A Truncation Mutant of the 95-Kilodalton Subunit of Transcription Factor IIIC Reveals Asymmetry in Ty3 Integration

Abstract

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 glutathioneS-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.

ACKNOWLEDGMENTS

We thank M. Snyder for providing the mTn3::lacZ/LEU2 library and C. Friddle for providing the vectorette PCR protocol. We also thank E. Chen for assistance with the mutant screen, M. H. Nymark-McMahon and L. Yieh for assistance with VLP and BR500 preparations, J. Steffan for assistance with GST pulldown assays, T. Menees for helpful discussions, and A. Sentenac for critical reading of the manuscript.

This work was supported by Public Health Service grant GM33281 to S.B.S. and by the Synthesis and Structure of Biological Macromolecules training grant GM07311-24 (M.A.).