Protein Splicing Activity of the Haloferax volcanii PolB-c Intein Is Sensitive to Homing Endonuclease Domain Mutations

Abstract

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.

Inteins in Science: Evolution to Application

“…In a direct completion assay performed on intein-positive and intein-negative strains, it was seen that the intein-negative strains outcompeted the intein-positive strains due to a faster growth rate [ 75 ]. A study conducted by Mills and coworkers in 2020 confirmed that mutations of the conserved residue in HED reduces splicing activity and depreciates growth in H. volcanii [ 76 ]. There is still an ongoing debate as to whether the molecular parasite’s selfishness is the driving force for its survival at a cost of the individual since it is highly likely that the molecular parasite is benefited but not the organism that ensures its survival.…”

Conditional Alternative Protein Splicing Promoted by Inteins from Haloquadratum walsbyi

“…43,44 The other is a full-length intein with an intervening homing endonuclease domain (Haloferax volcanii PolB-c). 45 H. walsbyi has 15 inteins, with four separate inteins invading the cdc21 mini-chromosome maintenance (MCM) gene, a DNA helicase key to replication. 39 Two of these inteins have nested homing endonuclease domains, and the two studied here are miniinteins.…”

“…Four inteins from halophiles have been purified as unspliced precursor fusion proteins in E. coli and induced to splice efficiently in vitro with incubation at high salt concentrations, another example of conditional protein splicing. − Three of these inteins are mini-inteins (Halobacterium salinarum PolII and cdc21 and Halorhabdus utahensis MCM2), with the Halorhabdus utahensis intein splicing at lower salt concentrations than the inteins from Halobacterium salinarum. , The other is a full-length intein with an intervening homing endonuclease domain (Haloferax volcanii PolB-c) H.…”