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.
Inteins are found in essential genes of several infectious bacteria and fungi, most notably the devastating human pathogen M. tuberculosis, and represent novel drug targets via splicing inhibition. We will investigate the regulation of recombinase function by protein splicing with archaeal RadA and M. tuberculosis RecA.
| Lennon, Christopher W; Stanger, Matthew; Banavali, Nilesh K et al. (2018) Conditional Protein Splicing Switch in Hyperthermophiles through an Intein-Extein Partnership. MBio 9: |
| Kelley, Danielle S; Lennon, Christopher W; Li, Zhong et al. (2018) Mycobacterial DnaB helicase intein as oxidative stress sensor. Nat Commun 9:4363 |
| Belfort, Marlene (2017) Mobile self-splicing introns and inteins as environmental sensors. Curr Opin Microbiol 38:51-58 |
| Lennon, Christopher W; Belfort, Marlene (2017) Inteins. Curr Biol 27:R204-R206 |
| Kelley, Danielle S; Lennon, Christopher W; SEA-PHAGES et al. (2016) Mycobacteriophages as Incubators for Intein Dissemination and Evolution. MBio 7: |
| Lennon, Christopher W; Stanger, Matthew; Belfort, Marlene (2016) Protein splicing of a recombinase intein induced by ssDNA and DNA damage. Genes Dev 30:2663-2668 |
