Ty1 Integration Research Articles

Structural basis of Ty1 integrase tethering to RNA polymerase III for targeted retrotransposon integration

The yeast Ty1 retrotransposon integrates upstream of genes transcribed by RNA polymerase III (Pol III). Specificity of integration is mediated by an interaction between the Ty1 integrase (IN1) and Pol III, currently uncharacterized at the atomic level. We report cryo-EM structures of Pol III in complex with IN1, revealing a 16-residue segment at the IN1 C-terminus that contacts Pol III subunits AC40 and AC19, an interaction that we validate by in vivo mutational analysis. Binding to IN1 associates with allosteric changes in Pol III that may affect its transcriptional activity. The C-terminal domain of subunit C11, involved in RNA cleavage, inserts into the Pol III funnel pore, providing evidence for a two-metal mechanism during RNA cleavage. Additionally, ordering next to C11 of an N-terminal portion from subunit C53 may explain the connection between these subunits during termination and reinitiation. Deletion of the C53 N-terminal region leads to reduced chromatin association of Pol III and IN1, and a major fall in Ty1 integration events. Our data support a model in which IN1 binding induces a Pol III configuration that may favor its retention on chromatin, thereby improving the likelihood of Ty1 integration.

A small targeting domain in Ty1 integrase is sufficient to direct retrotransposon integration upstream of tRNA genes

Integration of transposable elements into the genome is mutagenic. Mechanisms targeting integrations into relatively safe locations, hence minimizing deleterious consequences for cell fitness, have emerged during evolution. In budding yeast, integration of the Ty1 LTR retrotransposon upstream of RNA polymerase III (Pol III)‐transcribed genes requires interaction between Ty1 integrase (IN1) and AC40, a subunit common to Pol I and Pol III. Here, we identify the Ty1 targeting domain of IN1 that ensures (i) IN1 binding to Pol I and Pol III through AC40, (ii) IN1 genome‐wide recruitment to Pol I‐ and Pol III‐transcribed genes, and (iii) Ty1 integration only at Pol III‐transcribed genes, while IN1 recruitment by AC40 is insufficient to target Ty1 integration into Pol I‐transcribed genes. Swapping the targeting domains between Ty5 and Ty1 integrases causes Ty5 integration at Pol III‐transcribed genes, indicating that the targeting domain of IN1 alone confers Ty1 integration site specificity.

Genome-Wide Mapping of Yeast Retrotransposon Integration Target Sites.

Transposable elements (TEs) are present in virtually all organisms. TE integration into genomes contributes to their structure and evolution, but can also be harmful in some cases. Deciphering where and how TE integration is targeted is fundamental to understand their intricate relationship with their host and explore the outcome of TE mobility on genome evolution and cell fitness. In general, TEs display integration site preference, which differs between elements. High-throughput mapping of de novo insertions by deep sequencing has recently allowed identifying genome-wide integration preferences of several TEs. These studies have provided invaluable clues to address the molecular determinants of integration site preference. Here, we provide a step-by-step methodology to generate massive de novo insertion events and prepare a library of genomic DNA for next-generation sequencing. We also describe a primary bioinformatic procedure to map these insertions in the genome. The whole procedure comes from our recent work on the integration of Ty1 in Saccharomyces cerevisiae, but could be easily adapted to the study of other TEs.

Ty3, a Position-specific Retrotransposon in Budding Yeast.

Long terminal repeat (LTR) retrotransposons constitute significant fractions of many eukaryotic genomes. Two ancient families are Ty1/Copia (Pseudoviridae) and Ty3/Gypsy (Metaviridae). The Ty3/Gypsy family probably gave rise to retroviruses based on the domain order, similarity of sequences, and the envelopes encoded by some members. The Ty3 element of Saccharomyces cerevisiae is one of the most completely characterized elements at the molecular level. Ty3 is induced in mating cells by pheromone stimulation of the mitogen-activated protein kinase pathway as cells accumulate in G1. The two Ty3 open reading frames are translated into Gag3 and Gag3-Pol3 polyprotein precursors. In haploid mating cells Gag3 and Gag3-Pol3 are assembled together with Ty3 genomic RNA into immature virus-like particles in cellular foci containing RNA processing body proteins. Virus-like particle Gag3 is then processed by Ty3 protease into capsid, spacer, and nucleocapsid, and Gag3-Pol3 into those proteins and additionally, protease, reverse transcriptase, and integrase. After haploid cells mate and become diploid, genomic RNA is reverse transcribed into cDNA. Ty3 integration complexes interact with components of the RNA polymerase III transcription complex resulting in Ty3 integration precisely at the transcription start site. Ty3 activation during mating enables proliferation of Ty3 between genomes and has intriguing parallels with metazoan retrotransposon activation in germ cell lineages. Identification of nuclear pore, DNA replication, transcription, and repair host factors that affect retrotransposition has provided insights into how hosts and retrotransposons interact to balance genome stability and plasticity.

