Fig. 6: The C-terminal extension of the TAC toxin interacts with P-site fMet-tRNAfMet and is critical for its function.
a Contrary to other ribosome-dependent toxins, HigBTAC strongly interacts with the P-site tRNA and ribosomal protein S13 (uS13). Left, Overview of the contacts between HigBTAC [K95A], the P-site fMet-tRNAfMet, uS13, and the cspA mRNA. Right, Close up of the interactions observed between the third α-helix of HigBTAC [K95A], the P-site fMet-tRNAfMet, and uS13. Residues and nucleotides within 4 Å of each other are indicated, and the cryo-electron density map is displayed as a gray mesh. b Known toxins observed in pre-cleavage state with their respective mRNA targets and the P-site tRNA are shown here with a canonical translation reference. The structures are aligned on the P-site tRNA and presented in the same orientation as in a. Shown are (left to right): E. coli RelE [R45A-R81A] (PDB 4V7J); Proteus vulgaris HigB (PDB 4ZSN); E. coli YoeB dimer (PDB 4V8X); and a cognate tRNA observed in the A-site during canonical translation (PDB 7K00). As above, the P-site tRNA is orange and the mRNA is purple. c Positively charged residues of HigBTAC helix α3 in contact with P-site tRNA are important for its function. M. smegmatis transformed with pGMC-vector (-), HigBTAC wild-type or its mutant derivatives (Alanine substitution of residue R61, E106, D110, K113, or R117) were serial diluted, spotted on LB streptomycin agar plates with or without anhydrotetracycline (Atc ng/mL) inducer and incubated 3 days at 37 °C. Alanine substitution in R61 and K113 affects HigBTAC inhibition of CspA synthesis (d) and cleavage (e) in vitro. CspA was expressed in a cell-free translation system with or without HigBTAC wild-type, R61A, or K113A substitution, and analyzed as described in Fig. 3a for CspA protein synthesis and Fig. 3b for cspA cleavage. HigBTAC [K95A] is shown as inactive control. Arrows show the uncleaved (A, 126 nt) and cleaved (A*, 95 nt) cspA. Representative results of three independent experiments are shown.
