Site Selection of Pyrococcus horikoshi RadA Intein

Inteins are polypeptides translated within host proteins that undergo an autocatalytic protein splicing reaction to join the flanking sequences, termed N- and C- extiens. Long considered molecular parasites, recent work has demonstrated that protein splicing can be regulated by environmental signals, representing an exciting new form of post-translational regulation. A landmark example comes from the homologous recombinase RadA of the archaeon Pyrococcus horikoshii, which splices in response to substrate single-stranded DNA. Interestingly, while the P. horikoshii RadA intein splices efficiently when inserted into foreign exteins under all conditions tested, splicing is inhibited in native exteins in absence of ssDNA. In order to better understand this intriguing observation, P. horikoshii RadA intein site preference within the native extein context was studied by artificially moving the intein to different locations throughout the native extein. Surprisingly, splicing at alternate locations within the native extein context was not able to occur. These results help us to better understand why the P. horikoshii RadA intein selects this one specific threonine when there are many others throughout the extein.