Reverse transcriptase mediated binding of primer tRNA to the viral genome

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Retroviral replication relies on primer binding to the viral RNA genome.
• Transfer RNA (tRNA) serves as the primer for reverse transcription in many retroviruses.
• The specific interactions between tRNA and viral RNA, mediated by reverse transcriptase, are crucial for initiating DNA synthesis.

Purpose of the Study:

• To investigate the formation and function of a complex between beef tRNATrp and avian myeloblastosis virus 35S RNA.
• To elucidate the role of reverse transcriptase in the interaction between tRNA and viral RNA.
• To understand the structural requirements for tRNA primer selection and positioning on the RNA genome.

Main Methods:

• Formation of a tRNA-RNA complex in the presence of avian myeloblastosis virus reverse transcriptase.
• Assay of the complex's activity in initiating DNA synthesis.
• Nuclease digestion (RNAase T1 and micrococcal nuclease) to probe tRNA structural changes.
• Comparative analysis using a heterologous tRNA (E. coli tRNA2Glu) to assess primer specificity.

Main Results:

• A functional complex between beef tRNATrp and avian myeloblastosis virus 35S RNA was formed and actively initiated DNA synthesis.
• Reverse transcriptase interaction with tRNA caused partial unwinding of the tRNA acceptor stem.
• E. coli tRNA2Glu showed low incorporation at 35°C but high incorporation at 80°C, suggesting temperature-dependent primer annealing and selection.

Conclusions:

• The viral DNA polymerase (reverse transcriptase) is critical for correctly positioning the tRNA primer on the viral RNA genome.
• The interaction involves structural modifications of the tRNA primer, specifically the acceptor stem.
• Primer selection and annealing are influenced by both tRNA structure and reaction conditions, highlighting the enzyme's role in fidelity.