Abstract: Sub1 is implicated in transcriptional activation, elongation, and mRNA 3'-end formation in budding yeast. To gain more insight into its function, we performed a synthetic genetic array screen with SUB1 that uncovered genetic interactions with genes involved in the HOG osmoresponse pathway. We find that Sub1 and the HOG pathway are redundant for survival in moderate osmolarity. ChIP analysis shows that Sub1 is recruited to osmoresponse gene promoters during osmotic shock, and is required for full recruitment of TBP, TFIIB, and RNA polymerase (RNAP) II at a subset of these genes. Furthermore, we detect Sub1 at the promoter of every constitutively-transcribed RNAP II and, unexpectedly, at every RNAP III gene tested, but not at the RNAP I-transcribed rDNA promoter. Significantly, deletion of SUB1 reduced levels of promoter-associated RNAP II or III at these genes, but not TBP. Together these data suggest that in addition to a general role in polymerase recruitment at constitutive RNAP II and RNAP III genes, during osmotic shock, Sub1 facilitates osmoresponse gene transcription by enhancing pre-initiation complex formation.