of TBP and is responsible for the in vitro transcription of U6 snRNA (12). Second, TFIIIC2 primarily associates with the B-box but this by itself does not suffice to support efficient transcription in the presence of TFIIIB and the polymerase, implying that additional factors, which participate in transcription complex formation, are required (13,14). The mechanisms of complex formation are hitherto poorly understood in higher eukaryotes and until recently only one of these auxilliary components has been described. This activity was purified from crude fractions of TFIIIC (PC 0.6) and was hence designated as TFIIIC1 (13,15). TFIIIC1 alone does not bind to DNA, but extended the DNAse I footprint over the A-box when added to TFIIIC2 primarily bound to the B-box (13). In contrast to TFIIIC2, which is only required for transcription of type 1 and 2 pol III genes (13,16-18), TFIIIC1 is essentially required for transcription of all pol III genes (3,19). TFIIIC1 positively influences the DNA-binding activity of all three pol III assembly factors: TFIIIC2, TFIIIA and the PSE-binding protein PBP (18,19).
Although TFIIIC1 was consistently reported to be required for transcription, its binding properties to intragenic pol III promoter genes were discussed controversely. TFIIIC1, described by Wang and Roeder (18), was reported to show a strong interaction with the termination site of type 2 genes. In contrast, no termination site binding was found for this factor from the laboratory of Berk (13) and from our laboratory (19). Moreover, we have shown recently that TFIIIC1 is chromatographically and functionally distinct from an activity which binds specifically and strongly to the termination region of pol III genes with internal promoters. This DNA-binding activity was initially described to be contained in fractions designated as TFIIIC0 and this fraction could also partially substitute for TFIIIC1 activity (19). Until now, however, it was unclear whether the termination site binding activity (TBA) and the TFIIIC1 related activity (TFIIIC1-like) of these TFIIIC0 fractions were mediated by the same or different components. We show here that TBA can be separated from TFIIIC1 and TFIIIC1-like, demonstrating that the specific DNA-binding of TBA is independent of TFIIIC1 or related activities.
TFIIIC1 functions as a basal pol III factor and thus is also essentially required for transcription of the 5[prime] regulated 7SK RNA and U6 snRNA genes (3,19). However, Yoon et al. (3) reported that TFIIIC1 could only complement a heat-treated extract from human cells lacking TFIIIC activity but not a reconstituted system (consisting of PTF, Oct-1, TBP and TFIIIB) for 7SK transcription. From these findings the authors implied that additional factors, contained in the heat-treated extract, might be necessary. Using an in vitro transcription system consisting of PBP, TBP, TFIIIB-[alpha], RNA pol III (2,12) and TFIIIC1 we also find that the mouse U6 gene is poorly transcribed under such conditions. Employing this reconstituted system we identified a novel activity, TFIIIU, which is essentially required for transcription of the mammalian U6 snRNA gene. Although TFIIIU co-elutes with the termination site binding activity TBA upon MQ chromatography, we show in this report that these activities can be separated by further purification and that TFIIIU has no effect on VAI transcription.
MATERIALS AND METHODS
Plasmids
The plasmids pUmU60.34, pUVAI, pUhtimet and pUh5S were as previously described (2,20,21) and contained single copies of the genes coding for mouse U6 snRNA, VAI RNA, human methionine initiator tRNA or synthetic human 5S rRNA.
Preparation of nuclear extract and purification of transcription factors
Buffer A contained 20 mM HEPES, pH 7.9, 10% (v/v) glycerol, 3 mM DTT, 0.2 mM PMSF. Buffer B contained 20 mM Tris-HCl, pH 7.9, 10% (v/v) glycerol, 5 mM MgCl2, 3 mM DTT, 0.2 mM PMSF.
Nuclear extracts from human embryonic kidney (HEK) or HeLa cells were prepared from several batches of 35 l suspension cultures with an index of 7.4 × 105 cells/ml as described previously (22). The nuclear extract was dialyzed against buffer A including 100 mM KCl and was subsequently chromatographed through phosphocellulose (PC) as described previously (23).
