A SINGLE-STRANDED-DNA BINDING-PROTEIN THAT SPECIFICALLY RECOGNIZES CIS-ACTING SEQUENCES IN THE REPLICATION ORIGIN AND TRANSCRIPTIONAL PROMOTER REGION OF TETRAHYMENA RDNA

Type I repeat sequences are evolutionarily conserved sequence elements found in the replication origin and transcriptional promoter region o f the rRNA genes (rDNA) in Tetrahymena thermophila. An abundant single -stranded DNA binding protein, ssA-TIBF, specifically interacts with t he A-rich strand of the Type I repeat sequence. Quantitative binding c ompetition experiments performed with purified ssA-TIBF demonstrate th at the binding site for ssA-TIBF includes sequences both within the co nserved 33 nt element and in a 3' flanking region: addition of the 3' flanking sequence to the Type I repeat oligonucleotide increases the b inding affinity of ssA-TIBF by nearly 100-fold (apparent K-d = 3.0 x 1 0(-10) M). A mutation in the ssA-TIBF binding site previously shown to be the determinant of an rDNA replication defect in vivo results in a 25-fold decrease in ssA-TIBF binding affinity in vitro. ssA-TIBF also binds with high affinity to a copy of the Type I repeat sequence with in the essential promoter region defined by in vitro transcription ass ays. The affinity of ssA-TIBF for the promoter repeat, which differs f rom other copies of the repeat at 8 out of 33 positions, is at least e qual to its affinity for the Type I repeat sequences in the origin reg ion. The biochemical properties of ssA-TIBF in vitro suggest that it c ould play a role in both replication and transcription of Tetrahymena rDNA in vivo.