Identification of DNA-binding proteins that recognize a Conserved Type I repeat sequence in the replication origin region of Tetrahymena rDNA

Abstract

An origin of DNA replication has been mapped within the 5' non-transcribed spacer region of the amplified macronuclear rRNA genes (rDNA) of Tetrahymena thermophila. Mutations in 33 nt conserved AT-rich Type I repeat sequences located in the origin region cause defects in the replication and/or maintenance of amplified rDNA in vivo. Fe(II)EDTA cleavage footprinting of restriction fragments containing the Type I repeat showed that most of the conserved nucleotides were protected by proteins in extracts of Tetrahymena cells. Two classes of proteins that bound the Type I repeat were identified and characterized using synthetic oligonucleotides in electrophoretic mobility shift assays. One of these, ds-TIBF, bound preferentially to duplex DNA and exhibited only moderate specificity for Type I repeat sequences. In contrast, a single-stranded DNA-binding protein, ssA-TIBF, specifically recognized the A-rich strand of the Type I repeat sequence. Deletion of the 5' or 3' borders of the conserved sequence significantly reduced binding of ssA-TIBF. The binding properties of ssA-TIBF, coupled with genetic evidence that Type I sequences function as cis-acting rDNA replication control elements in vivo, suggest a possible role for ssA-TIBF in rDNA replication in Tetrahymena.

A single-stranded DNA binding protein that specifically recognizes cis-acting sequences in the replication origin and transcriptional promoter region of Tetrahymena rDNA

“…Nevertheless, the affinity of ssA-TIBF for the promoter Type Id repeat equals or exceeds its affinity for the extended Type lb repeat. Accurate initiation of rRNA transcription in both T. pyriformis (Miyahara et al, 1993) (Umthun et al, 1994), this study demonstrates that additional sequences which make a major contribution to ssA-TIBF binding affinity are located outside the conserved element. It should be noted, however, that although ssA-TIBF can distinguish between the C3-and B-rDNA alleles in vitro, several induced C3-rmm mutations are due to a single base pair deletion in the central tract of 11 A residues in copies of the Type 1 repeat (Larson et al, 1986;Yaeger et al, 1989 (Hofmann & Gasser, 1991;Kuno et al, 1990Kuno et al, .…”

“…An apparent K,i was calculated from the resultant linear double-reciprocal plot ( Figure 4E) (Freifclder, 1982). (Umthun et al, 1994). To more rigorously examine the binding characteristics of ssA-TIBF.…”

“…Type I repeat sequences are evolutionarily conserved sequence elements found within the 5' nontranscribed spacer region (5'NTS) of the rDNA, both in the replication origin region mapped by electron microscopy (Cech & Brehm. 1981) and in the transcriptional promoter region (Challoner et al, 1985;Niles et al, 1981 (Umthun et al, 1994 Figure 1C). …”

“…In the ciliated protozoan Tetrahymena thermophila, both duplex and single-stranded DNA binding proteins that interact with Type I repeat sequences upstream of the rRNA genes (rDNA) have been identified (Umthun et al, 1994 (Cech & Brehm, 1981;Cech, 1986). Domains 1 and 2 are nuclease-hypersensitive regions that lie within añ 420 bp imperfect duplication (Palen & Cech, 1984).…”

A Promoter Region Mutation Affecting Replication of the Tetrahymena Ribosomal DNA Minichromosome

“…1B) (7). Factors which bind the 33-nucleotide (nt) type I elements in vitro have been identified (17,46), although their function is unknown.…”