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RNA Polymerase III Interferes with Ty3 Integration

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RNA Polymerase III Interferes with Ty3 Integration FEBS 18345 FEBS Letters 405 (1997) 305-311 RNA polymerase III interferes with Ty3 integration Charles M. Connolly1, Suzanne B. Sandmeyer* Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, CA 92697-4025, USA Received 8 January 1997; revised version received 12 February 1997 verse transcription of Ty3 RNA in association with the VLP, Abstract Ty3, a gypsylike retrotransposon of budding yeast, integrates at the transcription initiation site of genes transcribed the DNA copy is integrated into the host genome in a process by RNA polymerase III (pol III). It was previously shown that mediated by Ty3 integrase (IN) [12]. Ty3 IN is a homologue integration in vitro requires intact promoter elements and the pol of retroviral IN. Recombinant retroviral IN has been demon- III transcription factors TFIIIB and TFIIIC. In order to test the strated to have in vitro 3'-end processing and strand-transfer effect of pol III on integration, increasing amounts of a pol Ill- activities (reviewed in [13]). Although Ty3 IN has been dem- containing fraction were added to Ty3 in vitro integration onstrated to be required in vivo for 3'-end processing and reactions. The pol Ill-containing fraction was inhibitory to integration of Ty3 DNA, integration activity has only been integration. These results are consistent with a model where the observed in vitro in association with a VLP fraction [14]. Ty3 integration complex and pol III recognize similar features of the stable transcription complex and compete with each other for Despite the many similarities between retroviruses and Ty3 access to the transcription initiation site. elements, neither retrovirus integration adjacent to tRNA © 1997 Federation of European Biochemical Societies. gene targets nor a bias for tRNA gene-containing regions has been reported. Although, retrovirus insertion has been Key words: Integration; Retro virus; RNA polymerase III; linked in past studies to genomic DNA which is transcription- Transposable element; tRNA gene; Ty3 ally active, more recent studies are consistent with widespread distribution of integration sites, including regions occupied by nucleosomes [15,16]. Features which target retroviral integra- tion have now been studied in vitro. These studies may also 1. Introduction provide further insight into the preferences of retroviruslike elements. Retrovirus IN strand transfer is enhanced by static A subset of retroelements have been identified which have and induced bends [17-19]. Bent regions within nucleosomes patterns of integration that reflect dramatic insertion bias for and DNA which is bent by specific DNA binding proteins are tRNA genes. These include DRE I [1], a nonLTR retrotrans- favored integration sites. Not all DNA bending proteins are poson from Dictyostelium discoideum, and the Tyl and Ty2 equally active in creating preferred sites, however, as DNA copialike and Ty3 gypsylike LTR retrotransposons from Sac- bending proteins which occupy the inside of the bend have charomyces cerevisiae (reviewed in [2]). DRE I elements are been shown to be effective in targeting integration, while those close to position —50 nt relative to tRNA gene sequence. Tyl which occupy the outer, distorted side of the bend can ac- and Ty2 genomic elements occur predominately within a re- tually inhibit integration [20]. gion of about 700 nt upstream of tRNA genes [3-5]. Ty3 is In vitro integration of the Ty3 element requires VLPs, and even more closely associated with tRNA genes than the other the pol III transcription factors TFIIIC and TFIIIB, in addi- elements [6-8]. Of the two staggered nicks introduced by Ty3 tion to a DNA target [21]. TFIIIC is comprised of six sub- strand transfer into the target, one occurs within 1-2 nt of the units and binds to the two internal promoter elements, box A transcription initiation site and the other 5 nt distal. The and box B, of the tRNA gene (reviewed in [22,23]). This target region spanned by the staggered cleavage therefore factor in turn mediates binding of TFIIIB, the pol III initia- overlaps the region contained in the open complex bubble tion factor. TFIIIB is composed of three subunits, a 70 kDa formed upon RNA polymerase III (pol III) transcription ini- subunit with similarity to the pol II transcription factor tiation [9]. In the case of the Saccharomyces cerevisiae ele- TFIIB; a 90 kDa subunit, and the TATA binding protein. ments Tyl and Ty3, tRNA gene targeting requires intact pro- Although TFIIIC is required for binding of TFIIIB, the moter elements and other genes transcribed by pol III such as TFIIIB-DNA complex is quite stable and can mediate multi- 5S and U6 genes are also targets [8,10]. Thus, the processes of ple rounds of pol III