The secondary structure of the truncated P5abc subdomain
(tP5abc, a 56-nucleotide RNA) of the Tetrahymena thermophila group I intron
ribozyme changes when its tertiary structure forms [Proc. Natl. Acad. Sci.
USA 95, 11555-11560 (1998)]. On addition of magnesium ions to tP5abc three
Watson-Crick base pairs and three G-U base pairs are changed, and an extrastable
tetraloop is lost. In collaboration with Prof. T. Cechís laboratory, we
have now made mutations in tP5abc to stabilize secondary structures corresponding
to different folded forms of the RNA. The secondary structure of a double
mutant D2 (U168C and U177G) has been determined by solution NMR spectroscopy.
It is different from the secondary structure of tP5abc in the tertiary
folded form and in the unfolded form. In contrast to tP5abc, the secondary
structure of the mutant D2 does not change in the presence of up to 5 mM
magnesium ions and a magnesium ion binding site on the P5c stem is perturbed
as revealed by heteronuclear NMR. However, nondenaturing gel electrophoresis
mobility experiments show that identical mutations in the context of the
whole P4-P6 domain do not affect the tertiary folding of P4-P6. These results
demonstrate that the metal ion binding site perturbation caused by mutations
can interfere with the tertiary folding of a relatively small RNA tP5abc;
while the tertiary folding can be rescued in the context of a larger RNA
P4-P6. This study provides insights into the tertiary folding of RNAs.
The three-dimensional structure determination of tP5abc in the absence
of magnesium is being analyzed. It should be solved in the near future,
and can serve as a model for functional analysis. |
