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 of the rRNA genes (rDNA) in Tetrahymena thermophila. An abundant single-stranded DNA binding protein, ssA-TIBF, specifically interacts with the A-rich strand of the Type I repeat sequence. Quantitative binding competition experiments performed with purified ssA-TIBF demonstrate that the binding site for ssA-TIBF includes sequences both within the conserved 33 nt element and in a 3' flanking region: addition of the 3' flanking sequence to the Type I repeat oligonucleotide increases the binding affinity of ssA-TIBF by nearly 100-fold (apparent Kd = 3.0 x 10(-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 within the essential promoter region defined by in vitro transcription assays. The affinity of ssA-TIBF for the promoter repeat, which differs from other copies of the repeat at 8 out of 33 positions, is at least equal to its affinity for the Type I repeat sequences in the origin region. The biochemical properties of ssA-TIBF in vitro suggest that it could play a role in both replication and transcription of Tetrahymena rDNA in vivo.
Hou
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Department of Zoology and Genetics, Iowa State University, Ames 50011-3213, USA.
Umthun
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AR
Dobbs
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D000818:Animals; D001483:Base Sequence; D004261:DNA Replication; D016054:DNA, Protozoan / Q000235:genetics / Q000378:metabolism*; D004275:DNA, Ribosomal / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012335:RNA, Ribosomal / Q000235:genetics; D018741:Replication Origin; D016808:Tetrahymena thermophila / Q000235:genetics*; D014158:Transcription, Genetic
D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D012335:RNA, Ribosomal