Mutational analysis of the transcription factor IIIB-DNA target of Ty3 retroelement integration.

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.