An in vitro RNA polymerase III system from S. cerevisiae: effects of deletions and point mutations upon SUP4 gene transcription

Abstract

A soluble cell-free extract containing RNA polymerase III and factors essential for selective transcription of the yeast SUP4-o tRNATyr gene was prepared from Saccharomyces cerevisiae cells. An intragenic promoter for yeast RNA polymerase III was identified within the yeast tRNATyr coding sequence by testing several sup4 genes with 5'- and 3'-terminal deletions in the homologous transcription system. Thirty-four different sup4 genes with spontaneous mutations were also tested in the in vitro system. Two point mutations drastically reduced transcription initiation and two other mutations caused premature termination. These mutations have nearly identical effects on SUP4 gene transcription by Xenopus RNA polymerase III (1), which demonstrates that the essential features of RNA polymerase III transcription initiation and termination signals have been conserved throughout the course of eukaryotic evolution.