Inteins are selfish genetic elements residing in open reading frames that can splice posttranslationally, resulting in the ligation of an uninterrupted, functional protein. Like other inteins, the DNA polymerase B (PolB) intein of the halophilic archaeon Haloferax volcanii has an active homing endonuclease (HEN) domain, facilitating its horizontal transmission. Previous work has shown that the presence of the PolB intein exerts a significant fitness cost on the organism compared to an intein-free isogenic H. volcanii. Here, we show that mutation of a conserved residue in the HEN domain not only reduces intein homing but also slows growth. Surprisingly, although this mutation is far from the protein splicing active site, it also significantly reduces in vitro protein splicing. Moreover, two additional HEN domain mutations, which could not be introduced to H. volcanii, presumably due to lethality, also eliminate protein splicing activity in vitro. These results suggest an interplay between HEN residues and the protein splicing domain, despite an over 35 Å separation in a PolB intein homology model. The combination of in vivo and in vitro evidence strongly supports a model of co-dependence between the self-splicing domain and the HEN domain that has been alluded to by previous in vitro studies of protein splicing with HEN domain-containing inteins.

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Protein splicing is a post‐translational process facilitated by an intervening polypeptide called an intein, or internal protein. The intein is located between two flanking polypeptides, called exteins. The intein catalyzes its excision from the extein concomitant with the ligation of the exteins. The extremophile Haloferax volcanii grows in high salt environments. We are interested in the salt‐dependence of the protein splicing and nuclease activity of the intein that has invaded its DNA Polymerase beta gene. We have shown that we can induce protein splicing of the intein in vitro as a function of salt concentration and time. The intein has an intervening homing endonuclease domain and we are interested to learn if mutations within this domain would influence splicing. Characterization of a conditionally‐active nuclease may have utility in biotechnology applications. Support or Funding Information This work was supported by the National Science Foundation (grant MCB‐1517138), National Institutes of Health (grant 1R15GM132817‐01), and by the Camille & Henry Dreyfus Foundation

Protein splicing is a post‐translational process facilitated by an intervening polypeptide, or intein. We studied inteins that interrupt the DNA Polymerase II from three different extremophiles, H. salinarum (Hsa), Pyrococcus abyssi (Pab), and Pyrococcus horikoshii (Pho). Intriguingly, all three of these inteins splice conditionally based on environmental cues. We hypothesized that since deep‐sea extremophiles such as P. abyssi and P. horikoshii live under moderately high pressures (150–200 atm), splicing will be unaffected or perhaps even enhanced under pressure. We also hypothesized that inteins from surface dwelling extremophiles like Hsa will not function as efficiently at moderate pressure. Lastly, we hypothesized high pressure (2000 atm and above), regardless of the native environment, will destabilize, unfold, and inactivate the activity. Results suggest that moderate pressures (200 atm and below) do not affect the splicing efficiency of inteins from either deep‐sea extremophile. While similarly unaffected at moderate pressure (100 atm), the function of Hsa inteins appear to be significantly diminished by higher pressures (1000 atm and above). In contrast, higher pressures appear to have no negative effect on the splicing of inteins from the deep‐seas extremophiles, and in some cases can even increase the splicing efficiency.Support or Funding InformationThis work was supported by the National Science Foundation (grant MCB‐1517138) (KVM), the Camille & Henry Dreyfus Foundation, and by Kim and Wendell P. Weeks P15 to the Holy Cross Alumni/Parent Summer Research Scholarship Fund (CKR).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.