Abstract MOT1 encodes an essential ATPase that functions as a general transcriptional regulator in vivo by modulating TATA-binding protein (TBP) DNA-binding activity. Although MOT1 was originally identified both biochemically and in several genetic screens as a transcriptional repressor, a combination of subsequent genetic, chromatin immunoprecipitation, and microarray analysis suggested that MOT1 might also have an additional role in vivo as a transcriptional activator. To better understand the role(s) of MOT1 in vivo, we selected for genomic suppressors of a mot1 temperature-sensitive mutation. This selection identified mutations in SPT15 (TBP) and BUR6, both of which are clearly linked with MOT1 at the functional level. The vast majority of the suppressor mutations, however, unexpectedly occurred in six genes that encode known components of the SUMO pathway and in two other genes with unknown functions, SLX5 and SLX8. Additional results presented here, including extensive synthetic lethality observed between slx5Δ and slx8Δ and SUMO pathway mutations, suggest that SLX5 and SLX8 are new components or regulators of the SUMO pathway and that SUMO modification might have a general role in transcriptional regulation as part of the TBP regulatory network.