The Brf and TATA-binding Protein Subunits of the RNA Polymerase III Transcription Factor IIIB Mediate Position-specific Integration of the Gypsy-like Element, Ty3
2000; Elsevier BV; Volume: 275; Issue: 38 Linguagem: Inglês
10.1074/jbc.m003149200
ISSN1083-351X
AutoresLynn Yieh, George A. Kassavetis, E. Peter Geiduschek, Suzanne Sandmeyer,
Tópico(s)RNA modifications and cancer
ResumoTy3 integrates into the transcription initiation sites of genes transcribed by RNA polymerase III. It is known that transcription factors (TF) IIIB and IIIC are important for recruiting Ty3 to its sites of integration upstream of tRNA genes, but that RNA polymerase III is not required. In order to investigate the respective roles of TFIIIB and TFIIIC, we have developed an in vitrointegration assay in which Ty3 is targeted to the U6 small nuclear RNA gene, SNR6. Because TFIIIB can bind to the TATA box upstream of the U6 gene through contacts mediated by TATA-binding protein (TBP), TFIIIC is dispensable for in vitro transcription. Thus, this system offers an opportunity to test the role of TFIIIB independent of a requirement of TFIIIC. We demonstrate that the recombinant Brf and TBP subunits of TFIIIB, which interact over the SNR6 TATA box, direct integration at theSNR6 transcription initiation site in the absence of detectable TFIIIC or TFIIIB subunit B". These findings suggest that the minimal requirements for pol III transcription and Ty3 integration are very similar. Ty3 integrates into the transcription initiation sites of genes transcribed by RNA polymerase III. It is known that transcription factors (TF) IIIB and IIIC are important for recruiting Ty3 to its sites of integration upstream of tRNA genes, but that RNA polymerase III is not required. In order to investigate the respective roles of TFIIIB and TFIIIC, we have developed an in vitrointegration assay in which Ty3 is targeted to the U6 small nuclear RNA gene, SNR6. Because TFIIIB can bind to the TATA box upstream of the U6 gene through contacts mediated by TATA-binding protein (TBP), TFIIIC is dispensable for in vitro transcription. Thus, this system offers an opportunity to test the role of TFIIIB independent of a requirement of TFIIIC. We demonstrate that the recombinant Brf and TBP subunits of TFIIIB, which interact over the SNR6 TATA box, direct integration at theSNR6 transcription initiation site in the absence of detectable TFIIIC or TFIIIB subunit B". These findings suggest that the minimal requirements for pol III transcription and Ty3 integration are very similar. base pair(s) polymerase III transcription factor TATA-binding protein virus-like particles polymerase chain reaction Integration site selection is a key step in the retroelement life cycle, potentially influencing both the effect of the insertion on the host genome and the expression of the element itself. Similar to retroviruses, yeast Ty elements transpose through reverse transcription of an almost full-length RNA copy into a full-length DNA copy, which is integrated into the host genome. Despite very similar molecular mechanisms of integration, budding yeast elements (Tys), both gypsy-like (Ty3) and copia-like (Ty1, 2, 4, and 5), differ from retroviruses in that they exhibit dramatic global integration site preferences (1Hani J. Feldmann H. Nucleic Acids Res. 1998; 26: 689-696Crossref PubMed Scopus (120) Google Scholar, 2Sandmeyer S. Genome Res. 1998; 8: 416-418Crossref PubMed Scopus (29) Google Scholar, 3Kim J.M. Vanguri S. Boeke J.D. Gabriel A. Voytas D.F. Genome Res. 1998; 8: 464-478Crossref PubMed Scopus (419) Google Scholar, 4Boeke J.D. Devine S.E. Cell. 1998; 93: 1087-1089Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Ty1 elements, for example, integrate preferentially