Temperature-sensitive control of protein activity by conditionally splicing inteins

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Temperature-sensitive (TS) alleles are crucial for studying gene function across various model systems.
• The scarcity and difficulty in identifying TS alleles have historically limited their widespread application.
• Existing methods for generating TS alleles are often inefficient or difficult to implement.

Purpose of the Study:

• To develop a novel system for generating conditional, temperature-sensitive alleles.
• To leverage conditionally active inteins for temporal gene regulation.
• To demonstrate the utility of this system in a heterologous model organism.

Main Methods:

• Engineered temperature-sensitive splicing variants of the yeast Saccharomyces cerevisiae vacuolar ATPase subunit (VMA) intein.
• Inserted these TS inteins into the Gal4 protein and subsequently transferred them into the Gal80 protein.
• Tested the functionality of the engineered Gal80-intein(TS) construct in Drosophila melanogaster for temperature-dependent gene regulation.

Main Results:

• Successfully generated temperature-sensitive alleles using conditionally active inteins.
• Demonstrated efficient temporal regulation of the Gal4/upstream activation sequence (UAS) system in Drosophila melanogaster.
• The Gal80-intein(TS) construct provided robust temperature-dependent control over gene expression.

Conclusions:

• The developed intein-based system provides a powerful new method for generating temperature-sensitive alleles.
• This technique offers a versatile approach for conditional protein function analysis in diverse model systems.
• The minimal host requirements suggest broad applicability for generating conditional inteins across multiple organisms and proteins.