Open AccessDetermination of the transcription initiation site ofTetrahymena pyriformisrDNA usingin vitrocapping of 35S pre-rRNA
Author(s) -
Hidetoshi Saiga,
Kiyohisa Mizumoto,
Takashi Mizutani,
Toru Higashinakagawa
Publication year - 1982
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/10.14.4223
Subject(s) - biology , tetrahymena pyriformis , tetrahymena , ribosomal rna , in vitro , protozoa , transcription (linguistics) , microbiology and biotechnology , genetics , gene , linguistics , philosophy
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.