RESUMO
The 39S ribosomal RNA (rRNA) precursor has been isolated from Acanthamoeba castellanii. In vitro capping of the isolated RNA verified that it is the primary transcript and identified the 5' nucleotide as pppA. The position of the 5' coding nucleotide on the rRNA repeat unit sequence was identified using Northern blot, R-loop, and S1 nuclease mapping techniques. Dinucleotide priming of an in vitro transcription system stalled because of low initiating nucleotide concentration revealed that ApA maximally stimulates initiation of transcription. All of these results show that the underlined A in the sequence 5'-TATATATAAAGGGAC (RNA-like strand) coincides with the 5' nucleotide of the primary transcript. This identification is compatible with in vitro transcription experiments mapping the promoter for this transcription unit. The initiation sequences of rRNA genes from 14 species are compared, and a weak consensus for the initiator derived: [Formula; see text].
