TFIIIC-independent in vitro transcription of yeast tRNA genes

The most peculiar transcriptional property of eukaryotic tRNA genes, as wel l as of other genes sen ed by RNA polymerase III, is their complete depende nce on the intragenic interaction platform provided by transcription factor IIIC (TFIIIC) for the productive assembly of the TBP-containing initiation factor TFIIIB. The sole exception, in yeast, is the U6 RNA gene, which is able to exploit a TATAAATA element, 30 bp upstream of the transcription sta rt site, for the TFIIIC-independent assembly of TFIIIB. To find out whether this extragenic core promoter organization and autonomous TFIIIB assembly capacity are unique features of the U6 gene or also apply to other genes tr anscribed by RNA polymerase III, we scanned the 5'-flanking; regions (up to position -100) of the entire tRNA gene set of Saccharomyces cerevisae sear ching for U6-like TATA motifs. Four tRNA genes harboring such a sequence mo tif around position -30 were identified and found to be transcribed in vitr o by a minimal system only composed of TFIIIB and RNA polymerase III. Ln th is system, start site selection is not at all affected by the absence of TF IIIC, which, when added, significantly stimulates transcription by determin ing an increase in the number, rather than in the efficiency of utilization , of productive initiation complexes. A specific TBP-TATA element interacti on is absolutely required for TFIIIC-independent transcription, but the nea rby sequence context also contributes to the efficiency of autonomous TFIII B assembly. The existence of a TFIIIB assembly pathway leading to the faith ful transcription of natural eukaryotic tRNA genes in the absence of TFIIIC provides novel insights into the functional flexibility of the eukaryotic tRNA gene transcription machinery and on its evolution from an ancestral RN A polymerase III system relying on upstream, TATA-centered control elements . (C) 2000 Academic Press.