Mn2+ suppressor mutations and biochemical communication between Ty1 reverse transcriptase and RNase H domains.

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• Ty1 reverse transcriptase/RNase H (RT/RH) activity is highly sensitive to manganese (Mn2+) concentrations.
• Elevated intracellular Mn2+ inhibits Ty1 retrotransposition and in vitro RT activity, affecting other RTs like HIV-1 RT.

Purpose of the Study:

• To characterize Mn2+ inhibition of Ty1 RT/RH.
• To identify suppressor mutations that restore Ty1 transposition in cells with high manganese levels.

Main Methods:

• Generated and analyzed Ty1 RT/RH suppressor mutants in pmr1Δ cells, which have elevated intracellular manganese.
• Mapped suppressor mutations and assessed in vitro RT and RNase H activities of the mutants.

Main Results:

• Suppressor mutations localized to the RNase H domain, not the RT domain.
• Mn2+ inhibition of in vitro RT activity was significantly reduced in suppressor mutants.
• RNase H activity and cleavage specificity were largely unaffected in the mutants.

Conclusions:

• Mn2+ inhibition effects are transmitted from the RNase H domain to the polymerase domain.
• Suggests biochemical communication between the RT and RH domains during reverse transcription.