A POSITION-DEPENDENT TRANSCRIPTION-ACTIVATING DOMAIN IN TFIIIA

Transcription of the Xenopus 5S RNA gene by RNA polymerase III require s the gene-specific factor TFIIIA. To identify domains within TFIIIA t hat are essential for transcriptional activation, we have expressed C- terminal deletion, substitution, and insertion mutants of TFIIIA in ba cteria as fusions with maltose-binding protein (MBP). The MBP-TFIIIA f usion protein specifically binds to the 5S RNA gene internal control r egion and complements transcription in a TFIIIA-depleted oocyte nuclea r extract. Random, cassette-mediated mutagenesis of the carboxyl regio n of TFIIIA, which is not required for promoter binding, has defined a 14-amino-acid region that is critical for transcriptional activation. In contrast to activators of RNA polymerase II, the activity of the T FIIIA activation domain is strikingly sensitive to its position relati ve to the DNA-binding domain. When the eight amino acids that separate the transcription-activating domain from the last zinc finger are del eted, transcriptional activity is lost. Surprisingly, diverse amino ac ids can replace these eight amino acids with restoration of full trans criptional activity, suggesting that the length and not the sequence o f this region is important. Insertion of amino acids between the zinc finger region and the transcription-activating domain causes a reducti on in transcription proportional to the number of amino acids introduc ed. We propose that to function, the transcription-activating domain o f TFIIIA must be correctly positioned at a minimum distance from the D NA-binding domain.