IDENTIFICATION OF THE CIS-ACTING DNA-SEQUENCE ELEMENTS REGULATING THETRANSCRIPTION OF THE SACCHAROMYCES-CEREVISIAE GENE ENCODING TBP, THE TATA BOX-BINDING PROTEIN

TBP, the TATA-box binding protein, plays a key role in eukaryotic gene transcription since it is required for transcription initiation by al l three eukaryotic nuclear DNA-dependent RNA polymerases. In order to gain insight into the mechanisms of regulation of this key basal trans cription factor, we undertook a mutational analysis of the sequences i nvolved in directing transcription of the gene encoding TBP in Sacchar omyces cerevisiae. An extensive family of mutations in the promoter of the gene encoding TBP were fused to the Escherichia coil reporter gen e lacZ, transferred back into yeast, and assayed for their ability to direct expression of beta-galactosidase. Levels of beta-galactosidase activity measured from yeast transformed with this family of construct s indicate that both positive- and negative-acting cis-elements locate d within 400 nucleotides of the transcription start site are involved in regulating transcription of the TBP-encoding gene. Analyses of RNA prepared from these same cells showed that specific transcription init iation is maintained in the mutant reporter constructs and that RNA le vels mirror beta-galactosidase levels. In order to corroborate the res ults of these mutational analyses of the TBP-encoding gene, in vivo ci s-element occupancy was examined using several different footprinting reagents. The patterns of protection observed demonstrated that the se quence elements implicated in the control of TBP gene transcription by reporter gene analyses appear to be bound by protein(s) in vivo. Inte resting sequence similarities were noted between two TBP-gene regulato ry elements and 5'-flanking sequences of genes encoding several other basal transcription factors.