Murray State Theses and Dissertations
Abstract
Inteins, intervening proteins, interrupt host protein sequences and are removed through an autocatalytic protein splicing reaction. Abundant in the microbial world, inteins are often found within essential genes involved in DNA replication, recombination, and repair. Recent work has illuminated examples of conditional protein splicing (CPS), whereby intein removal and subsequent host protein activation is highly dependent on environmental cues. The discovery of CPS suggests that some inteins may serve as post-translational regulatory elements for their host organisms. While previous work has confirmed this interaction, the specific factors and mechanisms that influence protein splicing in the cellular context are still widely unknown.
In this research, we establish that the autocatalytic splicing process for an intein in the essential DnaB helicase of Mycobacterium smegmatis (Msm DnaBi1) is temperature sensitive. Using a kanamycin intein splicing reporter (KISR) that requires protein splicing to provide resistance to the antibiotic kanamycin, we demonstrate that protein splicing is inhibited at elevated, yet still physiological, temperatures. In accordance with previous research, we see that overexpression of the chaperonin GroEL dramatically improved antibiotic resistance in a KISR-dependent manner when the Msm DnaBi1-KISR system was expressed in Escherichia coli. Using the Maltose-binding protein-intein-GFP (MIG) reporter we confirm that temperature inhibits Msm DnaBi1 splicing in vitro. We go on to show that the effect of GroEL is greater under heat-induced protein-folding stress. Western blotting illustrates that GroEL significantly increases the stability of the unspliced precursor protein.
This work is the first characterization of one protein promoting the splicing of another, demonstrating the ability of cellular factors to influence intein excision. From a physiological perspective, this work suggests Msm DnaBi1 may have a regulatory role to inhibit DNA replication under heat-stress. These results have important implications for the regulation of protein splicing in nature, in stress and non-stress conditions, as well as in intein-based technologies.
Year manuscript completed
2026
Year degree awarded
2026
Author's Keywords
Intein, GroEL, DnaB, Chaperone, Mycobacterium smegmatis, Protein Splicing
Degree Awarded
Master of Science
Department
Biology
College/School
Jesse D. Jones College of Science, Engineering and Technology
Thesis Advisor
Christopher Lennon
Committee Chair
Christopher Lennon
Committee Member
Chris Trzepacz
Committee Member
Alexey Arkov
Document Type
Thesis
Recommended Citation
Smetana, John S., "A Molecular Chaperone Can Promote Protein Splicing Through Precursor Stabilization" (2026). Murray State Theses and Dissertations. 449.
https://digitalcommons.murraystate.edu/etd/449