within a window of 750 bp,1upstream of genes transcribed by RNA polymerase III (pol III). Analysis of the yeast genome sequence shows that Ty2 and Ty4 also occupy this region upstream of a fraction of tRNA genes. Ty5 integrates into regions of silenced DNA, including the silent mating type loci and telomeres. Hence Ty1, Ty2, Ty4, and Ty5 exhibit regional integration specificity. Despite similarities with these other elements, Ty3 differs in that it integrates within a highly defined window, one or two base pairs (bp) upstream of pol III transcription initiation sites. Targeting of integration appears to be directed by the cooperative actions of Ty3 element- and cell-encoded factors. For example, in addition to the element-encoded integrase protein, Ty3 and Ty1 targeting requires the presence of a transcriptionally competent pol III promoter (5Chalker D.L. Sandmeyer S.B. Genes Dev. 1992; 6: 117-128Crossref PubMed Scopus (160) Google Scholar, 6Devine S.E. Boeke J.D. Genes Dev. 1996; 10: 620-633Crossref PubMed Scopus (179) Google Scholar), and Ty5 targeting requires factors involved in the establishment of silent chromatin (7Zou S. Voytas D.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7412-7416Crossref PubMed Scopus (83) Google Scholar). Class III genes, including plasmid-borne U6, 5S, and tRNA genes, are used in vivo for position-specific Ty3 integration. Comparison of integration sites suggests that Ty3 integration preference is not a direct function of specific local sequences. Each class of pol III-transcribed genes differs from the others in composition of promoter elements (8White R.J. RNA Polymerase III Transcription. Springer-Verlag, Berlin1998Crossref Google Scholar) and distances of common motifs from the integration site (5Chalker D.L. Sandmeyer S.B. Genes Dev. 1992; 6: 117-128Crossref PubMed Scopus (160) Google Scholar). Pol III promoter mutations that affect transcription factor binding at positions distant from the integration site also block Ty3 integration (5Chalker D.L. Sandmeyer S.B. Genes Dev. 1992; 6: 117-128Crossref PubMed Scopus (160) Google Scholar, 9Chalker D.L. Sandmeyer S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4927-4931Crossref PubMed Scopus (34) Google Scholar). These results lead to the hypothesis that interactions with the promoter-bound pol III transcription complex determine the selection of Ty3 integration sites. Pol III genes use transcription factors (TF) IIIC and IIIB to assemble pol III at the transcription initiation site (reviewed in Ref. 8White R.J. RNA Polymerase III Transcription. Springer-Verlag, Berlin1998Crossref Google Scholar). TFIIIC is composed of six subunits and interacts with the box A and box B promoter elements. TFIIIB is composed of the TATA-binding protein (TBP), a 68-kDa protein (B" or Tfc5, also referred to as TFIIIB90), and a 67-kDa protein (Brf, also called TFIIIB70). TBP and Brf are tightly associated in a complex referred to as B′, which is chromatographically separable from B". The contributions of these transcription factors to the specific integration of Ty3 have been investigated using an in vitrointegration assay. Reconstitution of specific integration into the initiation site of a tRNA gene requires Ty3 virus-like particles (VLPs), the plasmid-borne target gene, and DEAE-purified TFIIIB- and TFIIIC-containing fractions, consistent with DNA-bound TFIIIB, or TFIIIB and TFIIIC together, recruiting the Ty3 preintegration complex to its site of integration (10Kirchner J. Connolly C.M. Sandmeyer S.B. Science. 