Transcription of α-Specific Genes in Saccharomyces cerevisiae: DNA Sequence Requirements for Activity of the Coregulator αl

Abstract

Transcription activation of α-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and α1, which bind to DNA sequences, called P'Q elements, found upstream of α-specific genes. Neither MCM1 nor α1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for α1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for α-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-α1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of α1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of α-specific genes. In all natural α-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-α1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of α1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for α1.