The PCC fraction (600 mM KCl) was used to purify human TFIIIC0, TFIIIC1, TFIIIC2 and PBP as described before (19) with the following modifications.
PBP. The PCC fraction was dialyzed against buffer B containing 60 mM KCl, loaded onto a Mono Q column (`MQ', Pharmacia) at 10 mg protein/ml bed volume. After washing the column with the same buffer, bound proteins were eluted with a linear gradient from 60 to 450 mM KCl. Fractions eluting at 300-330 mM KCl contained PBP (0.2 mg protein/ml).hTFIIIC1. Fractions eluting from 250 to 290 mM KCl in buffer B from Mono Q (MQ0.27; 19) were diluted to 200 mM KCl in buffer B and applied to an EMD S03- Fractogel column (`ESF'; Merck, Darmstadt) at 5 mg protein/ml bed volume. After washing the column with 200 mM KCl in buffer B, bound proteins were eluted with a linear gradient from 200 to 600 mM KCl and a 1 M KCl step. hTFIIIC1 activity eluted with 450-500 mM KCl (ESF0.47; 0.2 mg protein/ml).hTFIIIC0. Fractions eluting from 150 to 190 mM KCl in buffer B from the MQ gradient (MQ0.17; 19) were applied to an EMD S03- Fractogel column (Merck) at 5 mg protein/ml bed volume. After washing the column with 200 mM KCl in buffer B, bound proteins were eluted with a linear salt gradient from 200 to 600 mM KCl and a 1 M KCl step. Termination region binding activity (TBA) eluted from 390 to 480 mM KCl (0.12 mg protein/ml), TFIIIU eluted from 365 to 415 mM KCl (0.09 mg protein/ml), TFIIIC1-like eluted from 500 to 545 mM KCl (0.04 mg protein/ml). Alternatively, MQ TFIIIC0 fractions (150-190 mM KCl) were dialyzed against buffer B with 60 mM KCl, applied to a Resource S column (`RS', Pharmacia), washed with buffer B containing 170 mM KCl and eluted with a linear gradient from 170 to 600 mM KCl. TBA eluted with 220-265 mM KCl (0.05 mg protein/ml), TFIIIC1-like eluted with 320 mM KCl (0.04 mg protein/ml) from this column. The flowthrough, containing TFIIIU, was directly applied onto an EMD S03- Fractogel column (Merck) and eluted with a linear gradient from 200 to 600 mM KCl in buffer B. Fractions eluting from 345 to 410 mM KCl contained TFIIIU.The PCB fraction (350 mM KCl) was used to purify human TFIIIB-[alpha], TFIIIB-[beta] and RNA polymerase III (pol III) as described before (12,19). Recombinant human TBP (rhTBP) was expressed in Escherichia coli and purified as described (24). Recombinant human TFIIIA (rhTFIIIA) was expressed in E.coli and purified as described (19,25).
In vitro transcription
The assays were as described previously (19,23). For type 1 and 2 pol III genes, 0.2 or 1 µg of plasmid DNA was used in 50 µl reactions. For U6 transcription, 0.5 or 2 µg of plasmid DNA was used in 70 µl reactions. All reactions were conducted in the presence of 10 U Ribonuclease Inhibitor (Stratagene, Heidelberg). In vitro synthesized RNA products were electrophoretically separated on 6% (VAI RNA) or 7.5% denaturing urea sequencing gels and autoradiographed for at least 1 day at -80°C with intensifying screens.
DNAse I footprint analysis
Fragments were generated by PCR amplification of the VAI gene from pUVAI with 5[prime] end-labelled 17mer sequencing primer, unlabelled 17mer reverse sequencing primer (M13/pUC; Boehringer, Mannheim) and Goldstar DNA Polymerase (Eurogentec). Individual protein fractions were pre-incubated with 2 µg poly (dIdC) and 0.5 µg pUC 9 DNA as non-specific competitor in a total volume of 50 µl for 15 min at 30°C. Subsequently 15 000 c.p.m. of the labelled fragment were added and incubation was prolonged for 90 min at 30°C. Samples were cleaved with 40 ng of DNAse I for 1 min at room temperature, reactions were stopped with 50 µl of stop solution and further processed as described before (19,21). An end-labelled MspI digest of pBR322 was used as a size marker to assign the protected regions.
