Post-Translational Regulation of Recombinase Function by Intein Splicing - NRSA Post Doc Fellowship

Abstract Inteins, or protein introns, invade genes at the level of DNA and are expressed within host proteins. Protein splicing occurs when the intein auto-catalytically excises itself from the host protein. Inteins are abundant in nature, present in approximately one half of archaeal and a fourth of bacterial genomes, but have been vastly understudied to date. Recent work has suggested that inteins may be more than selfish invaders, and in some instances may have evolved to regulate host protein function prior to splicing, representing a completely novel form of post-translational regulation. We have discovered that RadA from Pyrococcus horikoshii, a member of the highly conserved RecA/Rad51 recombinase family, splices dramatically faster in the presence of its substrate, ssDNA. This result represents the first example of a biomolecule critical to host protein function influencing splicing, and detailed study of this system may prove key to understanding the possible regulatory role of inteins. We seek to explore this provocative result further in vitro and in vivo both in E coli and the native organism P. horikoshii, using a combination of biochemistry, genetics, molecular modeling and structural biology. We also seek to exploit a previously described temperature- dependent splicing characteristic of P. horikoshii RadA to develop a fluorescent molecular thermometer, which will both inform our understanding of the interactions between intein and RadA prior to splicing and serve as a tool for biotechnology. These interdisciplinary approaches will occur in a collaborative environment with structural biologists and chemical engineers.