A nucleosomal surface defines an integration hotspot for the Saccharomyces cerevisiae Ty1 retrotransposon

Ty1, the most abundant retrotransposon in Saccharomyces cerevisiae, integrates preferentially upstream of genes transcribed by RNA polymerase III (Pol III). Targeting is likely due to interactions between the Ty1 integration complex and a feature of chromatin characteristic of sites of Pol III transcription. To better understand Ty1 targeting determinants, >150,000 Ty1 insertions were mapped onto the S. cerevisiae genome sequence. Logistic regression was used to assess relationships between patterns of Ty1 integration and various genomic features, including genome-wide data sets of histone modifications and transcription-factor binding sites. Nucleosomes were positively associated with Ty1 insertions, and fine-scale mapping of insertions upstream of genes transcribed by Pol III indicated that Ty1 preferentially integrates into nucleosome-bound DNA near the H2A/H2B interface. Outside of genes transcribed by Pol III, Ty1 avoids coding sequences, a pattern that is not due to selection, but rather reflects a preference for nucleosome-rich sites flanking genes. Ty1 insertion sites were also mapped in four mutant lines that affect Ty1 transposition frequency or integration specificity (rrm3Δ, hos2Δ, rtt109Δ, and rad6Δ). Patterns of integration were largely preserved in the mutants, although significantly more insertions into coding sequences were observed in the rad6Δ strain, suggesting a loosening of target specificity in this mutant that lacks an enzyme involved in ubiquitinating H2A. Overall, our data suggest that nucleosomes are necessary for Ty1 integration, and that a secondary factor, likely a histone modification or nucleosome-bound factor enriched at sites of Pol III transcription, determines preferred target sites.

Retrotransposon Ty1 integration targets specifically positioned asymmetric nucleosomal DNA segments in tRNA hotspots

The Saccharomyces cerevisiae genome contains about 35 copies of dispersed retrotransposons called Ty1 elements. Ty1 elements target regions upstream of tRNA genes and other Pol III-transcribed genes when retrotransposing to new sites. We used deep sequencing of Ty1-flanking sequence amplicons to characterize Ty1 integration. Surprisingly, some insertions were found in mitochondrial DNA sequences, presumably reflecting insertion into mitochondrial DNA segments that had migrated to the nucleus. The overwhelming majority of insertions were associated with the 5' regions of Pol III transcribed genes; alignment of Ty1 insertion sites revealed a strong sequence motif centered on but extending beyond the target site duplication. A strong sequence-independent preference for nucleosomal integration sites was observed, in distinction to the preferences of the Hermes DNA transposon engineered to jump in yeast and the Tf1 retrotransposon of Schizosaccharomyces pombe, both of which prefer nucleosome free regions. Remarkably, an exquisitely specific relationship between Ty1 integration and nucleosomal position was revealed by alignment of hotspot Ty1 insertion position regions to peak nucleosome positions, geographically implicating nucleosomal DNA segments at specific positions on the nucleosome lateral surface as targets, near the "bottom" of the nucleosome. The specificity is observed in the three tRNA 5'-proximal nucleosomes, with insertion frequency dropping off sharply 5' of the tRNA gene. The sites are disposed asymmetrically on the nucleosome relative to its dyad axis, ruling out several simple molecular models for Ty1 targeting, and instead suggesting association with a dynamic or directional process such as nucleosome remodeling associated with these regions.

