DIRECTING TRANSCRIPTION OF AN RNA-POLYMERASE-III GENE VIA GAL4 SITES

A yeast chimeric RNA polymerase III transcription system was construct ed to explore the ordered, multistep process of gene activation in viv o. A promoter deficient U6 RNA gene harboring GAL4-binding sites could be reactivated by fusing the GAL4 DNA-binding domain to components of the general transcription factor TFIIIC (tau) or TFIIIB. Expression o f chimeric tau 138 or tau 131 (but not tau 95) subunits activated tran scription from GAL4-binding sites located at various positions, includ ing upstream of or within the gene. The function(s) of the B block bin ding domain of TFIIIC was provided by the fused GAL4-(1-147) domain. T he GAL4-(1-147)-TFIIIB70 fusion protein acted at a distance like an ac tivator of transcription. In contrast, none of the 10 different GAL4-( 1-147)-polymerase subunit fusions was able to induce transcription, su ggesting that RNA polymerase recruitment is not sufficient to initiate transcription.