Genomic sequence analysis has shown that retroelements account for a significant proportion of the genomes of plants, animals, and microbes. Among various host organisms, the budding yeast Saccharomyces cerevisiae
offers one of the most genetically tractable model systems for studying these elements. Currently, five retrotransposons, Ty1 through Ty5, have been identified in S. cerevisiae
. Of these retroelements, Ty1, Ty2, Ty4, and Ty5 belong to the copialike family whereas Ty3 belongs to the gypsylike family. Although Ty3 is limited to an intracellular life cycle, It has many similarities to retroviruses, including a number of steps in its life cycle (34
). Retrotransposons and retroviruses rely on proteins encoded in the element or virus and in host cells. Full-length Ty3 DNA is approximately 5.4 kb in length (10
). It encodes Gag3p and Gag3-Pol3p polyproteins, which are processed into mature proteins in the context of the Ty3 viruslike particle by the Ty3 protease. Gag3p is processed into major structural proteins, capsid (CA) and nucleocapsid. Gag3p-Pol3p is processed into catalytic proteins PR, reverse transcriptase and integrase (IN) (17
). The life cycle requires host factors such as transcription and translation machinery and unknown factors involved in such processes as assembly, uncoating, nuclear import, and target site recognition.
Several host factors affect the efficiency or position of retroelement integration. For retroviral integration, in vitro experiments showed enhancement of integration by Ini1 (22
), HMG1 (1
), and HMG I(Y) (14
) and reduction of autointegration by barrier to autointegration factor (29
). For yeast retrotransposons, in vivo experiments have identified chromatin-associated proteins that enhance the efficiency of integration into promoter regions of RNA polymerase II (pol II)-transcribed genes or heterochromatic DNA or affect the general efficiency of integration. For example, Ubc2 and CAF-I affect the integration preference of Ty1 (20
). Ty5 targets heterochromatic DNA through contacts mediated by Sir proteins (42
). Most genomic Ty1 and Ty2 elements are found within 750 bp of the 5′ end of tRNA genes (24
), and this targeting presumably involves host proteins.
Ty3 is distinguished from other retroelements by its extreme integration specificity. It integrates within a few nucleotides of the transcription initiation sites of pol III genes on plasmids (9
) and of chromosomal tRNA genes (24
). There is no sequence similarity among pol III transcription initiation sites, suggesting that the structure of the transcription initiation complex, rather than a consensus DNA sequence, is responsible for the specificity of integration (9
). The tRNA and U6 gene transcription preinitiation complexes are composed of transcription factors IIIC and IIIB (TFIIIC and TFIIIB). TFIIIC binds to promoter elements, box B and box A, and recruits the initiation factor TFIIIB, which binds upstream of the initiation site. In the case of the tRNA and U6 genes, TFIIIC is required for transcription in vivo, but in defined in vitro systems TFIIIB can mediate TATA box-dependent transcription in the absence of TFIIIC (39
). Similar to what is observed for transcription, only TFIIIB is required for integration upstream of the U6 gene in vitro (40
). However, in vitro, chromatographic fractions containing TFIIIB and TFIIIC are required for position-specific integration of Ty3 upstream of a tRNA gene (27
). As is the case for transcription in vivo, a point mutation in box B that abolishes TFIIIC binding abrogates the activity of a tRNA or U6 in Ty3-targeting assays (9
). Although the in vitro studies suggest that direct contacts must occur between TFIIIB and the preintegration complex (PIC), they did not address whether the in vivo role of TFIIIC in transposition is indirect, that is via loading TFIIIB, or direct, through interactions with the PIC.
In this study, we identified a mutation that caused truncation of the 95-kDa subunit of TFIIIC (TFIIIC95) and severely reduced the recovery of Ty3 integrants in an in vivo assay. The mutant strain had no detectable defect in growth or transcription of tRNAs. Although intermediates of the Ty3 life cycle were not altered, Ty3 elements integrated between a pair of divergent tRNA genes in the mutant strain showed orientation bias. Furthermore, interaction observed between Ty3 IN and TFIIIC95 was attenuated for the mutant protein, suggesting that TFIIIC95 participates directly in docking the PIC. These results provide the first genetic evidence directly linking the targeting of a Ty element to a specific component of the pol III transcription initiation complex. The orientation bias observed here offers insights into the asymmetry of a retroelement PIC.