Fig. 4. Intein splicing defects affect in vivo levels of mTk-RadA.

(A) Tk-RadA allelic variants introduced into the T. kodakarensis genome at the native locus retain native extein and promoter sequences. Strains differ solely in the sequences encoding the Tk-RadA-intein. The full-length precursor of Tk-RadA^WT from the parental strain TS559 contains the unspliced intein (red and orange; amino acids 150 to 633) with an active HEN domain (orange; amino acids 286 to 585) embedded within N-extein and C-extein (blue; amino acids 1 to 149 and 634 to 839, respectively). AL002 was constructed from TS559 and encodes an inactive HEN (red, orange, and black; amino acids 373 to 381) with alanine substitution of amino acids 373 to 381 [Tk-RadA^A(373–381)]. AL003 (Tk-RadA^ΔIntein) encodes a Tk-RadA variant lacking the entire intein sequence (amino acids 150 to 633) introduced into AL002. Reintroduction of the allele lacking the HEN domain with a small loop derived from the mini-intein of P. horikoshii RadA protein into AL003 generated AL015 (Tk-RadA^{Δ270–592+P.ho. loop}). (B and E) In vivo Tk-RadA protein and intein levels were monitored by Western blotting using polyclonal antibodies against the pTk-RadA^WT. Strains were grown to mid-exponential phase in biological triplicate at 85°C (B) and 65°C (E). All Tk-RadA^WT isoforms were detected, including pTk-RadA (P), pTk-RadA lacking the HEN domain (P*), mature, ligated exteins (LEs) of Tk-RadA, and the spliced intein (I). (C and F) Tk-RadA splicing efficiencies at 85°C (C) and 65°C (F) were quantified from the ratio of LE/P or LE/P* intensities. (D and G) Relative mTk-RadA levels at 85°C (D) and 65°C (G) were quantified from the ratio of LE (variant strains)/LE (TS559). Error bars represent SE from the mean of a minimum of three biological replicates.