Abstract
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.
Highlights
Ty3 is a gypsy-like retroelement in Saccharomyces cerevisiae [1]
The Amino-terminal Half of Brf1 Contains Primary Determinants of Ty3 Integration into the SNR6 Gene—BЈ, comprised of the TFIIIB subunits Brf1 and TBP, was previously shown to be sufficient to mediate a low level of specific Ty3 integration
In the case of pol III transcription on the SNR6 template pU6LboxB, it has been shown that the amino-terminal half of Brf1(1–282) supports transcription in the context of TFIIIB on supercoiled but not linear templates [37]
Summary
Ty3 is a gypsy-like retroelement in Saccharomyces cerevisiae [1]. Despite similarities between the proteins encoded by Ty3 and other gypsy-like elements and retroviruses, Ty3 has the. Mutations in the box A and box B promoter elements of the tRNA and U6 RNA genes that interfere with transcription diminish transposition in vivo, suggesting that active targets in vivo must be able to bind pol III transcription factors [2]. The box A and box B promoter elements of the tRNA and SNR6 genes serve to bind transcription factor (TF) IIIC through sequence-specific interactions with two of the six TFIIIC subunits. The positions of the TFIIIC and TFIIIB subunits in promoter complexes have been mapped downstream and upstream, respectively, of the initiation site by cross-linking analysis [12, 13]. The in vitro requirements for Ty3 integration into tRNA genes have been probed using TFIIIC and TFIIIB. SNR6 can be transcribed by pol III, independently of TFIIIC [11, 20]
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