RESULTS
Purification of the termination site binding activity TBA from TFIIIC0 fractions
Starting from phosphocellulose fraction C (PCC), derived from nuclear extracts of human cells, we could recently enrich an activity by MQ FPLC chromatography which showed a strong interaction with the termination region of type 1 and 2 pol III genes (19). This activity eluted at 170 mM KCl from the MQ column and such MQ fractions were denoted as TFIIIC0 (Materials and Methods) to distinguish them from TFIIIC1 and TFIIIC2. TFIIIC1 activity eluting at 270 mM KCl from MQ columns did not bind to DNA and never showed DNase I protection on pol III genes with internal promoters (19).
Since the MQ TFIIIC0 fraction is comparatively crude, it was further purified in this report by FPLC chromatography on ESF columns. Bound proteins were eluted with a linear gradient from 200 to 600 mM KCl, and the DNA-binding activity of individual fractions was analyzed by footprint experiments. As shown in Figure
Figure 1. Purification of the MQ TFIIIC0 fraction by ESF chromatography. (A) DNAse I protection of the VAI termination region by TBA. A 321 bp fragment of the VAI gene, labelled at the 5[prime] end of the coding strand, was incubated with 2 µg of TFIIIC0 (MQ0.17) fraction (lane 1) or 40 µl of the individual fractions of the gradient (lanes 3-15) for footprint analysis as described in Materials and Methods. Lane 2 served as a control reaction without protein. The protected region of the VAI gene, spanning sequences from +154 to +182, is appropriately indicated. (B) TBA does not support transcription in a TFIIIC1 deficient system. Fractions of the ESF-gradient were assayed for their ability to complement VAI, htimet or h5S rRNA transcription. In vitro transcription was performed separately for the three genes as described in Materials and Methods, and the positions of the individual transcripts are indicated. One microgram of each template was incubated with 50 ng of pol III (ESF0.55) and 4 µg of TFIIIB-[beta] (ESF0.31, lanes 1-15). For lanes 2-15, 0.3 µg of TFIIIC2 (MQ0.5) and for lanes 3-15, 20 µl of the individual fractions of the ESF gradient were added as indicated. For in vitro transcription of the h5S gene all samples were supplemented with 1 µl rhTFIIIA.
The TFIIIC0 fraction additionally contains a TFIIIC1 related activity (TFIIIC1-like)
MQ TFIIIC0 fractions containing the DNA-binding activity TBA were initially described to be able to partially substitute for TFIIIC1 activity in transcription reactions (19). Therefore, individual fractions of the ESF gradient (Fig.
Figure 2. (A) TBA stimulates VAI transcription in a system containing TFIIIC1. ESF-gradient fractions were analyzed in a TFIIIC1 containing reconstitution system. The in vitro transcription of 0.2 µg of pUVAI was conducted in the presence of 10 ng of pol III (ESF0.55), 0.8 µg of TFIIIB-[beta] (ESF0.31), 60 ng of TFIIIC2 (MQ0.5) and 200 ng of TFIIIC1 (ESF0.47). For lane 1, TFIIIC1 was omitted. Five microlitres of individual ESF gradient fractions 2-13 were added as indicated (lanes 3-14) and in vitro transcription was performed as described in Materials and Methods. The position of the VAI transcript is indicated. (B) Transcriptional activity of TBA depends on the presence of TFIIIC1. In vitro transcription of 0.2 µg of pUVAI was performed in the presence of highly purified pol III, TFIIIB-[beta] and TFIIIC2 (lanes 1-16) as described for (A). For lanes 3-7, 0.2 µg of TFIIIC1 (ESF0.47) were analyzed in the presence of increasing amounts of TBA (0.13, 0.25, 0.5 and 1 µg of fraction ESF-Grad. 430 mM KCl, lanes 4-7). For lanes 2 and 8, 1 µg of TBA (ESF-Grad. 430 mM KCl) were added alone. For lanes 9-16, increasing amounts of TFIIIC1 (0.1, 0.25, 0.5 and 1 µg of fraction ESF0.47) were analyzed in the presence of 1 µg of TBA (ESF-Grad. 430 mM KCl, lanes 9-12) or were added alone to the basal transcription system (lanes 13-16). (C) TFIIIC1-like cannot fully substitute for TFIIIC1 activity. In vitro transcription of 0.2 µg of pUVAI was performed in the presence of highly purified pol III, TFIIIB-[beta] and TFIIIC2 (lanes 1-16) as described for (A). Increasing amounts of TBA [0.25 µg (lanes 8 and 12), 0.5 µg (lanes 9 and 13) and 1 µg (lanes 2, 10 and 14) of fraction ESF-Grad. 430 mM KCl] were analyzed in the presence of 0.24 µg (lanes 7-10) or 0.48 µg (lanes 11-14) of TFIIIC1-like (ESF-Grad. 520 mM KCl) or 0.5 µg of TFIIIC1 (ESF0.47, lane 16). For lanes 3-6, 0.06, 0.12, 0.24 and 0.48 µg of TFIIIC1-like (ESF-Grad. 520 mM KCl), for lane 15, 0.5 µg of TFIIIC1 (ESF0.47) were added alone to the basal transcription system.
