FIG. 6.

Template exclusion assays demonstrate that the S16E mutant form of TFIIIA supports the formation of an inactive complex on oocyte 5S rRNA genes. GV extract was immunodepleted of endogenous TFIIIA and then supplemented with wild-type (WT) or mutant TFIIIA. (A) A somatic 5S rRNA maxigene was challenged with increasing amounts of oocyte-type gene. The two DNA templates were added together at the beginning of the assay (Sim), or the oocyte-type gene was added to the extract first, followed after 20 min by the somatic-type gene (Seq). Transcription was initiated by the addition of ribonucleoside triphosphates (rNTPs). Transcripts were analyzed on a denaturing polyacrylamide gel. The positions of the somatic (S) and oocyte (O) transcripts are denoted by brackets. Lanes marked GV indicate assays in extract prior to immunodepletion, and GV(ID) indicates assays in depleted extract that was not supplemented with exogenous TFIIIA. (B) A tRNA gene was challenged with increasing amounts of oocyte-type gene to determine whether the S16E mutant form of TFIIIA recruits other pol III transcription factors. The two DNA templates were added together to extract, and after a 40-min incubation, the transcription assays were initiated by the addition of ribonucleoside triphosphates. Brackets denote the position of 5S rRNA and tRNA transcripts.