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

Approximately 700 nucleotide sequences surrounding the transcription initiation site were determined with a cloned rDNA fragment of Tetrahymena pyTiformis and the transcription initiation site was localized on these sequences using purified 35S pre-rRNA. A considerable portion of the 35S prerRNA was found to be capped in vitro. The P-labeled, capped 35S pre-rRNA, on nuclease PI 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 PI mapping localised the 5'-end of the 35S pre-rRNA at the same adenine nucleotide, 496 base pairs upstream from the Hindlll 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.