A protein chaperone can stabilize the intein-containing precursor to indirectly promote protein splicing.

Intervening proteins (inteins) interrupt host protein sequences and are removed by a self-mediated protein splicing reaction. Inteins are abundant in the microbial world and are often found within essential genes involved in DNA replication, recombination, and repair. Compelling examples of conditional protein splicing, where intein removal and subsequent host protein activation are highly dependent on environmental cues, suggest that some inteins serve as post-translational regulatory elements. Within the cellular context, however, the factors influencing protein splicing are still largely unknown. In this work, we demonstrate that the splicing of an intein from Mycobacterium smegmatis DnaB, an essential helicase, is temperature sensitive. Using two artificial extein systems-a kanamycin intein splicing reporter (KISR) and an in vitro splicing reporter-we show that splicing is inhibited at elevated, yet physiological, temperatures. In accordance with our results from M. smegmatis, we find that overexpression of GroEL in Escherichia coli expressing the KISR system drastically increased antibiotic resistance in a temperature-dependent manner. Under heat stress, we find that GroEL dramatically increases the levels of unspliced precursor protein. Mechanistically, GroEL does not appear to directly promote protein splicing, but rather increases the pool of unspliced precursor, which in turn results in more overall splicing. These findings represent the first description of a chaperone promoting protein splicing, demonstrating the ability of cellular factors to influence intein excision. From a physiological perspective, we hypothesize that a M. smegmatis DnaB intein may act as a switch to inhibit DNA replication under conditions of heat stress. Extending past this, our findings have broad implications for improving the efficiency of intein-based protein engineering technologies.