and complements transcription in a TFIIIA-depleted oocyte nuclear extract. Random, cassette-mediated mutagenesis of the carboxyl region 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 TFIIIA activation domain is strikingly sensitive to its position relative to the DNA-binding domain. When the eight amino acids that separate the transcription-activating domain from the last zinc finger are deleted, transcriptional activity is lost. Surprisingly, diverse amino acids can replace these eight amino acids with restoration of full transcriptional activity, suggesting that the length and not the sequence of this region is important. Insertion of amino acids between the zinc finger region and the transcription-activating domain causes a reduction in transcription proportional to the number of amino acids introduced. We propose that to function, the transcription-activating domain of TFIIIA must be correctly positioned at a minimum distance from the DNA-binding domain.Initiation of transcription by eukaryotic RNA polymerases requires the formation of specific protein complexes at gene promoter elements. RNA polymerase III transcribes a number of small genes, such as 5S RNA and tRNA genes, that have promoters within the transcription unit (reviewed in reference 20). Many of the transcription factors required for RNA polymerase III transcription have been identified, and their order of assembly on 5S RNA genes has been clearly established, particularly for Xenopus laevis and Saccharomyces cerevisiae, yet little is known of the actual mechanism of transcriptional activation (3,31,43). Although there are undoubtedly species-specific differences, some generalizations can be made regarding the assembly and properties of transcription complexes formed on 5S RNA genes. Assembly of a transcription complex on the 5S RNA gene is initiated by sequence-specific binding of the zinc finger protein TFIIIA to the internal control region (ICR) (15, 21, 34). Components of TFIIIC bind next and stabilize the binding of TFIIIA to the gene, committing the template for transcription. TFIIIC requires specific DNA base pairs within A-and C-box elements of the ICR (33). However, it does not bind to the ICR of 5S RNA genes in the absence of TFIIIA (7, 44). In contrast, independent sequence-specific DNA binding by factors in the TFIIIC fraction is observed at A-and B-box elements of tRNA genes (6,16,45). Assembly of the preinitiation complex is completed by TFIIIB. One component of TFIIIB is the TATA box-binding protein, which is required for transcription by RNA polymerases I, II, and III (9,29,32,46,49). TFIIIB specifically interacts with an upstream DNA-binding site only after assembly I into the transcription complex (2, 27, 30). The activation mechanism for DNA binding by TFIIIB is unknown but presumably results from protein-protein interactions. The * Corresponding author. completed compl...
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