Two DNA-binding factors from Saccharomyces cerevisiae have been characterized, GRFI (general regulatory factor I) and ABFI (ARS-binding factor I), that recognize specific sequences within diverse genetic elements.GRFI bound to sequences at the negative regulatory elements (silencers) of the silent mating type loci HML E and HMR E and to the upstream activating sequence (UAS) required for transcription of the MAT a genes. A putative conserved UAS located at genes involved in translation (RPG box) was also recognized by GRFI. In addition, GRFI bound with high affinity to sequences within the (Cl_3A)-repeat region at yeast telomeres.Binding sites for GRFI with the highest affinity appeared to be of the form 5'-(A/G)(A/C)ACCCAN NCA(T/C)(T/C)-3', where N is any nucleotide. ABFI-binding sites were located next to autonomously replicating sequences (ARSs) at controlling elements of the silent mating type loci HMR E, HMR 1, and HML I and were associated with ARS1, ARS2, and the 2,um plasmid ARS. Two tandem ABFI binding sites were found between the HIS3 and DEDI genes, several kilobase pairs from any ARS, indicating that ABFI-binding sites are not restricted to ARSs. The sequences recognized by ABFI showed partial dyad-symmetry and appeared to be variations of the consensus 5'-TATCATTNNNNACGA-3'. GRFI and ABFI were both abundant DNA-binding factors and did not appear to be encoded by the SIR genes, whose products are required for repression of the silent mating type loci. Together, these results indicate that both GRFI and ABFI play multiple roles within the cell.Eucaryotic chromosomes appear to be organized in domains governing such processes as gene expression, DNA replication, and chromosome condensation. In an effort to define the biochemical components directing these events, we have been attracted to the features of the mating type loci of Saccharomyces cerevisiae (reviewed in reference 33). Haploid yeast cells of the a or a mating type express different genes at MAT, in the middle of chromosome III (Fig. 1). The alternate forms of this locus, MATa and MA To, encode distinct regulatory factors al and a2 or al and oa2, but have extensive regions of homology, referred to as W, X, and Z (2, 61). There are, in addition, silent copies of genes specifying each mating type stored on the left and right arms of chromosome III at HMLa and HMRa, respectively. In homothallic strains, the mating type of successive generations switches in a tightly regulated manner (29,35,80). Switching is accomplished by gene conversion whereby sequences at MAT are replaced by those at HML or HMR (33,47). This process is initiated with the cleavage of MAT DNA in Z by a specific endonuclease that is the product of the HO gene (49, 50). The silent copies of the mating type loci are controlled by negative regulatory sequences located over 1,000 base pairs (bp) away from the promoters for these genes. Deletion analysis of HML and HMR has revealed cis-acting regulatory elements, E and I, that flank each locus on the left and right sides, respectivel...