Csy4 binds its substrate and product with high affinity and functions as a single-turnover enzyme. (A) Csy4 cleaves within pre-crRNA repeat sequences (black) to generate mature crRNAs that contain a spacer sequence (colored line) flanked by fragments of the repeat. The substrate sequence and cleavage site (red triangle) are indicated above, with the crRNA construct previously used for crystallography shown in bold. (B) A schematic depicts protein:RNA contacts revealed by a co-crystal structure of Csy4 bound to a fragment of the crRNA repeat (PDB ID: 2XLK). Important amino acid residues are shown in yellow, and RNA nucleotides are numbered as in A. Red circles, pentagons, boxes, and red dotted lines denote phosphates, ribose groups, bases, and hydrogen-bonding interactions, respectively. (C) EMSAs (top) were performed with Csy4-H29A and the substrate and product of the cleavage reaction. The resulting data for these and all subsequent binding assays were fit with a standard binding isotherm to yield equilibrium dissociation constants (solid lines; see Materials and Methods), and average K_d and standard error of the mean (SEM) values from at least three independent experiments are reported in Supplemental Table 1. (D) RNA cleavage assays were conducted at five different enzyme:substrate molar ratios, and the extent of the reaction at various time points was assessed by denaturing PAGE (top). The resulting data for these and all subsequent cleavage assays were fit with a single exponential to yield first-order rate constants (solid lines; see Materials and Methods), and average k_obs and SEM values from three independent experiments are reported in Supplemental Table 1. Error bars for each time point represent the standard deviation and are not always visible.

Importance of loop sequence for high-affinity RNA binding. (A) EMSAs demonstrate that Csy4 binds the reverse complement of the crRNA repeat (rc) >10⁵-fold more weakly than the WT-crRNA repeat. (B) Mutant RNA substrates were generated by changing the WT loop sequence (GUAUA) to a quintuple mutant (UAUAC), the highly stable UUCG tetraloop, or a poly(A) pentaloop, or by removing the loop through use of a substrate nicked between U12 and A13. EMSAs reveal substantial defects associated with binding these mutant RNAs.

Stem length dependence during substrate binding and cleavage. (A) One or two G–C base pairs were inserted at the top of the stem between the closing C–G base pair and the GUAUA pentaloop, and EMSAs were performed. (B) To test the hypothesis that longer stems prevent stable binding of the arginine-rich helix via their effect on major groove accessibility, a substrate was generated that contains five G–C base pairs inserted above the WT stem. Subsequently, asymmetric adenosine bulges were inserted on the 3′ side of the duplex between the five-base-pair WT stem and the five-base-pair insertion. EMSAs reveal that binding affinities increase monotonically (black arrow) with bulges of increasing size. (C) One or two G–C or A–U base pairs were inserted below the terminal C–G base pair, and cleavage time courses were performed. Additional A–U base pairs have negligible effects on k_obs, whereas two additional G–C base pairs result in ∼1500-fold slower kinetics.

Amino acid contributions to binding energy and cleavage kinetics. (A) Csy4 residues involved in base-pair recognition and phosphate backbone contacts were mutated to alanine in order to assess their energetic contributions to binding. EMSAs were performed with a noncleavable crRNA repeat substrate containing a deoxyribonucleotide substitution at G20, and binding defects relative to Csy4-H29A were determined and converted to ΔΔG values (T = 298 K). Plotted are the average and SEM from at least three independent experiments. (B) First-order rate constants (k_obs) for WT-crRNA repeat cleavage by each Csy4 mutant were determined. Cleavage data for R118A/R115A and R115A/R119A mutants showed biphasic kinetics and were fit with a double exponential decay to yield two rate constants (Supplemental Table 2), the faster of which is shown. Plotted are the average fold defects (relative to WT-Csy4) and SEM from three independent experiments. Average K_d, k_obs, and SEM values are reported in Supplemental Table 2.

Substrate specificity within the crRNA repeat stem. (A) A library of mutated crRNA repeat substrates was generated containing all possible Watson-Crick base-pair substitutions at each position in the double-stranded stem. EMSAs were performed with Csy4-H29A and these RNA substrates, and the resulting binding defects relative to WT-crRNA repeat were determined and converted to ΔΔG values (T = 298 K). The WT stem sequence is shown at the left, with data for base-pair substitutions at each position color-coded similarly. Binding experiments with RNAs mutated at the bottom C–G or U–A base pair were repeated with Csy4-R102A (middle) or Csy4-Q104A (right), respectively; ΔΔG values were calculated relative to WT-crRNA repeat binding by each Csy4 mutant. Shown above are chemical structures of the interactions made by Arg102 and Gln104 with the WT base pairs. (B) Single-turnover cleavage assays were performed with the same library of RNA mutants as in A, and the resulting defects in k_obs relative to WT-crRNA repeat were determined. The data are plotted as in A. (C) To investigate the importance of the terminal C–G base pair during cleavage, mismatched substrates were generated by mutating C6 or G20 individually and single-turnover cleavage assays were performed.