Fig. 3: Structural basis for tRNA capture by activated Ba1Cas12a3.
From: RNA-triggered Cas12a3 cleaves tRNA tails to execute bacterial immunity
a, Overview of Ba1Cas12a3 bound to a crRNA, target RNA and tRNAAla(UGC). The REC1 domain in the cryo-EM structure was not resolved. b, Atomic model of the Ba1Cas12a3 quaternary complex. The anticodon region of the model was predicted using AlphaFold3 owing to the flexibility of the region. c, Interactions between the REC2 domain and the T-arm. d, Interactions between the tRLD domain and the 3′ CCA tail. e, Impact of truncating tRNAAla(UGC) on in vitro cleavage by Ba1Cas12a3. A, anticodon; D, D-arm; T, T-arm. Open and closed circles represent the absence or presence, respectively, of the indicated component. f, Mutational analysis of the truncated tRNA substrate h1 as part of in vitro cleavage by Ba1Cas12a3 and Sm3Cas12a3. g, Schematic (left) and quantification (right) of the mutational analysis of tRNA recognition domains in Ba1Cas12a3 in TXTL reactions. The fold reduction in GFP fluorescence was calculated in comparison to a non-target control. The three residues shown in d plus a poorly resolved neighbouring residue (K257) were swapped with alanine residues (AS; R251A, N253A, K256A and K257A) or with residues with an altered charge (CS; R251E, N253D, K256E and K257E). dBa1, Ba1Cas12a3 with one RuvC active site mutation (E1032A); WT, wild type. h, Schematic (left) and quantification (right) of the mutational analysis of tRNA recognition domains in Ba1Cas12a3 as part of in vitro cleavage of the truncated tRNA substrate h1. dBa1, Ba1Cas12a3 with three RuvC active-site mutations (D712A, E1032A and D1137A). i, Schematic (left) and quantification (right) of in vitro cleavage of an arbitrary RNA sequence by selected Ba1Cas12a3 mutants. Images in e and f are representative of independently prepared cleavage reactions (n = 3). Bars and error bars represent the mean ± s.d. of independently mixed TXTL or in vitro reactions. Statistical analysis was performed using two-tailed Welch’s t-tests with all biological replicates (n = 4 for g, n = 3 for h and i). P values that are not significant (P ≥ 0.05) are shown in grey. For gel source data, see Supplementary Fig. 1.
