FIG.4.

TFIIIA with serine-16 replaced with glutamic acid cannot restore transcription of oocyte 5S rRNA genes in immunodepleted GV extract. Each transcription assay mixture includes 5 μl of extract and 100 ng of DNA (containing a 4:1 mixture of oocyte-type to somatic-type maxigene). After preincubation for 15 min, transcription was initiated by the addition of ribonucleoside triphosphates, including [α-³²P]CTP. Reactions were stopped after 90 min, and radiolabeled tRNA was added in order to normalize the recovery of RNA from each sample, which were analyzed on a polyacrylamide gel containing 7 M urea. Lane 1 is an assay with GV extract prior to immunodepletion of endogenous TFIIIA, and lane 2 is depleted extract to which DNA template, but no TFIIIA, was added. (A) Lanes 3 to 16 contain pairs of assays containing 20 or 100 ng of the indicated variant of TFIIIA. (B) Lanes 3 to 7, 8 to 12, and 13 to 17 contain 5, 20, 40, 100, or 300 ng of the indicated variant of TFIIIA. S and O indicate the positions of transcripts from the somatic- and oocyte-type genes, respectively. (C) The autoradiograph in panel B was scanned with a laser densitometer. The S/O transcript ratios were normalized relative to the exogenous tRNA added to each assay and are presented as bar graphs. WT, wild type.