Two large-scale analyses of Ty1 LTR-retrotransposon de novo insertion events indicate that Ty1 targets nucleosomal DNA near the H2A/H2B interface

BackgroundOver the years, a number of reports have revealed that Ty1 integration occurs in a 1-kb window upstream of Pol III-transcribed genes with an approximate 80-bp periodicity between each integration hotspot and that this targeting requires active Pol III transcription at the site of integration. However, the molecular bases of Ty1 targeting are still not understood.FindingsThe publications by Baller et al. and Mularoni et al. in the April issue of Genome Res. report the first high-throughput sequencing analysis of Ty1 de novo insertion events. Their observations converge to the same conclusion, that Ty1 targets a specific surface of the nucleosome at he H2A/H2B interface.ConclusionThis discovery is important, and should help identifying factor(s) involved in Ty1 targeting. Recent data on transposable elements and retroviruses integration site choice obtained by large-scale analyses indicate that transcription and chromatin structure play an important role in this process. The studies reported in this commentary add a new evidence of the importance of chromatin in integration selectivity that should be of interest for everyone interested in transposable elements integration.

Chromatin-Associated Genes Protect the Yeast Genome From Ty1 Insertional Mutagenesis

Chromosomal genes modulate Ty retrotransposon movement in the genome of Saccharomyces cerevisiae. We have screened a collection of 4739 deletion mutants to identify those that increase Ty1 mobility (Ty1 restriction genes). Among the 91 identified mutants, 80% encode products involved in nuclear processes such as chromatin structure and function, DNA repair and recombination, and transcription. However, bioinformatic analyses encompassing additional Ty1 and Ty3 screens indicate that 264 unique genes involved in a variety of biological processes affect Ty mobility in yeast. Further characterization of 33 of the mutants identified here show that Ty1 RNA levels increase in 5 mutants and the rest affect mobility post-transcriptionally. RNA and cDNA levels remain unchanged in mutants defective in transcription elongation, including ckb2Delta and elf1Delta, suggesting that Ty1 integration may be more efficient in these strains. Insertion-site preference at the CAN1 locus requires Ty1 restriction genes involved in histone H2B ubiquitination by Paf complex subunit genes, as well as BRE1 and RAD6, histone H3 acetylation by RTT109 and ASF1, and transcription elongation by SPT5. Our results indicate that multiple pathways restrict Ty1 mobility and histone modifications may protect coding regions from insertional mutagenesis.

Hos2 and Set3 promote integration of Ty1 retrotransposons at tRNA genes in Saccharomyces cerevisiae.

The yeast LTR retrotransposon Ty1 integrates preferentially into regions upstream of tRNA genes. The chromatin structure of transcriptionally active tRNA genes is known to be important for Ty1 integration, but specific chromatin factors that enhance integration at tRNA genes have not been identified. Here we report that the histone deacetylase, Hos2, and the Trithorax-group protein, Set3, both components of the Set3 complex (Set3C), enhance transposition of chromosomal Ty1 elements by promoting integration into the upstream region of tRNA genes. Deletion of HOS2 or SET3 reduced the mobility of a chromosomal Ty1his3AI element about sevenfold. Despite the fact that Ty1his3AI RNA, total Ty1 RNA, and total Ty1 cDNA levels were not reduced in hos2delta or set3delta mutants, transposition of endogenous Ty1 elements into the upstream regions of tRNA(Gly) genes was substantially decreased. Furthermore, when equivalent numbers of Ty1HIS3 mobility events launched from a pGAL1:Ty1his3AI plasmid were analyzed, only one-quarter to one-half as many were found upstream of tRNA(Gly) genes in a hos2delta or set3delta mutant than in a wild-type strain. Chromatin immunoprecipitation analysis revealed that Hos2 is physically associated with tRNA genes. Taken together, our results support the hypothesis that Hos2 and Set3 function at tRNA genes to promote Ty1 integration.

Genome-wide identification of Isw2 chromatin-remodeling targets by localization of a catalytically inactive mutant

Isw2 ATP-dependent chromatin-remodeling activity is targeted to early meiotic and MATa-specific gene promoters in Saccharomyces cerevisiae. Unexpectedly, preferential cross-linking of wild-type Isw2p was not detected at these loci. Instead, the catalytically inactive Isw2p-K215R mutant is enriched at Isw2 targets, suggesting that Isw2p-K215R, but not wild-type Isw2p, is a sensitive chromatin immunoprecipitation (ChIP) reagent for marking sites of Isw2 activity in vivo. Genome-wide ChIP analyses confirmed this conclusion and identified tRNA genes (tDNAs) as a new class of Isw2 targets. Loss of Isw2p disrupted the periodic pattern of Ty1 integration upstream of tDNAs, but did not affect transcription of tDNAs or the associated Ty1 retrotransposons. In addition to identifying new Isw2 targets, our localization studies have important implications for the mechanism of Isw2 association with chromatin in vivo. Target-specific enrichment of Isw2p-K215R, not wild-type Isw2p, suggests that Isw2 is recruited transiently to remodel chromatin structure at these sites. In contrast, we found no evidence for Isw2 function at sites preferentially enriched by wild-type Isw2p, leading to our proposal that wild-type Isw2p cross-linking reveals a scanning mode of the complex as it surveys the genome for its targets.

