Conditional Protein Splicing Switch in Hyperthermophiles through an Intein-Extein Partnership

• Published in: mBio Vol. 9; no. 1
• Main Authors: Lennon, Christopher W, Stanger, Matthew, Banavali, Nilesh K, Belfort, Marlene
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 30-01-2018
• Subjects: Archaeal Proteins - genetics; Archaeal Proteins - metabolism; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Exteins; hyperthermophile; Inteins; mobile genetic elements; Models, Biological; Models, Molecular; Posttranslational regulation; Protein Splicing; Pyrococcus horikoshii - metabolism; Pyrococcus horikoshii - radiation effects; recombinase; Sequence Deletion; Temperature; Thermococcus - metabolism; Thermococcus - radiation effects; mobile genetic elements; recombinase; Posttranslational regulation; hyperthermophile
• ISSN: 2161-2129; 2150-7511
• DOI: 10.1128/mBio.02304-17

Inteins are intervening proteins that undergo an autocatalytic splicing reaction that ligates flanking host protein sequences termed exteins. Some intein-containing proteins have evolved to couple splicing to environmental signals; this represents a new form of posttranslational regulation. Of particular interest is RadA from the archaeon
, for which long-range intein-extein interactions block splicing, requiring temperature and single-stranded DNA (ssDNA) substrate to splice rapidly and accurately. Here, we report that splicing of the intein-containing RadA from another archaeon,
, is activated by significantly lower temperatures than is
RadA, consistent with differences in their growth environments. Investigation into variations between
and
RadA inteins led to the discovery that a nonconserved region (NCR) of the intein, a flexible loop where a homing endonuclease previously resided, is critical to splicing. Deletion of the NCR leads to a substantial loss in the rate and accuracy of
RadA splicing only within native exteins. The influence of the NCR deletion can be largely overcome by ssDNA, demonstrating that the splicing-competent conformation can be achieved. We present a model whereby the NCR is a flexible hinge which acts as a switch by controlling distant intein-extein interactions that inhibit active site assembly. These results speak to the repurposing of the vestigial endonuclease loop to control an intein-extein partnership, which ultimately allows exquisite adaptation of protein splicing upon changes in the environment.
Inteins are mobile genetic elements that interrupt coding sequences (exteins) and are removed by protein splicing. They are abundant elements in microbes, and recent work has demonstrated that protein splicing can be controlled by environmental cues, including the substrate of the intein-containing protein. Here, we describe an intein-extein collaboration that controls temperature-induced splicing of RadA from two archaea and how variation in this intein-extein partnership results in fine-tuning of splicing to closely match the environment. Specifically, we found that a small sequence difference between the two inteins, a flexible loop that likely once housed a homing endonuclease used for intein mobility, acts as a switch to control intein-extein interactions that block splicing. Our results argue strongly that some inteins have evolved away from a purely parasitic lifestyle to control the activity of host proteins, representing a new form of posttranslational regulation that is potentially widespread in the microbial world.