TBA stimulates VAI transcription only in a system containing TFIIIC1
Since TBA which binds with high affinity to the termination region of the VAI gene fails to restore transcription of this gene in the absence of TFIIIC1 (Fig.
The effect on transcription mediated by TBA strictly depends on and is related to the quantity of TFIIIC1 present. Results from titration experiments depicted in Figure
As demonstrated in lanes 3-6 of Figure
Identification of an activity necessary for U6 snRNA transcription
TFIIIC1 is essentially required for transcription of all pol III genes including the U6 gene. However, expression of the mouse U6 snRNA gene cannot be fully restored by TFIIIC1 in the presence of PBP, pol III, TFIIIB-[alpha] and rTBP as will subsequently be dealt with in more detail (Fig.
Figure 3. A novel component, TFIIIU, is involved in in vitro transcription of the mU6 snRNA gene. (A) Influence of ESF gradient fractions on reconstituted U6 transcription in vitro. Ten microlitres of the individual ESF-gradient fractions 2-15 (lanes 2-15) were analyzed for their ability to reconstitute U6 transcription in the presence of 15 ng of pol III (ESF0.55), 2 µg of TFIIIB-[alpha] (ESF0.5), 0.5 µg of PBP (MQ0.32), 0.12 µg of TFIIIC1 (ESF0.47) and 20 ng of rhTBP (lanes 1-15). In vitro transcription was carried out as described in Materials and Methods using 0.5 µg pUmU60.34 as template DNA. The position of the U6 transcript is indicated. (B) TFIIIU strongly enhances U6 transcription. In vitro transcription of 0.5 µg of pUmU60.34 was performed in a basal system containing highly purified pol III, TFIIIB-[alpha], PBP and rhTBP (lanes 2-13) as described for (A). For lane 1, PBP was omitted. For reconstitution of U6 transcription, TFIIIU activity (0.9 µg of ESF-Grad. 370 mM KCl, lane 9) was analyzed in the presence of increasing amounts of TFIIIC1 (0.2, 0.4 and 0.8 µg of fraction ESF0.47, lanes 6-8) or of TFIIIC1-like (0.06, 0.12 and 0.24 µg of fraction ESF-Grad. 520 mM KCl, lanes 10-12). For lanes 3-5, 0.2, 0.4 and 0.8 µg of TFIIIC1 (ESF0.47), for lane 13, 0.24 µg of TFIIIC1-like (ESF-Grad. 520 mM KCl) were added alone to the basal reconstitution system. (C) TFIIIU is required for efficient U6 transcription. To verify whether TFIIIU is distinct from the hitherto established and required human U6 transcription factors, each component of a fully reconstituted U6 transcription system was individually omitted as indicated above each lane. The reactions were incubated and processed as described in Materials and Methods using 2 µg of pUmU60.34, 60 ng of pol III (ESF0.55, lanes 1-4 and 6-10); 8 µg of TFIIIB-[alpha] (ESF0.5, lanes 1-5 and 7-10); 2 µg of PBP (MQ0.32; lanes 2-6, 8 and 10); 0.4 µg of TFIIIC1 (ESF0.47, lanes 1-6 and 9); 0.9 µg (lane 3) and 1.8 µg (lanes 4-7 and 9-10) of TFIIIU (ESF-Grad. 370 mM KCl). All reactions were supplemented with 80 ng of rhTBP. We found previously that transcription of the mU6 snRNA gene was more efficiently reconstituted by the MQ TFIIIC0 