RNA: Catalysis, Splicing, Evolution by M. Belfort and D.A. Shub (Eds.)

By M. Belfort and D.A. Shub (Eds.)

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Extra info for RNA: Catalysis, Splicing, Evolution

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27 28 29 30 31 Studies on the hammerhead RNA self-cleaving domain * (RNA enzymes; viroids; R N A secondary structure; processing of R N A ) Duane £ . Ruffner, SueAnn C. Dahm and Olke C. ) Received by M. Belfort: 24 September 1988 Accepted: 27 February 1989 SUMMARY Nine different hammerhead R N A self-cleaving domains consistent with the consensus secondary structure proposed by Keese and Symons (1987) were prepared and tested for cleavage. Each hammerhead was constructed from two oligoribonucleotides in two different configurations.

In view of the discussion in the previous section, one must consider the possibility that the mutation may also destabilize (or stabilize) the self-structure of 1-S such that the net effect is to leave the equilibrium between reactants and a dimer enzyme-substrate complex un­ changed. To further characterize the extent of involvement of a dimer hammerhead, we prepared two mutants, 1-S(U8) and 1-S(A17) designated as in the legend to Fig. 1. Both were unable to undergo cleavage in the presence of 1-E (Fig.

An aliquot of the enzyme in H 20 was heated t o 90 ° C for 1 min and slowly cooled t o 25 °C. Reactions were initiated by the addition of enzyme to the substrate and buffer at 25 ° C. Reactions were terminated by the addition of an equal volume of 7 M urea/50 m M E D T A / 0 . 02% xylene cyanol. Reaction products were separated by electrophoresis into 2 0 % polyacrylamide - 7 M urea gels and located by autoradiography. T h e bands were excised and counted to determine the fraction of cleavage. The t l 2/is the time required for 5 0 % cleavage to occur.

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