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A Single-Stranded Promoter for RNA Polymerase III

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A Single-Stranded Promoter for RNA Polymerase III A single-stranded promoter for RNA polymerase III Oliver Schro¨ der*, E. Peter Geiduschek, and George A. Kassavetis Division of Biological Sciences and Center for Molecular Genetics, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0634 Contributed by E. Peter Geiduschek, December 3, 2002 Single strands of DNA serve, in rare instances, as promoters for the purified single-stranded components and purified by native transcription; duplex DNA promoters with individual strands that gel electrophoresis and, when necessary, electrophoresis through also have a promoter capacity͞function have not been described. gel-immobilized oligonucleotides to trap and separate partially We show that the nontranscribed strand of the Saccharomyces annealed and͞or unannealed contaminants (8). cerevisiae U6 snRNA gene directs transcription initiation factor IIIB-requiring and accurately initiating transcription by RNA poly- Proteins. Purification and quantification of wild-type TFIIIB merase III. The nontranscribed strand promoter is much more subunits and TBPm3 have been described (6, 9). TBP and Bdp1 extended than its duplex DNA counterpart, comprising the U6 gene were estimated to be nearly 100%, and Brf1 was 20% active TATA box, a downstream T7 tract, and an upstream-lying segment. in formation of heparin-resistant promoter complexes. -A requirement for placement of the 3؅ end of the transcribed Bdp1(⌬355–372) was purified under native conditions as de (template) strand within the confines of the transcription bubble is scribed for Bdp1(138–594) (11). N⌬68Brf1 was purified as seen as indicating that the nontranscribed strand provides a described (10). TFIIIC and pol III were purified as described; scaffold for RNA polymerase recruitment but is deficient at a quantities of pol III are specified as fmol of enzyme active for subsequent step of transcription initiation factor IIIB’s direct in- specific transcription (12, 13). volvement in promoter opening. Transcription and 5؅ End Mapping. TFIIIB–DNA complexes were formed for 40 min at 20°C in 20 ␮l of reaction buffer containing udding yeast (Saccharomyces cerevisiae) RNA polymerase ⅐ ␮ ͞ 40 mM Tris HCl (pH 8.0), 7 mM MgCl2,3mMDTT,100 g ml BIII (pol III) is brought to its promoters by its transcription ␮ ͞ ͞ factor (TF)-IIIB, which is composed of three subunits, TATA- BSA, 5 g ml poly(dG-dC), 5–7% (vol vol) glycerol, 80–90 mM binding protein (TBP), Brf1, and Bdp1. S. cerevisiae TFIIIB, in NaCl with 20 fmol DNA template, 52 fmol TFIIIC (as indicated), 200 fmol TBP (or TBPm3), 100 fmol Brf1 (or Brf1N⌬68), and turn, is brought to its binding site upstream of the transcriptional ⌬ start by TFIIIC, but also can be directed to the promoter by TBP 150 fmol Bdp1 (or Bdp1 355–372). Two microliters (5 fmol) of pol III were added for an additional 20 min. Multiple rounds of subunit-directed binding to a TATA box (1–3). Only a few ␮ budding yeast pol III genes, among them the U6 snRNA gene, transcription at 20°C were started by adding 5 l of reaction have sufficiently strong TATA boxes to allow TFIIIC- buffer containing 1 mM ATP, 1 mM CTP, 1 mM GTP, and 125 ␮M[␣-32P]UTP and stopped after 30 min by adding 155 ␮lof independent and solely TFIIIB-directed transcription of DNA ⅐ ͞ ͞ (4). In contrast, fission yeast (Saccharomyces pombe) genes stop solution (10 mM Tris HCl 3 mM EDTA 0.2% SDS). transcribed by pol III have TATA boxes as absolutely required Samples were precipitated, processed for denaturing gel elec- promoter elements (4, 5). trophoresis, and quantified by phosphorimage plate analysis. Our recent analysis of the functions of TFIIIB in initiation of Primer extension (reverse transcription) analysis of unlabeled transcription has exploited certain deletion mutants of Brf1 and transcripts to determine transcription start sites was carried out as described (10). Bdp1 that generate distinctive defects of promoter opening in linear DNA. The search for conditions under which activity Results would be restored to these defective TFIIIB assemblies has The analysis that is reported here has its origin in an observation involved the exploitation of DNA templates with partially made during the course of experiments on the effects of DNA opened promoters or with breaks in either DNA strand. Explor- strand breaks at defined locations on transcription by RNA pol ing the architecture of the pol III promoter complex by manip- III (7, 8). The duplex DNA for this analysis was assembled from ulating its DNA scaffold in this manner has provided insights three purified oligonucleotides, and partial assemblies contain- into the mechanism of promoter opening, including its general ing single-stranded tails were removed carefully. Control DNA upstream 3 downstream polarity, and into the selective role of preparations with long 5Ј and 3Ј overhanging tails (assembled the transcribed (template) strand in specifying͞measuring the with only two oligonucleotides) were made to assess inhibition of distance from