1995; 267: 1488-1491Crossref PubMed Scopus (182) Google Scholar). Because TFIIIC is required to load TFIIIB onto DNA at the tRNA gene promoter, the respective roles of these factors could not be distinguished using the tRNA gene target. The roles of TFIIIB and TFIIIC in pol III transcription have been examined in detail. In vivo, TFIIIC recruits TFIIIB to the region upstream of the transcription initiation site of all yeast (Saccharomyces cerevisiae) pol III genes (11Eschenlauer J.B. Kaiser M.W. Gerlach V.L. Brow D.A. Mol. Cell. Biol. 1993; 13: 3015-3026Crossref PubMed Scopus (80) Google Scholar, 12Burnol A.-F. Margottin F. Schultz P. Marsolier M.-C. Oudet P. Sentenac A. J. Mol. Biol. 1993; 233: 644-658Crossref PubMed Scopus (58) Google Scholar). TFIIIB then recruits pol III for initiation of transcription (13Kassavetis G.A. Braun B.R. Nguyen L.H. Geiduschek E.P. Cell. 1990; 60: 235-245Abstract Full Text PDF PubMed Scopus (360) Google Scholar, 14Kassavetis G.A. Joazeiro A.C.P. Pisano M. Geiduschek E.P. Colbert T. Hahn S. Blanco J. Cell. 1992; 71: 1055-1064Abstract Full Text PDF PubMed Scopus (182) Google Scholar). Two sets of observations show that TFIIIB is the central initiation factor of pol III: 1) TFIIIC can be removed from tRNA genes after TFIIIB is assembled at the promoter without loss of transcription activity (13Kassavetis G.A. Braun B.R. Nguyen L.H. Geiduschek E.P. Cell. 1990; 60: 235-245Abstract Full Text PDF PubMed Scopus (360) Google Scholar). 2) SNR6, which does not require TFIIIC for loading TFIIIBin vitro, can be transcribed by pol III in the presence of TFIIIB only (15Margottin F. Dujardin G. Gerard M. Egly J.-M. Huet J. Sentenac A. Science. 1991; 251: 424-426Crossref PubMed Scopus (117) Google Scholar, 16Joazeiro C.A.P. Kassavetis G.A. Geiduschek E.P. Mol. Cell. Biol. 1994; 14: 2798-2808Crossref PubMed Scopus (72) Google Scholar); TFIIIB components have been demonstrated to contact pol III subunits directly (17Khoo B. Brophy B. Jackson S.P. Genes Dev. 1994; 8: 2879-2890Crossref PubMed Scopus (108) Google Scholar, 18Werner M. Chaussivert N. Willis I.M. Sentenac A. J. Biol. Chem. 1993; 268: 20721-20724Abstract Full Text PDF PubMed Google Scholar, 19Andrau J.-C. Sentenac A. Werner M. J. Mol. Biol. 1999; 288: 511-520Crossref PubMed Scopus (46) Google Scholar, 20Flores A. Briand J.F. Gadal O. Andrau J.C. Rubbi L. Van M., V Boschiero C. Goussot M. Marck C. Carles C. Thuriaux P. Sentenac A. Werner M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7815-7820Crossref PubMed Scopus (128) Google Scholar, 21Ferri M.L. Peyroche G. Siaut M. Lefebvre O. Carles C. Conesa C. Sentenac A. Mol. Cell. Biol. 2000; 20: 488-495Crossref PubMed Scopus (66) Google Scholar). The ability to bind TFIIIB directly to the SNR6 gene in the absence of TFIIIC provides an in vitro system in which TFIIIB-bound DNA can be tested for TFIIIC-independent Ty3 targeting. In the experiments that are presented here, distinct roles for TFIIIB and TFIIIC in Ty3-specific integration have been investigated by developing a variation of a previously devised in vitro integration assay. The use of the three recombinant TFIIIB subunits, TBP, Brf, and B" (22Kassavetis G.A. Nguyen S.T. Kobayashi R. Kumar A. Geiduschek E.P. Pisano M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9786-9790Crossref PubMed Scopus (91) Google Scholar), allows a precise definition of the minimal protein requirements for specific integration of Ty3. We demonstrate that TFIIIB recruits the Ty3 preintegration complex to its site of integration. Recombinant B′ (i.e. TBP + Brf) is sufficient to direct specific integration of Ty3 in vitro and B" contributes strongly to the efficiency of integration, conceivably through the DNA distortion that it generates near the integration site. TFIIIC contributes to targeting specificity and selection by determining the orientation of TFIIIB on the promoter. Standard methods were used for culturing