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Evidence that a downstream pseudoknot is required for translational read-through of the Moloney murine leukemia virus
N M Wills1, R F Gesteland, J F Atkins
1Howard Hughes Medical Institute, University of Utah, Salt Lake City 84112.
Abstract:
Approximately 5% of the ribosomes translating the gag gene of murine leukemia viruses read through the UAG terminator and translate the in-frame pol gene to produce the gag-pol fusion polyprotein, the sole source of the pol gene products. We show that a pseudoknot located eight nucleotides 3' of the UAG codon in the Moloney murine leukemia virus is required for read-through. This requirement is markedly different from that known to be involved in other cases of read-through but surprisingly similar to some stimulatory sequences known to promote ribosomal frameshifting.
Insights
Murine leukemia viruses use a pseudoknot RNA structure to enable ribosomal read-through of a UAG terminator codon. This process is essential for producing the gag-pol fusion polyprotein, crucial for viral replication.
Area of Science:
- Molecular Biology
- Virology
- RNA Structure
Background:
- Murine leukemia viruses (MLVs) require a gag-pol fusion polyprotein for replication.
- This polyprotein is produced by ribosomal read-through of a UAG terminator codon within the gag gene.
- Understanding the mechanisms of translational read-through is key to understanding viral gene expression.
Purpose of the Study:
- To investigate the RNA elements involved in ribosomal read-through of the UAG terminator in Moloney murine leukemia virus (MMLV).
- To determine the role of a specific RNA pseudoknot structure in facilitating gag-pol polyprotein synthesis.
Main Methods:
- Analysis of MMLV gag gene sequences and predicted RNA structures.
- Mutagenesis studies to assess the requirement of the pseudoknot for read-through.
- Ribosomal run-off assays to quantify gag-pol polyprotein production.
Main Results:
- A pseudoknot structure located 8 nucleotides downstream of the UAG stop codon in MMLV is essential for efficient ribosomal read-through.
- Deletion or disruption of this pseudoknot significantly reduces the production of the gag-pol fusion protein.
- The mechanism of read-through mediated by this pseudoknot differs from other known read-through mechanisms but shares similarities with sequences that promote ribosomal frameshifting.
Conclusions:
- The MMLV pseudoknot is a critical cis-acting element that regulates translation termination and promotes read-through.
- This finding reveals a novel mechanism for viral polyprotein synthesis and highlights the diverse roles of RNA structures in gene expression.
- The pseudoknot's unique function provides insights into the interplay between RNA structure, ribosomes, and viral genome expression.