Identification and characterization of a minimal substrate of RNase III

Copyright information:Taken from "Characterization of RNA sequence determinants and antideterminants of processing reactivity for a minimal substrate of ribonuclease III"Nucleic Acids Research 2006;34(13):3708-3721.Published online 8 Aug 2006PMCID:PMC1540722.© 2006 The Author(s) () Sequence and secondary structure of R1.1[WC] RNA and smaller variants, including μR1.1 RNA. The scissile bonds in R1.1[WC] RNA are indicated ([1] and [2]), and the db and two pb are outlined. Time course assays of cleavage were performed using ∼2.5 nM 5′ P-labeled RNA, 10 nM RNase III and buffer containing 10 mM Mg (see Materials and Methods). In the gel electropherogram shown on the right, lanes 1, 3, 5, 7 and 9 represent incubation of substrate for 4 min with RNase III in the absence of Mg, while lanes 2, 4, 6, 8 and 10 show complete reactions. ‘S’ and ‘5′’ refer to the uncleaved substrates and 5′ end-containing cleavage products, respectively. The asterisks indicate minor products of nonspecific, nonenzymatic cleavages. The relative reactivities are provided below the RNA structures, and represent the average of three or more independent experiments, with the standard error of the mean of ±9%. The cleavage of μR1.1[Δ2] RNA was not detectable (ND), so a relative reactivity could not be determined. Since μR1.1 RNA is the reference substrate (see Results), its relative reactivity is 1.0. The μR1.1 RNA cleavage site was mapped by comparison of the gel electrophoretic mobilities of the products of cleavage of internally P-labeled μR1.1 RNA with the cleavage products of R1.1[WC] RNA (data not shown). This experiment demonstrated the comigration of the 28 bp upper stem–loop products, created by cleavage at sites 1 and 2; see also (). () Examination of the involvement of the tetraloop as a reactivity epitope. In the gel electropherogram on the right, lanes 1, 3, 5, 7 and 9 represent incubation of substrate for 4 min with RNase III in the absence of Mg, while lanes 2, 4, 6, 8 and 10 represent complete reactions. ‘S’ and ‘5′’ refer to uncleaved substrate and 5′ end-containing cleavage product, respectively. The asterisks indicate minor products of nonspecific, nonenzymatic cleavage. The relative reactivities are provided below the RNA structures, and are the average of three or more independent experiments, with the average standard error of the mean of ±9%.