REPLICATION OF AN RIBOSOMAL-RNA GENE ORIGIN PLASMID IN THE TETRAHYMENA-THERMOPHILA MACRONUCLEUS IS PREVENTED BY TRANSCRIPTION THROUGH THE ORIGIN FROM AN RNA-POLYMERASE-I PROMOTER

In the somatic macronucleus of the ciliate Tetrahymena thermophila, th e palindromic rRNA gene (rDNA) minichromosome is replicated from an or igin near the center of the molecule in the 5' nontranscribed spacer, The replication of this rDNA minichromosome is under both cell cycle a nd copy number control. We addressed the effect on origin function of transcription through this origin region, A construct containing a pai r of 1.9-kb tandem direct repeats of the rDNA origin region, containin g the origin plus a mutated (+G), but not a wild type, rRNA promoter, is initially maintained in macronuclei as an episome, Later, linear an d circular replicons with long arrays of tandem repeats accumulate (W. -J. Pan and E. H. Blackburn, Nucleic Acids Res, in press), We present direct evidence that the +G mutation inactivates this rRNA promoter, I t lacks the footprint seen on the wild-type promoter and produces no d etectable in vivo transcript, Independent evidence that the failure to maintain wild-type 1.9-kb repeats was caused by transcription through the origin came from placing a short DNA segment containing the rRNA gene transcriptional termination region immediately downstream of the wild-type rRNA promoter, Insertion of this terminator sequence in the correct, but not the inverted, orientation restored plasmid maintenanc e, Hence, origin function was restored by inactivating the rRNA promot er through the +G mutation or causing termination before transcripts f rom a wild-type promoter reached the origin region, We propose that tr anscription by RNA polymerase I through the rDNA origin inhibits repli cation by preventing replication factors from assembling at the origin .