Fig. 3: Pairing interactions with 3′ RCCA limit formation of ES*.

a Analysis of ptRNA_AU and ptRNA_GG dissociation kinetics. 5′ ³²P-labeled ptRNA_AU (top) and ptRNA_GG (bottom) was bound to 150 nM RNase P in 5 mM Ca²⁺ and then the complexes were challenged with an excess (500 nM) of non-radiolabeled ptRNA_GG. The dissociation of ptRNA from ES/ES* was quantified by native EMSA in three independent experiments with essentially identical results. b Quantification of ptRNA remaining in stable ES* complexes fraction. c Schematic of different ptRNA substrate constructs used to examine the effect of 5′ leader length and 3′RCCA sequence on enzyme kinetics. T5: truncation of the ptRNA 5′ leader to two nucleotides; D3: 3′ RCCA deletion. The presence of mis-cleavage for the GG_D3 substrate is indicated by an asterisk. d–f Cleavage of 5′ ³²P-labeled AU_D3 and GG_D3. Mis-cleavage of GG_D3 was observed in all reactions used for analysis of kinetic parameters. Two independent experiments specifically analysing cleavage products demonstrated similar results. Reactions without and with RNase P are marked by minus (−) and plus (+) symbols, respectively. Source data are provided as a Source Data file.