and transforming Escherichia coli andS. cerevisiae and for recombinant DNA constructions (23Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing Associates/Wiley-Interscience, New York1998Google Scholar). All plasmids were amplified in, and prepared from, E. coliHB101. Single-stranded DNA for site-directed oligonucleotide mutagenesis was prepared from E. coli RZ1032. Plasmid pU6LboxB (24Whitehall S.K. Kassavetis G.A. Geiduschek E.P. Genes Dev. 1995; 9: 2974-2985Crossref PubMed Scopus (59) Google Scholar) was the pol III transcription template and target for Ty3 integration in vitro. Plasmids pDLC370 (5Chalker D.L. Sandmeyer S.B. Genes Dev. 1992; 6: 117-128Crossref PubMed Scopus (160) Google Scholar) and pLY1842 served as PCR controls for integration into r-U6 and l-U6, respectively. Plasmid pDLC370 has a Ty3 insertion with Ty3 sequence beginning at position −5 relative to the transcription start site r-U6. Plasmid LY1842 was constructed by cloning a PCR fragment generated with primers 242 and 411 (see below) and pU6LboxB containing a Ty3 insertion at bp −2 relative to the l-U6 TATA transcription start site (Fig. 2 A) into pCRII-TOPO. The pLY1855 plasmid was generated by removing the δ TATA box from pU6LboxB by site-directed mutagenesis using an oligonucleotide with sequence 5′-GCTGGAGATACAGAACTATTATGG-3′. Plasmid pU6LboxB-G56 was constructed by changing the conserved C56 of the SNR6 boxB to G using an oligonucleotide with sequence 5′-GGGGGGAGTCCAACGCCCGATTGC-3". Mutations were confirmed by DNA sequence analysis. Ty3 VLPs were prepared from S. cerevisiae strain AGY9 (pEGTy3-1) cells as described (25Hansen L.J. Chalker D.L. Orlinsky K.J. Sandmeyer S.B. J. Virol. 1992; 66: 1414-1424Crossref PubMed Google Scholar). Highly purified TFIIIC (oligobox B+ fraction) and pol III (MonoQ fraction), rTBP, rBrf, and rB", were prepared and quantitated as described or referenced (13Kassavetis G.A. Braun B.R. Nguyen L.H. Geiduschek E.P. Cell. 1990; 60: 235-245Abstract Full Text PDF PubMed Scopus (360) Google Scholar, 16Joazeiro C.A.P. Kassavetis G.A. Geiduschek E.P. Mol. Cell. Biol. 1994; 14: 2798-2808Crossref PubMed Scopus (72) Google Scholar, 26Kassavetis G.A. Kumar A. Ramirez E. Geiduschek E.P. Mol. Cell. Biol. 1998; 18: 5587-5599Crossref PubMed Scopus (60) Google Scholar, 27Kassavetis G.A. Riggs D.L. Negri R. Nguyen L.H. Geiduschek E.P. Mol. Cell. Biol. 1989; 9: 2551-2566Crossref PubMed Scopus (185) Google Scholar) and are specified as active molecules in specifically initiating transcription (pol III) or specific DNA binding (TBP, Brf, B", TFIIIC). TBP and B" were fully active; Brf was ∼20% active. Under standard conditions (Figs. Figure 2, Figure 3, Figure 4), samples for in vitro integration contained, in 50 μl of reaction buffer (40 mm Tris-HCl, pH 8, 7 mm MgCl2, 3 mmdithiothreitol, 100 μg/ml bovine serum albumin, and 50 mmNaCl), 1.0 nm TBP, 0.7 nm Brf, 1.5 nm B", and 3.6 nm target plasmid DNA. These components were preincubated for 30 min at 23 °C, shifted to 15 °C, and then 5 μg (protein) of Ty3 VLP fraction was added for 15 min. When purified TFIIIC was also present, it was preincubated with DNA for 30 min before adding TFIIIB components. Samples contained 10.5 nm TFIIIC for the experiment shown in Fig. 3. For Fig. 4, samples contained the noted multiples of 0.5 nm TFIIIC. For Fig. 5 A, factors were preincubated with DNA for 1 h at 23 °C prior to adding VLPs. For Fig. 5 B, B′-DNA complexes were allowed to form for 30 min prior to an additional 15-min incubation with subsequently added B". Reactions were stopped by adding proteinase K, SDS, and EDTA, pH 8.0, to final concentrations of 0.2 mg/ml, 0.2% (w/v), and 20 mm, respectively, and incubating at 37 °C for 30 min. Reaction products were extracted with phenol/chloroform, and DNA was precipitated and redissolved in 10 mm Tris-HCl, pH 8.0, 1 mm EDTA.Figure 4TFIIIC affects Ty3 integration by directing the assembly of TFIIIB on the U6 promoter. Integration reactions were performed with 0.7–1.5 nm rTFIIIB subunits and 0.5–4.0 nm TFIIIC (shown as multiples of 0.5 nm). PCR analysis of the integration products was performed as described in the legend to Fig. 2. Reactants are indicated at thetop of the panel. Negative (N) and positive (P) controls are as described in the legend to Fig. 