Fig. 2: HTS-Kin analysis of RNase P 5’ leader determinants and anti-determinants due to 3’ RCCA pairing.
a High-throughput sequencing kinetics (HTS-Kin) involves generation of a randomized pool of substrate RNAs, ptRNAs for RNase P, the residual substrate population at different time points are purified and subject to Illumina sequencing. Internal competition kinetics is used to calculate relative rate constants for each sequence from the read data yielding a distribution from which comprehensive specificity determinants can be interpreted. b Sequence and 5′ leader structure of the ptRNAmet(N(−6) to N(−1)) randomized pool. The RNase P cleavage site is indicated by an orange arrow, the randomized positions are green. Red and purple boxes indicate the interactions between the substrate and P RNA, rnpA protein, respectively. c Rate constant distribution show as a plot of the number of sequences binned according to their krel values calibrated to the genomically encoded leader sequence (krel = (kcat/Km(NNNNNN))/(kcat/Km(AAAAAG))). y-axis on the left shows the frequencies of total population and y-axis on the right shows frequencies of ptRNA_AU and ptRNA_GG subsets. d Optimal sequence logo for the fastest 1% of sequences from the 21C rate constant distribution (top) And the sequence logo calculated from endogenous E. coli ptRNA leader sequences (bottom). e Dot plot of krel values comparing the effect of an A(−2) to G(−2) mutation in the context of all other sequence combinations. This plot shows only the subset with an optimal C(−4), the plots for other N(−4) nucleotides is included in Supplementary Fig. 3. f Distribution of nucleotides of optimal and non-optimal sequences at N(−1)N(−2) for endogenous E. coli ptRNAs. Source data are provided as a Source Data file.