Identification and characterization of critical cis-acting sequences within the yeast Ty1 retrotransposon.

The yeast long terminal repeat (LTR) retrotransposon Ty1, like retroviruses, encodes a terminally redundant RNA, which is packaged into virus-like particles (VLPs) and is converted to a DNA copy by the process of reverse transcription. Mutations predicted to interfere with the priming events during reverse transcription and hence inhibit replication are known to dramatically decrease transposition of Ty1. However, additional cis-acting sequences responsible for Ty1 replication and RNA dimerization and packaging have remained elusive. Here we describe a modular mini-Ty1 element encoding the minimal sequence that can be retrotransposed by the Ty1 proteins, supplied in trans by a helper construct. Using a mutagenic screening strategy, we recovered transposition-deficient modular mini-Ty1-HIS3 elements with mutations in sequences required in cis for Ty1 replication and integration. Two distinct clusters of mutations mapped near the 5'-end of the Ty1 RNA. The clusters define a GAGGAGA sequence at the extreme 5'-end of the Ty1 transcript and a complementary downstream UCUCCUC sequence, 264 nt into the RNA. Disruption of the reverse complementarity of these two sequences decreased transposition and restoration of complementarity rescued transposition to wild-type levels. Ty1 cDNA was reduced in cells expressing RNAs with mutations in either of these short sequences, despite nearly normal levels of Ty1 RNA and VLPs. Our results suggest that the intramolecular interaction between the 5'-GAGGAGA and UCUCCUC sequences stabilizes an RNA structure required for efficient initiation of reverse transcription.

Host factors that affect Ty3 retrotransposition in Saccharomyces cerevisiae.

The retrovirus-like element Ty3 of Saccharomyces cerevisiae integrates at the transcription initiation region of RNA polymerase III. To identify host genes that affect transposition, a collection of insertion mutants was screened using a genetic assay in which insertion of Ty3 activates expression of a tRNA suppressor. Fifty-three loci were identified in this screen. Corresponding knockout mutants were tested for the ability to mobilize a galactose-inducible Ty3, marked with the HIS3 gene. Of 42 mutants tested, 22 had phenotypes similar to those displayed in the original assay. The proteins encoded by the defective genes are involved in chromatin dynamics, transcription, RNA processing, protein modification, cell cycle regulation, nuclear import, and unknown functions. These mutants were induced for Ty3 expression and assayed for Gag3p protein, integrase, cDNA, and Ty3 integration upstream of chromosomal tDNA(Val(AAC)) genes. Most mutants displayed differences from the wild type in one or more intermediates, although these were typically not as severe as the genetic defect. Because a relatively large number of genes affecting retrotransposition can be identified in yeast and because the majority of these genes have mammalian homologs, this approach provides an avenue for the identification of potential antiviral targets.

Ty3 requires yeast La homologous protein for wild-type frequencies of transposition.

The Saccharomyces cerevisiae retrovirus-like element Ty3 inserts specifically into the initiation sites of genes transcribed by RNA polymerase III (pol III). A strain with a disruption of LHP1, which encodes the homologue of autoantigen La protein, was recovered in a screen for mutants defective in Ty3 transposition. Transposition into a target composed of divergent tRNA genes was decreased eightfold. In lhp1 mutants, Ty3 polyproteins were produced at wild-type levels, assembled into virus-like particles (VLPs) and processed efficiently. The amount of cDNA associated with these particles was about half the amount in a wild-type control at early times, but approached the wild-type level after 48 h of induction. Ty3 integration was examined at two genomic tRNA gene families and two plasmid-borne tRNA promoters. Integration was significantly decreased at one of the tRNA gene families, but was only slightly decreased at the second tRNA gene family. These findings suggest that Lhp1p contributes to Ty3 cDNA synthesis, but might also act at a target-specific step, such as integration.

