Conserved cis- and trans-acting determinants for replication initiation and regulation of replication fork movement in tetrahymenid species

Abstract

The rDNA minichromosomes of Tetrahymena thermophila and Tetrahymena pyriformis share a high degree of sequence similarity and structural organization. The T. thermophila 5' non-transcribed spacer (5' NTS) is sufficient for replication and contains three repeated sequence elements that are conserved in T. pyriformis, including type I elements, the only known determinant for replication control. To assess the role of conserved sequences in replication control, structural and functional studies were performed on T. pyriformis rDNA. Similar to T. thermophila, replication initiates exclusively in the 5' NTS, localizing to a 900 bp segment. Elongating replication forks arrest transiently at one site which bears strong similarity to a tripartite sequence element present at fork arrest sites in T. thermophila rDNA. An in vitro type I element binding activity indistinguishable from the T. thermophila protein, ssA-TIBF, was detected in T. pyriformis extracts. The respective TIBF proteins bind with comparable affinity to type I elements from both species, suggesting that in vivo recognition could cross species boundaries. Despite these similarities, the T. pyriformis 5' NTS failed to support replication in transformed T. thermophila cells, suggesting a more complex genetic organization than previously realized.