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5TBP and Brf bound to DNA constitute the minimal integration target for Ty3 and B" promotes efficient integration. A, integration into plasmid pLY1855 (SNR6 TATA only) as a function of TBP, Brf, and B" concentration (indicated at the top in multiples of the standard integration assay described under "Experimental Procedures"). VLPs were present in all reactions at the standard 5 μg/sample. Negative (N) plasmid control: pLY1855; positive (P) controls as described in the legend to Fig. 3. Specific integration sites are indicated at the right side. B, placing a limit on the contamination of VLPs with B". Integration into pLY1855. The concentrations of B′ and B" are shown as multiples of concentrations in a standard reaction. Negative and positive controls (lanes 1 and 8) as inpanel A. Lanes 2–5, B′ was preincubated with target DNA for 30 min prior to the addition of B"; lanes 6and 7, only B′ was added.View Large Image Figure ViewerDownload Hi-res image Download (PPT) PCR was performed essentially as described (28Menees T.M. Sandmeyer S.B. Mol. Cell. Biol. 1994; 14: 8229-8240Crossref PubMed Scopus (40) Google Scholar), with the following changes to make detection specific to the SNR6 target: primer 242 (5′-GGAACTGCTGATCATCTCT-3′) (200 ng) and primer 411 (5′-CGAAACACAAGACAACCC-3′) (164 ng) were used for amplification of 40 ng (18 fmol) of DNA from the integration reactions (first incubation for 2.5 min at 95 °C, followed by 40 cycles of denaturation at 94 °C for 1 min and renaturation/extension at 65 °C for 1 min, followed by 5 min at 72 °C). The standard amplification reactions in a total of 40 ng of target plasmid DNA yielded products equivalent to those of reactions containing 1.6 to 8 fg of Ty3-positive target plasmid in a total of 40 ng of target plasmid. Thus, the products are highly specific to positive plasmid templates. To control for consistent DNA recovery from the integration reaction and for consistent operation of the above PCR, primers 679 (5′-ACTCCCCGTCGTGTAGATAACTACG-3′) and 680 (5′-AAGCCATACCAAACGACGAGC-3′) were used to amplify the β-lactamase gene carried by the target plasmids. PCR amplification of 100 pg of DNA (5 μl of 1:200 reaction dilution) was performed using 200 ng of each primer (first denaturation for 2.5 min at 95 °C, followed by 18 cycles of denaturation at 94 °C for 45 s, renaturation at 55 °C for 30 s, and extension at 72 °C for 30 s, with a final 5 min at 72 °C). PCR products were resolved by electrophoresis on nondenaturing, 8% polyacrylamide gel and visualized by staining with ethidium bromide. The fluorescence video image was quantified using a gel documentation program. PCR fragments representing specific integrations were separated as described above. Bands containing specific fragments were excised from the gel, DNA was eluted, ligated into the vector pCRII-TOPO according to the supplier's instructions, and transformed into E. coli. Individual transformants, selected on LB + ampicillin, were picked for analysis. DNA was prepared from five transformants per band and sequenced with oligonucleotide 242 primer. Recombinant B" (5–80 fmol), the crude pol