Transcriptional interactions between yeast tRNA genes, flanking genes and Ty elements: a genomic point of view.

Retroelement insertion can alter the expression of nearby genes. The Saccharomyces cerevisiae retrotransposons Ty1-Ty4 are transcribed by RNA polymerase II (pol II) and target their integration upstream of genes transcribed by RNA polymerase III (pol III), mainly tRNA genes. Because tRNA genes can repress nearby pol II-transcribed genes, we hypothesized that transcriptional interference may exist between Ty1 insertions and pol III-transcribed genes, the preferred targets for Ty1 integration. Ty1s upstream of two pol III-transcribed genes (SNR6 and SUP2) were recovered and analyzed by RNA blot analysis. Ty1 insertions were found to exert a neutral or modest stimulatory effect on the expression of these genes. Further RNA analysis indicated a modest tRNA position effect on Ty1 transcription. To investigate the possible genomic relevance of these expression effects, we compiled a comprehensive tRNA gene database. This database allowed us to analyze a genome's worth of tRNA genes and Ty elements. It also enabled the prediction and experimental confirmation of tRNA gene position effects at native chromosomal loci. We provide evidence supporting the hypothesis that tRNA genes exert a modest inhibitory effect on adjacent pol II promoters. Direct analysis of PTR3 transcription, promoted by sequences very close to a tRNA gene, shows that this tRNA position effect can operate on a native chromosomal gene.

Mutational Analysis of the Transcription Factor IIIB-DNA Target of Ty3 Retroelement Integration

The Ty3 retrovirus-like element inserts preferentially at the transcription initiation sites of genes transcribed by RNA polymerase III. The requirements for transcription factor (TF) IIIC and TFIIIB in Ty3 integration into the two initiation sites of the U6 gene carried on pU6LboxB were previously examined. Ty3 integrates at low but detectable frequencies in the presence of TFIIIB subunits Brf1 and TATA-binding protein. Integration increases in the presence of the third subunit, Bdp1. TFIIIC is not essential, but the presence of TFIIIC specifies an orientation of TFIIIB for transcriptional initiation and directs integration to the U6 gene-proximal initiation site. In the current study, recombinant wild type TATA-binding protein, wild type and mutant Brf1, and Bdp1 proteins and highly purified TFIIIC were used to investigate the roles of specific protein domains in Ty3 integration. The amino-terminal half of Brf1, which contains a TFIIB-like repeat, contributed more strongly than the carboxyl-terminal half of Brf1 to Ty3 targeting. Each half of Bdp1 split at amino acid 352 enhanced integration. In the presence of TFIIIB and TFIIIC, the pattern of integration extended downstream by several base pairs compared with the pattern observed in vitro in the absence of TFIIIC and in vivo, suggesting that TFIIIC may not be present on genes targeted by Ty3 in vivo. Mutations in Bdp1 that affect its interaction with TFIIIC resulted in TFIIIC-independent patterns of Ty3 integration. Brf1 zinc ribbon and Bdp1 internal deletion mutants that are competent for polymerase III recruitment but defective in promoter opening were competent for Ty3 integration irrespective of the state of DNA supercoiling. These results extend the similarities between the TFIIIB domains required for transcription and Ty3 integration and also reveal requirements that are specific to transcription.

Multiple regulators of Ty1 transposition in Saccharomyces cerevisiae have conserved roles in genome maintenance.

Most Ty1 retrotransposons in the genome of Saccharomyces cerevisiae are transpositionally competent but rarely transpose. We screened yeast mutagenized by insertion of the mTn3-lacZ/LEU2 transposon for mutations that result in elevated Ty1 cDNA-mediated mobility, which occurs by cDNA integration or recombination. Here, we describe the characterization of mTn3 insertions in 21 RTT (regulation of Ty1 transposition) genes that result in 5- to 111-fold increases in Ty1 mobility. These 21 RTT genes are EST2, RRM3, NUT2, RAD57, RRD2, RAD50, SGS1, TEL1, SAE2, MED1, MRE11, SCH9, KAP122, and 8 previously uncharacterized genes. Disruption of RTT genes did not significantly increase Ty1 RNA levels but did enhance Ty1 cDNA levels, suggesting that most RTT gene products act at a step after mRNA accumulation but before cDNA integration. The rtt mutations had widely varying effects on integration of Ty1 at preferred target sites. Mutations in RTT101 and NUT2 dramatically stimulated Ty1 integration upstream of tRNA genes. In contrast, a mutation in RRM3 increased Ty1 mobility >100-fold without increasing integration upstream of tRNA genes. The regulation of Ty1 transposition by components of fundamental pathways required for genome maintenance suggests that Ty1 and yeast have coevolved to link transpositional dormancy to the integrity of the genome.

