SELF-SPLICING OF THE TETRAHYMENA PRE-RIBOSOMAL-RNA IS DECREASED BY MISFOLDING DURING TRANSCRIPTION

RNA processing depends in part on the ability of nascent transcripts t o fold into the desired conformation. Self-splicing of the group I int ron from Tetrahymena was used to assess the folded state of preribosom al RNA transcripts when synthesized in vitro. A simple method for isol ating nondenatured RNA from a T7 RNA polymerase reaction was tested. T he intron alone is fully active when transcribed at 30-degrees-C, sugg esting that the active structure is both kinetically and thermodynamic ally favored. Longer precursor RNAs, however, were less than completel y active in self-splicing. Full activity, as judged by both the initia l rate and the extent of product formation, was restored by brief incu bation at 95-degrees-C and rapid cooling in the presence of magnesium ion. This result did not depend on the length of the precursor RNA in any simple way, but correlated loosely with the presence of intact exo n domains. When transcribed in the absence of cellular proteins, a sig nificant portion of the pre-RNA appears to be trapped in a conformatio n that does not readily undergo the first step of splicing.