III transcription-competent fraction BR500 (7 μg; BRα (29Braun B.R. Riggs D.L. Kassavetis G.A. Geiduschek E.P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2530-2534Crossref PubMed Scopus (75) Google Scholar)), and Ty3 VLPs were resolved on 9% polyacrylamide-SDS gel and transferred to polyvinylidene difluoride membrane. Polyclonal rabbit antiserum directed against B" () (22Kassavetis G.A. Nguyen S.T. Kobayashi R. Kumar A. Geiduschek E.P. Pisano M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9786-9790Crossref PubMed Scopus (91) Google Scholar) and125I-protein A were used to probe the blot, essentially as described (30Kamps M.P. Sefton B.M. Oncogene. 1988; 2: 305-315PubMed Google Scholar). Bands were quantified by PhosphorImager analysis. Photochemical cross-linking was performed with an 88-bp DNA probe containing the SNR6 TATA box (31Kassavetis G.A. Kumar A. Letts G.A. Geiduschek E.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9196-9201Crossref PubMed Scopus (54) Google Scholar) with 5-[N′-(p-azidobenzoyl)-3-aminoallyl]dUMP incorporated at bp −39 and −38 and [α-32P]dCMP at bp −37 (26Kassavetis G.A. Kumar A. Ramirez E. Geiduschek E.P. Mol. Cell. Biol. 1998; 18: 5587-5599Crossref PubMed Scopus (60) Google Scholar). The TATA box was modified to TGTAAATA to provide a unique orientation of TFIIIB-DNA complexes in conjunction with the TBP mutant TBPm3 (24Whitehall S.K. Kassavetis G.A. Geiduschek E.P. Genes Dev. 1995; 9: 2974-2985Crossref PubMed Scopus (59) Google Scholar). Protein-DNA complexes were formed as described for in vitro integration but in a 20-μl reaction volume, with β-mercaptoethanol in place of dithiothreitol, 10 fmol of photoprobe, 200 ng of poly(dG-dC)·poly(dG-dC), and NaCl at 70 mm. Where indicated, B′-DNA complexes were formed with 400 fmol of TBPm3 and 144 fmol of Brf for 60 min at ∼20 °C. B", in the indicated amounts, 5 μg of VLPs or B" and 5 μg of VLPs (preincubated together for 15 min at 0 °C) was added for an additional 20 min, followed by 2 min of UV irradiation. Reaction mixtures were treated with nucleases and resolved on 9% polyacrylamide-SDS gel as described (32Bartholomew B. Tinker R.L. Kassavetis G.A. Geiduschek E.P. Methods Enzymol. 1995; 262: 476-494Crossref PubMed Scopus (34) Google Scholar). In order to investigate whether TFIIIB suffices for Ty3 position-specific integration, a previously used in vitro tRNA gene integration assay (10Kirchner J. Connolly C.M. Sandmeyer S.B. Science. 1995; 267: 1488-1491Crossref PubMed Scopus (182) Google Scholar) was modified to utilizeSNR6 as a target. Integration reactions contained Ty3 VLPs, the S. cerevisiae SNR6 gene in plasmid pU6LboxB (24Whitehall S.K. Kassavetis G.A. Geiduschek E.P. Genes Dev. 1995; 9: 2974-2985Crossref PubMed Scopus (59) Google Scholar), and combinations of TFIIIB and TFIIIC. Pol III, previously shown to inhibit Ty3 integration (33Connolly C.M. Sandmeyer S.B. FEBS Lett. 1997; 405: 305-311Crossref PubMed Scopus (25) Google Scholar), was omitted. TFIIIC was supplied either as a DEAE column fraction or as highly purified protein (27Kassavetis G.A. Riggs D.L. Negri R. Nguyen L.H. Geiduschek E.P. Mol. Cell. Biol. 1989; 9: 2551-2566Crossref PubMed Scopus (185) Google Scholar); TFIIIB was added either as a DEAE fraction or as recombinant protein (rTFIIIB). Integration of Ty3 into the target plasmid was detected using a polymerase chain reaction (PCR)-based assay (Fig.1 A). One PCR primer anneals to the SNR6 gene and the other to a unique sequence in Ty3. A diagnostic PCR template sequence is created when Ty3 integrates into the SNR6 target in one of the two possible orientations, and the length of the corresponding PCR-amplified fragment specifies the position of integration. This assay differs from the previously described tRNA gene assay (10Kirchner J. Connolly C.M. Sandmeyer S.B. Science. 