the TATA box to the transcriptional start site transcription by attached single-stranded DNA. Finding that one (6–8). of these partially duplex DNAs, presenting only the nontran- That analysis also has led (by a path that is specified below) to scribed strand of its essential TATA box, could serve as a the surprising finding that is elaborated in this work: the template for factor-dependent and specifically initiating tran- nontranscribed strand of the U6 promoter suffices for TFIIIB- scription prompted the further exploration that is recounted dependent and accurately initiating transcription by pol III. We below. show that the single-stranded U6 promoter includes a TATA element, with which TFIIIB interacts in a distinctive way, and TFIIIB-Dependent Transcription with Partially Single-Stranded Pro- also involves additional promoter elements that are required to moters. We constructed partially single-stranded templates de- be single-stranded but are not part of the duplex DNA promoter. rived from the yeast U6 snRNA (SNR6) gene, presenting only Ј Methods the 5 overhanging nontranscribed (Fig. 1A Upper) strand of the upstream promoter region. The 3Ј end of the transcribed DNA Templates. Transcription templates for this work are based 3 (Fig. 1A Lower) strand was placed at different positions relative on plasmid pU6RboxB (9), with a single G A substitution at base pair Ϫ22 reversing the original A 3 G mutation. Fully duplex DNA (base pairs Ϫ60 to ϩ138 relative to the transcrip- Abbreviations: TFIIIB, transcription factor IIIB; TBP, TATA-binding protein; pol III, RNA tional start site) was generated as described (10). The 5Ј over- polymerase III; o͞h, 5Ј overhang. hang templates and gap templates were generated by annealing *To whom correspondence should be addressed. E-mail: [email protected]. 934–939 ͉ PNAS ͉ February 4, 2003 ͉ vol. 100 ͉ no. 3 www.pnas.org͞cgi͞doi͞10.1073͞pnas.242735699 Downloaded by guest on September 28, 2021 Fig. 1. (A) The single-stranded SNR6 promoter. The 198-nt nontranscribed (Upper) strand comprises the U6 snRNA gene sequence from Ϫ60 to ϩ138 relative to the normal transcription start site (bold͞underlined; designated as ϩ1). The TATA box and the terminator sequence are in bold letters; the T7-stretch is boxed; 3Ј ends of the transcribed strand are indicated by arrows. The extent of the transcription bubble is noted at the bottom. (B) Transcription activity of the single-stranded promoter. Pol III transcription was carried out in the presence of TFIIIB on duplex DNA or 5Ј overhang templates, with transcribed-strand 3Ј ends located as indicated. Because of the potential inhibitory effect of single-stranded DNA, templates were titrated, as indicated below each lane. (C) Transcriptional activity at 20 fmol 5Ј overhang DNA relative to duplex DNA. Error bars are SEM of at least five independent experiments. The distribution of transcriptional start sites, as determined by primer extension, is specified for each construct: open boxes, Ϫ2; black, ϩ1; gray, ϩ5. Relative activities of promoters with transcribed strand 3Ј ends at base pairs Ϫ18, Ϫ14, and Ϫ12, regarded as indistinguishable from background, were 0.008, 0.008, and 0.009, respectively. (D) TFIIIB dependence of transcription. Transcription was assessed on duplex DNA and the Ϫ6 overhang template in the presence of single, any two, or all three TFIIIB subunits. rm, recovery marker. to the normal transcription start site, and activity was monitored stream of the U6 gene’sT7 segment (T7:A7 in duplex DNA), that in multiple rounds of transcription. Only templates with tran- is, within the bounds of the transcription bubble. The experi- scribed strand 3Ј ends placed downstream of base pair Ϫ12 ments that follow explore the promoter sequence requirements (relative to the normal transcription start site as ϩ1) were found of these constructs and also exploit previously analyzed TBP, to be active (Fig. 1B and data not shown). At the optimal Bdp1, and Brf1 mutations to gain further insight into the mode concentration (Fig. 1B), 5Ј overhang DNA was seen to yield of action of TFIIIB in this special context. approximately one-fifth as many transcripts as duplex DNA (Fig. 1C). Excess DNA diminished activity (perhaps by nonspecifically Transcriptional Activity of Partially Single-Stranded Promoters Is and unproductively sequestering TBP or pol III). TFIIIC did not Sequence-Dependent. Mutations in the TATA box and the T7:A7 restore transcription activity to the inactive Ϫ12 5Ј overhang stretch were introduced into the transcriptionally active 5Ј (o͞h) construct (data not shown). Transcription from these 5Ј overhang construct Ϫ6o͞h, the inactive construct Ϫ12 o͞h, and overhang templates strictly required each TFIIIB subunit, in- the corresponding duplex DNA (Fig. 2A) and tested for tran- cluding Bdp1 (Fig. 1D). Transcriptional start sites, mapped by scriptional activity with TFIIIB and pol III (Fig. 2B). Destruc- primer extension, were shifted slightly upstream to base pair Ϫ2 tion of the TATA box eliminated transcription of duplex DNA for constructs Ϫ10 o͞htoϪ4o͞h, remained at base pair ϩ1 for (Fig.
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