Post-translational environmental switch of RadA activity by extein–intein interactions in protein splicing

Abstract

Post-translational control based on an environmentally sensitive intervening intein sequence is described. Inteins are invasive genetic elements that self-splice at the protein level from the flanking host protein, the exteins. Here we show in Escherichia coli and in vitro that splicing of the RadA intein located in the ATPase domain of the hyperthermophilic archaeon Pyrococcus horikoshii is strongly regulated by the native exteins, which lock the intein in an inactive state. High temperature or solution conditions can unlock the intein for full activity, as can remote extein point mutations. Notably, this splicing trap occurs through interactions between distant residues in the native exteins and the intein, in three-dimensional space. The exteins might thereby serve as an environmental sensor, releasing the intein for full activity only at optimal growth conditions for the native organism, while sparing ATP consumption under conditions of cold-shock. This partnership between the intein and its exteins, which implies coevolution of the parasitic intein and its host protein may provide a novel means of post-translational control.

Impact of a homing intein on recombination frequency and organismal fitness

“…Our findings show that under conditions that include occasional growth of the population, the high fitness cost of the intein guarantees the survival of alleles with the empty target site, even in the absence of inteins without HEN activity. Although here we showed a fitness cost for the intein, several reports have indicated a potential positive role for intein presence, claiming a regulatory function under stress conditions (36)(37)(38)(39)(40). However, our finding that natural H. volcanii populations in the same sampling location tend to be mixed, having both inteinpositive and intein-negative strains sharing the same niche, does not support a strong selective pressure for intein retention in this case.…”

Protein splicing of a recombinase intein induced by ssDNA and DNA damage

“…The archaeal Pho RadA intein provides an impressive example of conditional protein splicing, in which the native exteins have a strong influence on reaction rate (Topilina et al 2015b). Interactions between the intein and C-terminal extein (C-extein) inhibit splicing, and either increased temperature or solvent conditions that disrupt proteinprotein interactions are required for splicing to proceed (Topilina et al 2015b).…”

“…Interactions between the intein and C-terminal extein (C-extein) inhibit splicing, and either increased temperature or solvent conditions that disrupt proteinprotein interactions are required for splicing to proceed (Topilina et al 2015b). Whereas temperature represents a general environmental condition important for growth of the hyperthermophilic Pho, we asked whether another layer of regulation might exist in which splicing is influenced by a factor directly related to RadA function.…”

“…RadA is the archaeal homolog of Rad51 in eukaryotes and RecA in prokaryotes (Seitz et al 1998). The RadA intein from the hyperthermophilic archaeon Pyrococcus horikoshii (Pho) provides a striking example of conditional protein splicing, in which increased temperature is required for the reaction to proceed (Topilina et al 2015b). …”

SufB intein of Mycobacterium tuberculosis as a sensor for oxidative and nitrosative stresses

“…Bacterial strains, growth conditions, plasmid methodology, restriction enzymes, and oligonucleotides are found in SI Methods and Tables S1 and S3, following several protocols previously described in ref. 36.…”