TRANSCRIPTION OF ALPHA-SPECIFIC GENES IN SACCHAROMYCES-CEREVISIAE - DNA-SEQUENCE REQUIREMENTS FOR ACTIVITY OF THE COREGULATOR ALPHA-1

Transcription activation of alpha-specific genes in saccharomyces cere visiae is regulated by two proteins, MCM1 and alpha1, which bind to DN A sequences, called P'Q elements, found upstream of alpha-specific gen es. Neither MCM1 nor alpha1 alone binds efficiently to P'Q elements. T ogether, however, they bind cooperatively in a manner that requires bo th the P' sequence, which is a weak binding site for MCM1, and the Q s equence, which has been postulated to be the binding site for alpha1. 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 alp ha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription act ivation 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 d irect correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha1 complexes and the degree to which transcriptio n was activated in vivo. Thus, the ability of alpha1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-spec ific genes. In all natural alpha-specific genes the Q sequence is adja cent to the degenerate side of P'. To test the significance of this ge ometry, we created several novel juxtapositions of P, P', and Q sequen ces. When the Q sequence was opposite the degenerate side, the composi te QP' element was inactive as a promoter element in vivo and unable t o form stable ternary QP'-MCM1-alpha1 complexes in vitro. We also foun d that addition of a Q sequence to a strong MCM1 binding site allows t he addition of alpha.1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex f ormation but not the weak binding of MCM1 alone, supports the idea tha t the Q sequence serves as a binding site for alpha1.