transcription in the absence of TFIIIC transcription and integration are superficially linked, if not [24]. Whether TFIIIC is associated with the transcribing actually interdependent. tRNA gene has not been directly tested, although genomic The Ty3 element is composed of GAG3 and POL3 genes footprints of tRNA genes suggest that only TFIIIB may be which encode proteins with similar sequence and function to bound [25]. Features which potentially contribute to the tar- those encoded by the retroviral gag and pol genes [11]. Ty3 geting of Ty3 to the site of tRNA gene transcription initiation proteins condense around the genomic RNA to form a nucle- therefore include TFIIIC and TFIIIB, as well as topograph- oprotein complex referred to as the viruslike particle (VLP), ical features of the tRNA gene, such as novel chromatin struc- which is analogous to the retroviral core particle. After re- ture or the bend associated with the transcription initiation site [26]. A previous study showed that pol III did not appear *Corresponding author. Fax: (1) (714) 824-8598. to be essential in vitro for integration to occur [21]. However, E-mail: [email protected] it was possible that low levels of pol III present in IIIB and 1 IIIC fractions were sufficient for transposition. In such a sce- Present address: Department of Genetics, Box 357360, University of nario, open complex formation in that event could participate Washington, Seattle, WA 98105, USA. 0014-5793/97/S17.00 © 1997 Federation of European Biochemical Societies. All rights reserved. P//S0014-5793(97)00200-7 306 CM. Connolly, S.B. Sandmeyer IFEBS Letters 405 (1997) 305-311 in display of the target site to the integration complex. An the method of Bradford [34]; the concentration of protein in the alternative possibility is that the features of the stable complex pooled fraction was typically 2-5 mg/ml. The BR500 was further fractionated using DEAE-Sephadex. Typi- could function alternatively to target pol III or the integration cally, 1 mg of protein was loaded per ml of column resin. The BR500 complex. These models make distinct predictions regarding was adjusted to 10 mM MgCl2, and applied to the column. The the effect of pol III on integration. The former model would TFIIIB-, IIIC-, and pol Ill-containing fractions were eluted with buf- predict that transposition is dependent on pol III, while the fer E (buffer D with 10 mM MgCl2 instead of 5 mM) containing 100, latter would suggest that transposition might be inhibited by 250, and 500 mM NaCl, respectively. The peak protein fractions were identified and pooled. The fractions were dialyzed against buffer C as pol III. The current study was undertaken to determine the above, and stored at —70°C. The protein concentration of pooled effect of pol III on in vitro integration. fractions was determined by the method of Bradford [34]. The 100 mM NaCl fraction was typically 1-3 mg/ml, the 250 mM NaCl frac- tion was typically 0.5-1.5 mg/ml, and the 500 mM NaCl fraction was 2. Materials and methods typically 0.2-0.5 mg/ml. 2.1. Strains and culture conditions 2.4. In vitro pol HI transcription assays Construction, maintenance, and propagation of plasmids in E. coli In vitro transcriptions were performed using the yeast extract gen- were performed according to standard methods [27]. Plasmid manip- erated as above. The reactions contained 110 mM NaCl, 8 mM ulations in E. coli were carried out using strain HB101 (F~ hsdS20 MgCl2, 20 mM HEPES pH 7.8, 250 uM ATP, CTP, UTP, 15 uM r 32 [rB- mB-] recA13 !euB6 ara-14 proA2 lacYl galK2 rpsL20 [Sm ] xyl-5 [a- P]GTP (16.7 Ci/mmol), 75 fmol template tRNA gene, and 800 ng mtl-1 supE44). Transformation and culturing of 5*. cerevisiae strains pIBI20 carrier DNA in a volume of 50 ul. The reactions were typically were according to standard methods [28]. The S. cerevisiae strains incubated for 30 min at 30°C. Some were incubated for 15 min at used in this study were AGY9 (MATa ura3-52 his4-539 lys2-801 15°C, as noted in the text. The reactions were terminated by the trpl-A63 !eu2Al sptS) and yTM442 {MATa trpl-H3 ura3-52 his3- addition of 150 ul of stop buffer (27 mM EDTA, 0.3% SDS, 0.3 A200 ade2-101 lys2-l !eu2-Al canl-100) (unpublished data, T. Menees ug/ul salmon sperm DNA, and 1.3 M LiCl) [35]. The products were and S.B. Sandmeyer). Plasmids were transformed into yeast by the extracted with phenol/chloroform, and precipitated with ethanol in method of Ito [29]. Synthetic medium used for plasmid selection was the presence of 2 M NH4OAC. After a 70% ethanol wash, the prod- made according to standard protocols. ucts were dried and resuspended in 5 JJ.1 formamide containing 0.1% bromophenol blue, 0.1% xylene cyanol FF. The products were sepa- 2.2. Plasmid construction rated by electrophoresis on a denaturing 8% polyacrylamide gel (20:1 Plasmids were constructed by standard techniques.
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