Nucleic Acids Research, 1992, Vol. 20, No. 24 6613-6619
© 1992
ENZYMOLOGY |
Mutations at the guanosine-binding site of the Tetrahymena ribozyme also affect site-specific hydrolysis
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado Boulder, CO 80309-0215, USA
*To whom correspondence should be addressed
Received September 3, 1992. Revised November 20, 1992. Accepted November 20, 1992.
Self-splicing group I introns use guanosine as a nucleophile to cleave the 5' splice site. The guanosinebinding site has been localized to the G264–C311 base pair of the Tetrahymena intron on the basis of analysis of mutations that change the specificity of the nucleophile from G (guanosine) to 2AP (2-aminopurine ribonucleoside) (F. Michel et al. (1989) Nature 342, 391–395). We studied the effect of these mutations (GU, A-C and A-U replacing G264–C311) in the L-21 Scal version of the Tetrahymena ribozyme. In this enzymatic system (kcat/Km)G monitors the cleavage step. This kinetic parameter decreased by at least 5x103 when the G264–C311 base pair was mutated to an A-U pair, while (kcat/Km)2AP increased at least 40-fold. This amounted to an overall switch in specificity of at least 2x10g. The nucleophile specificity (G > 2AP for the G-C and G-U pairs, 2AP > G for the A-U and A-C pairs) was consistent with the proposed hydrogen bond between the nucleotlde at position 264 and N1 of the nucleophile. Unexpectedly, the A-U and A-C mutants showed a decrease of an order of magnitude in the rate of ribozyme-catalyzed hydrolysis of RNA, in which H2O or OH– replaces G as the nucleophlie, whereas the G-U mutant showed a decrease of only 2-fold. The low hydrolysis rates were not restored by raising the Mg2+ concentration or lowering the temperature. in addition, the mutant ribozymes exhibited a pattern of cleavage by Fe(II)-EDTA indistinguishable from that of the wild type, and the [Mg2+]
for folding of the A-U mutant ribozyme was the same as that of the wild type. Therefore the guanosine-binding site mutations do not appear to have a major effect on RNA folding or stability. Because changing G264 affects the hydrolysis reaction without perturbing the global folding of the RNA, we conclude that the catalytic role of this conserved nucleotide is not limited to guanosine binding.
+Present address: Department of Biochemistry, Beckman Center, Stanford University, Stanford, CA 94305-5307 USA
Present address: Department of Microbiology, University of Illinois, Urbana, IL 61801, USA