1995; 267: 1488-1491Crossref PubMed Scopus (182) Google Scholar) in that the target primer does not overlap with the Ty3 insertion, so that detection of integration is not limited to a single location; additional rounds of PCR are used to increase assay sensitivity. The integration assay uses the plasmid pU6LboxB, which contains a modified SNR6 gene with altered flanking DNA sequence (24Whitehall S.K. Kassavetis G.A. Geiduschek E.P. Genes Dev. 1995; 9: 2974-2985Crossref PubMed Scopus (59) Google Scholar) as its Ty3 target. A duplication of the SNR6transcriptional start site and terminator in pU6LboxB allows TFIIIB-DNA complexes oriented to promote transcription away from theSNR6 gene (leftward in Fig. 1 B) to be monitored in a transcription assay. In constructing pU6LboxB, theSNR6 TATA box was inverted to preferentially orient TFIIIB for leftward transcription, and the boxB promoter element was moved closer to the natural SNR6 boxA promoter element in order to optimize the ability of TFIIIC to direct TFIIIB binding at the SNR6 TATA box. TFIIIC shifts the preferred direction of transcription from leftward to rightward (Fig. 1 B) on this template. In constructing pU6LboxB, genomic sequence upstream of theSNR6 TATA box containing a Ty1 LTR (or δ) element was deleted, positioning a second TATA box (which originally flanked theSNR6 gene-distal end of the δ element) between theSNR6 TATA box and the terminator for leftward transcription. This "δ TATA box" (identical in sequence and orientation to theSNR6 TATA box in pU6LboxB) generates its own pair of divergent transcription units. Thus, TFIIIB binding autonomously to either TATA box of pU6LboxB in either orientation generates four primary transcripts (l-U6, r-U6, l-δ, and r-δ in Fig. 1 B). By selecting the site and orientation of TFIIIB binding, TFIIIC favors the formation of the r-U6 transcript and restricts the formation of the others (Ref. 24Whitehall S.K. Kassavetis G.A. Geiduschek E.P. Genes Dev. 1995; 9: 2974-2985Crossref PubMed Scopus (59) Google Scholar and data not shown). The ability to reconstitute specific integration of Ty3 upstream ofSNR6 in vitro was first tested using DEAE column fractions enriched for TFIIIB and TFIIIC. Plasmid pDLC370 ("Experimental Procedures"), which contains a Ty3 insertion, was used as a template for primers 411 and 242 in PCR to amplify a 442-bp fragment that served as a positive control for integration into the r-U6 initiation site (9Chalker D.L. Sandmeyer S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4927-4931Crossref PubMed Scopus (34) Google Scholar). As a control for the quantity of DNA in each integration reaction, primers 679 and 680 were also used to amplify the β-lactamase gene on pU6LboxB. PCR products were separated by nondenaturing polyacrylamide gel electrophoresis. Reconstitution of integration at the SNR6gene in these preliminary trials (data not shown) depended on the presence of Ty3 VLPs, as PCR amplification of reactions lacking VLPs was equivalent to amplification of the target plasmid alone. Integration reactions performed with TFIIIB and TFIIIC generated a predominant PCR product, whose length was consistent with specific integration close to the r-U6 transcription initiation site (Fig.2 A). A random (or at least complex and dispersed) pattern of integration was observed with Ty3 VLPs alone (Fig. 2 B, lane 2). The advantage of the relatively complex pU6LboxB construct is that it permits a determination of whether TFIIIB alone suffices to mediate Ty3-specific integration, while also examining whether TFIIIC exerts an effect on integration site selection. The binding