A DIFFERENTIAL RESPONSE OF WILD-TYPE AND MUTANT PROMOTERS TO TFIIIB70OVEREXPRESSION IN-VIVO AND IN-VITRO

TFIIIB, the initiation factor for transcription by RNA polymerase III (pol III) is, in yeast, composed of three subunits: TBP, TFIIIB70/Brf1 and TFIIIB90. To determine the extent to which each of these subunits is limiting for pol III transcription, the effect of overexpressing e ach subunit was assessed on the expression of wild-type and promoter m utant pol III genes both in vivo and in vitro. In vivo, we find that t he synthesis of wild-type pol III genes is not limited to a significan t extent by the level of any TFIIIB subunit, There is, however, a two- fold increase in the synthesis of the promoter mutant gene, sup9-e A19 -supS1, in strains overexpressing TFIIIB70. The findings suggest that overexpression of TFIIIB70 has a differential effect on the expression of pol III genes with strong versus weak promoters. In vitro transcri ption assays support this conclusion and reveal an inverse correlation between the transcriptional response to TFIIIB70 overexpression and p romoter strength. The individual TFIIIB subunits are nuclear by immuno fluorescence and are calculated to have nuclear concentrations in the low micromolar range. In comparison, the factors are diluted 100-fold or more in whole cell extracts. This dilution accounts for the general ly limiting nature of IFIIIB70 in pol III gene transcription in vitro.