Conditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria

Area of Science:

• Molecular Biology
• Biochemistry
• Microbiology

Background:

• Inteins are protein splicing elements that regulate protein function post-translationally.
• Conditional protein splicing (CPS) allows protein function to be tuned by environmental cues.
• Inteins are found in conserved proteins, including the mycobacterial replicative helicase DnaB.

Purpose of the Study:

• To investigate the environmental regulation of DnaB intein splicing in its native host.
• To develop a reporter system for monitoring intein splicing activity.
• To elucidate the mechanism by which zinc affects intein splicing.

Main Methods:

• Construction of a kanamycin intein splicing reporter (KISR) in Escherichia coli.
• Analysis of splicing-dependent resistance (SDR) phenotypes.
• Probing DnaB splicing in native Mycobacterium smegmatis.
• In vitro inhibition assays with zinc.
• X-ray crystallography of zinc-bound intein.

Main Results:

• Zinc was identified as a potent and reversible inhibitor of DnaB intein splicing in M. smegmatis.
• A reporter system demonstrated splicing-dependent kanamycin resistance.
• Crystal structure revealed zinc binding to the catalytic cysteine, inhibiting splicing.
• Intein insertion site influences splicing-dependent host protein activation.

Conclusions:

• Mycobacterial DnaB protein splicing is responsive to environmental zinc.
• Zinc-mediated regulation of DnaB splicing impacts DNA replication.
• This provides a potential mechanism for mycobactericidal activity and applications in CPS.