Protein splicing in cis and in trans

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Intein-mediated protein splicing is a natural, self-catalytic process.
• It involves the removal of an intein sequence and ligation of flanking extein segments.
• Conserved motifs and specific residues within the intein direct the splicing reaction.

Purpose of the Study:

• To elucidate the mechanistic details of the four-step protein splicing pathway.
• To gain insights into the roles of specific residues and conserved motifs in intein function.
• To understand the rate-limiting steps in protein splicing.

Main Methods:

• Structural analysis of precursor proteins with mutations in catalytic residues.
• Kinetic analysis using conditional trans-splicing.
• Crystallographic studies to determine protein structures.

Main Results:

• Mutated precursor proteins were stabilized for structural studies.
• Kinetic studies suggest the initial acyl rearrangement is rapid.
• Asn cyclization (step 3) appears to be the rate-limiting step in the splicing pathway.

Conclusions:

• While the basic pathway is known, uniform mechanistic details are still under investigation.
• Structural and kinetic data are progressively clarifying the protein splicing mechanism.
• Understanding protein splicing has enabled its application in biotechnology.