of rTFIIIB to the target plasmid was verified by its ability to direct transcription by pol III in vitro. The four expected transcription products were generated, with preferential production of the l-U6 transcript, as expected (data not shown). Integration at the four transcription initiation sites is predicted to produce four different classes of PCR products (Fig. 2 A). Integrations were performed under conditions tested for transcription activity, and with comparable concentrations and proportions of rTFIIIB and plasmid DNA (Fig. 2 B and data not shown). The combination of rTFIIIB and TFIIIC yielded a single PCR-amplified integration product (Fig.2 B, lane 3). Integration in the presence of rTFIIIB alone generated three major PCR products (lane 4) each consistent in size with integration at one of the four transcription initiation sites (Fig. 2 A); the size of the smallest PCR fragment corresponded with an integration event near the initiation site of r-U6 transcription (Fig. 2 B, compare lane 4 tolanes 3 and 5). The sizes of the remaining products corresponded to PCR templates generated by specific integration very close to the l-δ (lane 4, largest fragment, ∼552 bp), and r-δ and l-U6 (middle size fragment, ∼492 and ∼501 bp) transcription initiation sites, respectively. To determine whether these PCR products indeed report specific integration events, DNA fragments contained in the bands marked at the side of Fig. 2 B were recovered from a gel (cf. Fig. 2 B, lane 4) and cloned into the vector pCRII-TOPO. Sequences were determined for the Ty3-SNR6 junction in five clones from each fragment preparation using the SNR6internal PCR primer (Fig. 2 A). Sequencing of these clones demonstrated that the smallest fragment corresponds to Ty3 sequence beginning at positions −6 and −7 relative to the r-U6 transcription start site (Fig. 2 C). Because Ty3 integrates by means of a staggered strand transfer that is repaired, resulting in repeats of the intervening sequence on either end of the Ty3, this corresponds to a gene-proximal strand transfer between positions 1 and −1 and between −1 and −2, respectively, relative to the start site of transcription. The middle fragment appears to represent integration events at the l-U6 and/or r-δ transcription initiation sites. The identified integration sites in this region showed Ty3 sequence positioned at −2 and +5 relative to the l-U6 transcription initiation site, but cannot be correlated unambiguously with a specific partner TFIIIB-DNA complex because these two initiation sites are located very close together. Analysis of clones derived from the largest product yielded Ty3 sequence at each transcription initiation site, including +3, relative to l-δ. We interpret this as due to contamination in the region of this weaker and slower migrating band in the gel by shorter PCR products. However, the fragment size, together with the sequence of one of the five analyzed clones (Fig. 2 C), suggests that Ty3 integration was also associated with the l-δ transcription start site. The patterns of integration directed by rTFIIIB alone (Fig. 2B, lane 4) and TFIIIB plus TFIIIC (lane 3) were quite distinctive. The rTFIIIB-mediated integration products represented each of the four initiation sites, as just described, but TFIIIB and TFIIIC together generated PCR products consistent with integration primarily at the r-U6 transcription start site. Although the preceding experiment indicates that TFIIIB is the only pol III factor that is absolutely required for specific integration of Ty3, it is necessary to eliminate the possibility that the Ty3 VLP fraction contains and contributes TFIIIC. This seems unlikely because
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