Yeast precursor ribosomal RNA. Molecular cloning and probing the higher-order structure of the internal transcribed spacer I by kethoxal and dimethylsulfate modification

Plasmids were constructed in which a HindIII fragment of rDNA (6.4 × 10³ base-pairs) was inserted into vectors pGEM-1 and 2 in both orientations. The DNA insert encoded the yeast 35 S precursor rRNA beginning 180 bases upstream from the 5′ end of the mature 18 S rRNA and ending 289 bases beyond the 3′ end of the mature 25 S rRNA. The precursor rRNA molecules produced in vitro consisted of 6430 nucleotides, with about 15 residues derived from the Gemini vector on both ends. The general extent of secondary structure of the precursor rRNA was examined by ethidium fluorescence and compared to that of the mature rRNAs. Both precursor and mature rRNAs responded similarly to changes in magnesium ion concentration and to digestion by cobra venom and T₁ ribonucleases. The higher-order structure of the internal transcribed spacer-1 (ITS-1) region of the 35 S rRNA molecule was further examined by kethoxal and dimethylsulfate modifications and primer extension. Accessible adenine and guanine residues were located by primer extension analysis with avian myeloblastosis virus reverse transcriptase. On the basis of experimental data and computer-generated structures, a secondary structure model was proposed for the ITS-1 region. In this model, six hairpin stems involving adjacent nucleotides are present. A long-range interaction between nucleotides at the middle of the ITS-1 region and an, as yet, unidentified sequence located at another region of the precursor rRNA is suggested also. A candidate for this interacting sequence is that previously proposed, on a theoretical basis, to be involved in the removal of the precursor 18 S rRNA species for 35 S precursor molecule.