A truncation mutant of the 95-kilodalton subunit of transcription factor IIIC reveals asymmetry in Ty3 integration

Position-specific integration of the retroviruslike element Ty3 near the tr anscription initiation sites of tRNA genes requires transcription factors I IIB and IIIC (TFIIIB and TFIIIC). Using a genetic screen, we isolated a mut ant with a truncated 95-kDa subunit of TFIIIC (TFIIIC95) that reduced the a pparent 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 th e mutant. Steps of the Ty3 life cycle, such as protein expression, proteoly tic processing, viruslike particle formation, and reverse transcription, we re not affected by the mutation. However, Ty3 integration into a divergent tDNA target occurred exclusively in one orientation in the mutant strain. I nvestigation of this orientation bias showed that TFIIIC95 and Ty3 integras e interacted in two-hybrid and glutathione S-transferase pulldown assays an d that interaction with the mutant TFIIIC95 protein was attenuated. The ori entation bias observed here suggests that even for wild-type Ty3, the prote in complexes associated with the long terminal repeats are not equivalent i n vivo.