STIMULATION OF TRANSCRIPTION BY MUTATIONS AFFECTING CONSERVED REGIONSOF RNA-POLYMERASE-II

Mutations that increase the low-level transcription of the Saccharomyc es cerevisiae HIS4 gene, which results from deletion of the genes enco ding transcription factors BAS1, BAS2, and GCN4, were isolated previou sly in SIT1 (also known as RPO22, RPB1, and SUA8), the gene encoding t he largest subunit of RNA polymerase II (RNAPII). Here we show that si t1 substitutions cluster in two conserved regions of the enzyme which form part of the active site. Six sit1 mutations, affect region F, a r egion that is involved in transcriptional elongation and in resistance to alpha-aminatin. Four sit1 substitutions lie in another region invo lved in transcriptional elongation, region D, which binds Mg2+ ions es sential for RNA catalysis. One region D substitution is lethal unless suppressed by a substitution in region G and interacts genetically wit h PPR2, the gene encoding transcription elongation factor LIS. Some si t1 substitutions affect the selection of transcriptional start sites a t the CYC1 promoter in a manner reminiscent of that of sua8 (srm stand s for suppression of upstream ATG) mutations. Together, with previous findings which indicate that regions D and G are in close proximity to the 3' end of the nascent transcript and that region F is involved in the translocation process, our results suggest that transcriptional a ctivation by the sit1 mutations results from alteration of the RNAPII active center.