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A long-range pseudoknot in Qbeta RNA is essential for replication.

J Klovins¹, J van Duin

¹Department of Biochemistry Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden, 2300 RA, The Netherlands.

Journal of Molecular Biology

|December 10, 1999

Summary

Bacteriophage Qbeta RNA replication involves a surprising long-range pseudoknot. This RNA structure is essential for function, holding the 3' end near the replicase binding site.

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Area of Science:

Molecular Biology
Virology
RNA Structure and Function

Background:

Bacteriophage Qbeta RNA replication is initiated by replicase binding to an internal M-site, distant from the 3' end.
The mechanism for this long-range interaction and the role of RNA structure have remained unclear.

Purpose of the Study:

To investigate the structural basis for the interaction between the 3' end of bacteriophage Qbeta RNA and the internal replicase binding site.
To elucidate the role of a newly identified long-range pseudoknot in Qbeta RNA replication.

Main Methods:

RNA structure probing and analysis.
Site-directed mutagenesis to disrupt and restore the pseudoknot.
Replication assays to assess the functional impact of mutations.

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Main Results:

A long-range pseudoknot was identified, involving base-pairing between the 3' terminal hairpin loop and an upstream sequence near the M-site.
Disruption of this pseudoknot by a single mismatch abolished RNA replication.
Restoration of base-pairing through a second mutation fully rescued replication activity.
The pseudoknot appears to stabilize the 3' end in proximity to the replicase binding site.

Conclusions:

The RNA's three-dimensional shape, mediated by the long-range pseudoknot, is critical for bacteriophage Qbeta RNA replication.
This structural element likely positions the 3' end for efficient replication initiation.
Findings may inform understanding of replication mechanisms in other viral RNAs.