| Ty3 is gypsy-like retrotransposon in Saccharomyces cerevisiae. It has many similarities to retroviruses in genomic structure and lifecycle except no extracellular phase. Ty3 integrates specifically within 1 or 2 nucleotides (nt) of the transcription start sites (TSS) of RNA polymerase III-transcribed genes. The Pol III transcription machinery comprises three transcription factors: TFIIIA, TFIIIB, and TFIIIC. The three-subunit TFIIIB: Bdp1, Brf1 and TBP, or the fusion of Brf1n (1-382 aa)-TBPc (60-241 aa)-Brf1c (439-596 aa) is sufficient, in vitro, for Ty3 targeting the U6, snRNA, SNR6. Brf1 is Pol III-specific while TBP is not and binds upstream of genes that are not targeted by Ty3. This suggests a role for Brf1 in recruiting Ty3 to the target sites.;The objective of this dissertation was to study what required and influenced Ty3 specific and preferential targeting as related to Brf1 and chromosomal features. In vitro, a series of GST-IN subfragments and TBPc-Brf1 subdomains were constructed to map in vitro Ty3 IN targeting domain (TD), IN-IN interacting domain and IN-interacting domain within Brf1 by GST pull-down assay. The interaction between Ty3 IN-CTD and the Pol III-binding region of the Brf1-CTD is required for Ty3 specific targeting. In vivo, using Illumina high throughput sequencing (HTS) technology analyzed and identified the integration sites of approximately 20,000 de novo Ty3 integrations and a small subset of highly or rarely used targets. The distribution of mapped insertions and associated motifs identified by bioinformatics agree with observations of site selection specific to known targets. Differences of local features such as Pol III promoter and enrichment of transcription factors at the upstream of TSS are not likely the sole explaination of differential Ty3 targeting. We propose rather that Ty3 targeting preference is influenced by a combination of many factors and chromosomal features including: Pol III promoter elements specifically boxA and the upstream region of TSS, which are subsequently influenced by the enrichment of Pol III subunits and transcription factors; the competition between Pol III and Ty3 IN for accessing the transcription unit; density of Pol III genes, LTRs, and ARS; however, no influence by GC content, condensin or nucleosome spacing. |
