Determination of the transcription initiation site of Tetrahymena pyriformis rDNA using in vitro capping of 35S pre-rRNA.

Approximately 700 nucleotide sequences surrounding the transcription initiation site were determined with a cloned rDNA fragment of Tetrahymena pyriformis and the transcription initiation site was localized on these sequences using purified 35S pre-rRNA. A considerable portion of the 35S pre-rRNA was found to be capped in vitro. The 32P-labeled, capped 35S pre-rRNA, on nucleus P1 protection mapping, gave the protection band which is identical in size with that obtained with bulk 35S pre-rRNA. Both reverse transcription extension and nuclease P1 mapping localized the 5'-end of the 35S pre-rRNA at the same adenine nucleotide, 496 base pairs upstream from the HindIII site of the cloned rDNA fragment. Furthermore, sequencing of the 5'-terminal region of the in vitro capped 35S pre-rRNA unambiguously confirmed the above result. The strategy adopted in the present experiment could serve as a general procedure for determining the transcription initiation point even in cases where the concentration of the primary transcript is low.