fraction than by the MQ TFIIIC1 fraction (19). In order to check whether the U6 gene requires an additional activity besides TFIIIC1, which could possibly be contained in the TFIIIC0 fraction of the MQ gradient, individual fractions of the ESF gradient were analyzed with respect to their ability to reconstitute transcription of the mU6 gene in vitro in the presence of TFIIIC1, PBP, pol III, TFIIIB-[alpha] and rTBP. Under such conditions U6 snRNA synthesis was strongly stimulated by an activity, eluting from 365 to 415 mM KCl (fractions 5-7) from this gradient (Fig. In order to further analyze whether TFIIIU is essentially required for transcription of the mU6 snRNA gene, appropriate controls were conducted in Figure Additional controls were conducted in Figure 
TFIIIU is distinct from TBA
Although TFIIIU eluted at 365-415 mM KCl from the ESF column (Fig.
  |
Figure 4. TFIIIU is distinct from TBA. (A) Footprint analysis of fractions obtained by RS chromatography. A 321 bp fragment of the VAI gene, labelled at the 5[prime] end of the coding strand, was incubated with either TFIIIC0 (2 µg of the input MQ0.17 fraction, lane 1), 25 or 50 µl of the 170 mM KCl flowthrough of the RS column (lanes 2 and 3) or with 40 µl of the RS eluate fractions 1-5 (lanes 5-9) for footprint analysis as described in Materials and Methods. Lane 4 served as a control reaction without protein. The protected region is appropriately indicated relative to the VAI gene. (B) The flowthrough of the RS column contains TFIIIU activity. Individual fractions of the RS gradient were tested in U6 snRNA transcription in vitro for TFIIIU activity. All reactions contained 25 ng of pol III (ESF0.55), 4 µg of TFIIIB-[alpha] (ESF0.5), 1 µg of PBP (MQ0.32) and 40 ng rhTBP. Reactions shown in lanes 2-9 additionally contained 0.4 µg of TFIIIC1 (ESF0.47). 10 and 20 µl of the flowthrough of the RS column (lanes 3 and 4) or 15 µl of the fractions 1-5 (lanes 5-9) eluted from the RS column were added and reactions were performed as described in Materials and Methods using 1 µg pUmU60.34. The position of the U6 transcript is indicated. (C) TFIIIU activity does not complement VAI transcription. To verify the specifity of TFIIIU for U6 transcription, the flowthrough of the RS column was analyzed for its ability to reconstitute or stimulate VAI transcription in vitro in the absence or presence of TFIIIC1. In vitro transcription of 0.2 µg of pUVAI was performed in the presence of highly purified pol III, TFIIIB-[beta], TFIIIC2 (lanes 1-11) as described for (A). Reactions shown in lanes 5-11 additionally contained 0.2 µg of TFIIIC1 (ESF0.47). Further components were added as indicated above each lane: 0.2 µg (lane 6), 0.4 µg (lanes 2 and 7) and 0.8 µg (lanes 3 and 8) of TFIIIU (flowthrough of the RS column); 0.25 µg (lane 9), 0.5 µg (lane 10) and 1 µg (lanes 4 and 11) of TBA (ESF-Grad. 430 mM KCl).
It should be emphasized that TFIIIU had no effect on VAI transcription, neither in a TFIIIC1 deficient (Fig.
