Functional analysis of internal transcribed spacer 2 of Saccharomyces cerevisiae ribosomal DNA

Using the previously described “tagged ribosome” (pORCS) system for in vivo mutational analysis of yeast rDNA, we show that small deletions in the 5′-terminal portion of ITS2 completely block maturation of 26 S rRNA at the level of the 29 S b precursor (5.8 S rRNA-ITS2–26 S rRNA). Various deletions in the 3′-terminal part, although severely reducing the efficiency of processing, still allow some mature 26 S rRNA to be formed. On the other hand, none of the ITS2 deletions affect the production of mature 17 S rRNA. Since all of the deletions severely disturb the recently proposed secondary structure of ITS2, these findings suggest an important role for higher order structure of ITS2 in processing. Analysis of the effect of complete or partial replacement of S. cerevisiae ITS2 with its counterpart sequences from Saccharomyces rosei or Hansenula wingei, points to helix V of the secondary structure model as an important element for correct and efficient processing. Direct mutational analysis shows that disruption of base-pairing in the middle of helix V does not detectably affect 26 S rRNA formation. In contrast, introduction of clustered point mutations at the apical end of helix V that both disrupt base-pairing and change the sequence of the loop, severely reduces processing. Since a mutant containing only point mutations in the sequence of the loop produces normal amounts of mature 26 S rRNA, we conclude that the precise (secondary and/or primary) struture at the lower end of helix V, but excluding the loop, is of crucial importance for efficient removal of ITS2.