Recombinant reverse transcriptase of Rous sarcoma virus: characterization of DNA polymerase and RNAase H activities

Area of Science:

• Molecular Biology
• Enzymology
• Virology

Background:

• Rous sarcoma virus (RSV) reverse transcriptase is crucial for retroviral replication.
• Efficient production of recombinant viral enzymes is essential for molecular biology applications.

Purpose of the Study:

• To isolate and purify RSV reverse transcriptase from a recombinant E. coli expression system.
• To characterize the enzyme's properties, including subunit composition, optimal activity conditions, and fidelity.
• To evaluate its utility in complementary DNA (cDNA) synthesis.

Main Methods:

• Isolation and purification of RSV reverse transcriptase from engineered E. coli.
• Biochemical characterization of enzyme subunits (97.4 kDa and 61.3 kDa).
• Assays to determine optimal conditions for DNA polymerase and RNAase H activities.
• Fidelity assessment of DNA synthesis using a homogeneous RNA template.
• Evaluation of azidothymidine triphosphate inhibition.
• Application in cDNA synthesis.

Main Results:

• Homogeneous RSV reverse transcriptase was successfully purified, revealing a heterodimeric structure.
• Optimal conditions for DNA polymerase and RNAase H activities were determined.
• High fidelity of DNA synthesis was observed.
• Azidothymidine triphosphate showed inhibitory effects.
• The enzyme demonstrated efficacy in cDNA synthesis.

Conclusions:

• Recombinant RSV reverse transcriptase can be produced in E. coli and purified to homogeneity.
• The characterized enzyme possesses desirable properties for molecular biology, particularly for cDNA synthesis.
• This provides a valuable tool for research involving RNA-to-DNA conversion.