DISCUSSION
In yeast cells the same six polypeptides of the multiprotein complex TFIIIC are sufficient for DNA-binding and transcription (31-35). The mammalian counterpart of this factor, however, appears to be more complex. Human TFIIIC activity can be separated by MQ chromatography into three functionally distinct activities: TFIIIC0, TFIIIC1 and TFIIIC2 (13,18,19). MQ TFIIIC0 fractions show a strong interaction with the termination region of several pol III genes and can also partially substitute for TFIIIC1 activity (19). In the course of further purification studies of such TFIIIC0 fractions presented here, we identified three chromatographically and functionally distinct activities: the termination region binding activity TBA, a transcriptional activity which resembles TFIIIC1 (TFIIIC1-like) and an activity stimulating U6 transcription (TFIIIU).
The termination site binding activity (TBA) is a distinct entity and can be completely separated from TFIIIC1 and related activities
TFIIIC1 can already be completely separated from TBA by MQ chromatography (19) and such fractions of TFIIIC1 are transcriptionally fully active (Fig.
TBA co-elutes with a transcriptional activity upon MQ chromatography which can partially substitute for TFIIIC1 and which we hence designated as TFIIIC1-like. However, further purification by ESF (Fig.
In conclusion, our studies showed that TBA, the activity which binds to the termination region, is distinct from TFIIIC1 and TFIIIC1-like. Neither TFIIIC1 nor TFIIIC1-like bind primarily to DNA and both reconstitute pol III transcription. Consequently, TBA does not represent an integral part of these transcriptional activities.
In agreement with our results, no termination binding activity was described for TFIIIC1 or its related activity by Yoshinaga et al. (13). Hence we suppose that TFIIIC1-like reported here is related to the TFIIIC1 related activity described by Yoshinaga et al. (13), because both activities stem from phosphocellulose fraction C, reveal a similar elution profile from MQ chromatography and complement VAI transcription to a much lesser extent than TFIIIC1.
In contrast, TFIIIC1 and a functionally fully equivalent isoform TFIIIC1[prime] were described by Wang and Roeder (18), both of which correlated with strong binding activity to the termination region on VAI and tRNA genes and it is hence conceivable that these fractions contained TBA in addition to TFIIIC1.
TBA stimulates VAI transcription only in the presence of TFIIIC1 but not TFIIIC1-like
TBA by itself cannot complement a TFIIIC1 deficient transcription system (Fig.
Since TFIIIC1 alone is able to fully restore transcription of type 1 and 2 pol III genes in the presence of TFIIIC2, TFIIIB-[beta] and pol III (Fig.
However, template commitment experiments which we performed have not yet elucidated whether the binding of TBA to the termination region of the VAI gene is strong enough to strictly commit enhanced transcription to the first template (data not shown). Since these experiments revealed that DNA-binding of TBA per se is not sufficient, it is possible that protein-protein contacts between TBA and (an)other component(s) of the transcription complex are required and that such interactions must precede the event of DNA-binding to establish an activated transcription complex. Alternatively, it is possible that the dissociation rate of the binding of TBA is too high for template commitment.
Chu et al. (36) recently reported, for the human system, that palindromic sequences immediately preceding the termination region increased pol III transcription activity. Possibly these palindromic sequences are involved in stimulation of VAI transcription which we observed upon addition of the termination region binding factor TBA. Additionally, Dieci and Sentenac (37) found that the termination element of yeast pol III genes appears to be required to enable polymerase III to enter the facilitated reinitiation pathway. It is possible that the enhanced transcription level supported by TBA is related to these findings. Likewise, a specific binding activity for U-terminated nascent RNA was described for the human autoimmune antigen La (38) which was also discussed to clear the template for subsequent rounds of transcription (39). However, western blot analysis revealed that La did not co-chromatograph with TBA activity (data not shown).
A novel activity, TFIIIU, stimulates U6 snRNA transcription
We have described recently that transcription of the U6 snRNA gene is more effectively reconstituted by MQ TFIIIC0 fractions than by MQ fractions containing TFIIIC1 (19). Using an in vitro U6 transcription system comprising purified PBP, TFIIIB-[alpha], pol III and rTBP (2,12), addition of highly purified TFIIIC1 only resulted in a low level of U6 snRNA synthesis (Fig.