A truncation mutant of the 95-kilodalton subunit of transcription factor IIIC reveals asymmetry in Ty3 integration.

Position-specific integration of the retroviruslike element Ty3 near the transcription initiation sites of tRNA genes requires transcription factors IIIB and IIIC (TFIIIB and TFIIIC). Using a genetic screen, we isolated a mutant with a truncated 95-kDa subunit of TFIIIC (TFIIIC95) that reduced the apparent retrotransposition of Ty3 into a plasmid-borne target site between two divergently transcribed tRNA genes. Although TFIIIC95 is conserved and essential, no defect in growth or transcription of tRNAs was detected in the mutant. Steps of the Ty3 life cycle, such as protein expression, proteolytic processing, viruslike particle formation, and reverse transcription, were not affected by the mutation. However, Ty3 integration into a divergent tDNA target occurred exclusively in one orientation in the mutant strain. Investigation of this orientation bias showed that TFIIIC95 and Ty3 integrase interacted in two-hybrid and glutathione S-transferase pulldown assays and that interaction with the mutant TFIIIC95 protein was attenuated. The orientation bias observed here suggests that even for wild-type Ty3, the protein complexes associated with the long terminal repeats are not equivalent in vivo.

The Sgs1 helicase of Saccharomyces cerevisiae inhibits retrotransposition of Ty1 multimeric arrays.

Ty1 retrotransposons in the yeast Saccharomyces cerevisiae are maintained in a genetically competent but transpositionally dormant state. When located in the ribosomal DNA (rDNA) locus, Ty1 elements are transcriptionally silenced by the specialized heterochromatin that inhibits rDNA repeat recombination. In addition, transposition of all Ty1 elements is repressed at multiple posttranscriptional levels. Here, we demonstrate that Sgs1, a RecQ helicase required for genome stability, inhibits the mobility of Ty1 elements by a posttranslational mechanism. Using an assay for the mobility of Ty1 cDNA via integration or homologous recombination, we found that the mobility of both euchromatic and rDNA-Ty1 elements was increased 32- to 79-fold in sgs1Delta mutants. Increased Ty1 mobility was not due to derepression of silent rDNA-Ty1 elements, since deletion of SGS1 reduced the mitotic stability of rDNA-Ty1 elements but did not stimulate their transcription. Furthermore, deletion of SGS1 did not significantly increase the levels of total Ty1 RNA, protein, or cDNA and did not alter the level or specificity of Ty1 integration. Instead, Ty1 cDNA molecules recombined at a high frequency in sgs1Delta mutants, resulting in transposition of heterogeneous Ty1 multimers. Formation of Ty1 multimers required the homologous recombination protein Rad52 but did not involve recombination between Ty1 cDNA and genomic Ty1 elements. Therefore, Ty1 multimers that transpose at a high frequency in sgs1Delta mutants are formed by intermolecular recombination between extrachromosomal Ty1 cDNA molecules before or during integration. Our data provide the first evidence that the host cell promotes retrotransposition of monomeric Ty1 elements by repressing cDNA recombination.

Correct integration of model substrates by Ty1 integrase.

The retrovirus-like mobile genetic element of Saccharomyces cerevisiae, Ty1, transposes to new genomic locations via the element-encoded integrase (IN). Here we report that purified recombinant IN catalyzed correct integration of a linear DNA into a supercoiled target plasmid. Ty1 virus-like particles (VLPs) integrated donor DNA more efficiently than IN. VLP and IN-mediated insertions occurred at random sites in the target. Mg(2+) was preferred over Mn(2+) for correct integration, and neither cation enhanced nonspecific nuclease activity of IN. Products consistent with correct integration events were also obtained by Southern analysis. Recombinant IN and VLPs utilized many, but not all, linear donor fragments containing non-Ty1 ends, including a U3 mutation which has been shown to be defective for transposition in vivo. Together, our results suggest that IN is sufficient for Ty1 integration in vitro and IN interacts with exogenous donors less stringently than with endogenous elements.

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

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 vitro integration 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 the SNR6 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.