By analyzing individual fractions of the ESF gradient derived from MQ TFIIIC0 fractions for their ability to further reconstitute U6 transcription in the presence of TFIIIC1, we identified an activity eluting with 365-415 mM KCl which strongly stimulated U6 transcription (Fig.
The mechanism by which TFIIIU acts is as yet unknown, but we demonstrate that this activity is probably not related to any of the other established proteins required for formation of the U6 transcription complex (Fig.
We recently found (27) that human TFIIIB-[alpha] is composed of at least two activities which could be functionally analogous to yeast TFIIIB[prime][prime]90 and yeast TFIIIE (41,42). It is, however, very unlikely that yTFIIIE and hTFIIIU are functionally related, because the fractions of TFIIIB-[alpha] which we employed for our experiments were not depleted from the TFIIIE analogous activity described by Teichmann et al. (27). It is furthermore unlikely that TFIIIU is related to the Xenopus zinc finger protein Staf, a transcription activator of snRNA and snRNA-type genes of various species by RNA polymerases II and III (43,44). The potential Staf binding sites are located in the DSEs of these genes. Moreover, these authors reported a synergistic activating effect of octamer and staf motifs on the transcription by pol III. U6 in vitro transcription reported here was performed with sequences of the mouse U6 gene from -150 to +190, thus containing the PSE and TATA promoter sequences but lacking the DSE element (2). Therefore, TFIIIU acts in a way which is independent of a potential Staf binding sequence.
Importantly, TFIIIU is also distinct from TBA. Although the elution profiles of these components overlapped when ESF chromatography of MQ TFIIIC0 fractions was employed (compare Figs
ACKNOWLEDGEMENTS
We thank K. G. Becker (National Institutes of Health, Bethesda, MD) for kindly providing the clone C2H2-34.10 encoding human TFIIIA. We thank M. Bachmann (Mainz) for the kind donation of anti-human La monoclonal antibodies. We also thank Ulla Kopiniak and Frauke Seifart for expert technical assistance. This work was supported by grants of the Deutsche Forschungsgemeinschaft and the European Union.
REFERENCES
This article has been cited by other articles:
This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 19 Sep 1998
Copyright©Oxford University Press, 1998.
CiteULike 
Connotea 
Del.icio.us What's this?
S. Weser, C. Gruber, H. M. Hafner, M. Teichmann, R. G. Roeder, K. H. Seifart, and W. Meissner
Transcription Factor (TF)-like Nuclear Regulator, the 250-kDa Form of Homo sapiens TFIIIB'', Is an Essential Component of Human TFIIIC1 Activity
J. Biol. Chem.,
June 25, 2004;
279(26):
27022 - 27029.
[Abstract]
[Full Text]
[PDF]
L. Schramm and N. Hernandez
Recruitment of RNA polymerase III to its target promoters
Genes & Dev.,
October 15, 2002;
16(20):
2593 - 2620.
[Full Text]
[PDF]
W. Meissner, R. Thomae, and K. H. Seifart
The Activity of Transcription Factor IIIC1 Is Impaired during Differentiation of F9 Cells
J. Biol. Chem.,
February 22, 2002;
277(9):
7148 - 7156.
[Abstract]
[Full Text]
[PDF]
W. Meissner, H. Rothfels, B. Schafer, and K. Seifart
Development of an inducible pol III transcription system essentially requiring a mutated form of the TATA-binding protein
Nucleic Acids Res.,
April 15, 2001;
29(8):
1672 - 1682.
[Abstract]
[Full Text]
[PDF]
S. S. Chong, P. Hu, and N. Hernandez
Reconstitution of Transcription from the Human U6 Small Nuclear RNA Promoter with Eight Recombinant Polypeptides and a Partially Purified RNA Polymerase III Complex
J. Biol. Chem.,
June 1, 2001;
276(23):
20727 - 20734.
[Abstract]
[Full Text]
[PDF]
This Article 
Abstract
Print PDF (242K)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (9)
Request Permissions
Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Articles by Oettel, S.
Articles by Seifart, K. H.
Search for Related Content
PubMed
PubMed Citation
Articles by Oettel, S.
Articles by Seifart, K. H.
Social Bookmarking
What's this?