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RNA folding kinetics regulates translation of phage MS2 maturation gene
R A Poot1, N V Tsareva, I V Boni
1Leiden Institute of Chemistry, Department of Biochemistry, Gorlaeus Laboratories, University of Leiden, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
Abstract:
The gene for the maturation protein of the single-stranded RNA coliphage MS2 is preceded by an untranslated leader of 130 nt, which folds into a cloverleaf, i.e., three stem-loop structures enclosed by a long distance interaction (LDI). This LDI prevents translation because its 3' moiety contains the Shine-Dalgarno sequence of the maturation gene. Previously, several observations suggested that folding of the cloverleaf is kinetically delayed, providing a time window for ribosomes to access the RNA. Here we present direct evidence for this model. In vitro experiments show that ribosome binding to the maturation gene is faster than refolding of the denatured cloverleaf. This folding delay appears related to special properties of the leader sequence. We have replaced the three stem-loop structures by a single five nt loop. This change does not affect the equilibrium structure of the LDI. Nevertheless, in this construct, the folding delay has virtually disappeared, suggesting that now the RNA folds faster than ribosomes can bind. Perturbation of the cloverleaf by an insertion makes the maturation start permanently accessible. A pseudorevertant that evolved from an infectious clone carrying the insertion had overcome this defect. It showed a wild-type folding delay before closing down the maturation gene. This experiment reveals the biological significance of retarded cloverleaf formation.
Insights
Ribosome binding to MS2 phage RNA is faster than cloverleaf structure refolding. This kinetic delay, crucial for translation, is linked to the leader sequence
Area of Science:
- Molecular Biology
- Virology
- RNA Structure and Function
Background:
- The MS2 phage maturation gene is regulated by an untranslated leader sequence.
- This leader folds into a cloverleaf structure with a long-distance interaction (LDI) that inhibits translation by sequestering the Shine-Dalgarno sequence.
- Previous studies suggested a kinetic delay in cloverleaf folding creates a window for ribosome binding.
Purpose of the Study:
- To provide direct evidence for the kinetic delay model of MS2 phage RNA translation.
- To investigate the role of the leader sequence's structural properties in this folding delay.
- To demonstrate the biological significance of retarded cloverleaf formation.
Main Methods:
- In vitro experiments measuring ribosome binding rates versus RNA refolding rates.
- RNA engineering by replacing stem-loop structures in the leader sequence.
- Analysis of a pseudorevertant evolved from an insertion-mutated infectious clone.
Main Results:
- Ribosome binding to the maturation gene was observed to be faster than the refolding of the denatured cloverleaf structure.
- Altering the cloverleaf structure by replacing stem-loops with a single loop eliminated the folding delay.
- An insertion disrupting the cloverleaf made the start site accessible, but a pseudorevertant restored the wild-type folding delay.
Conclusions:
- Direct evidence supports a kinetic delay in MS2 phage RNA cloverleaf folding, enabling ribosome access for translation.
- The specific structural features of the leader sequence, not just the equilibrium LDI, are responsible for this crucial folding delay.
- Retarded cloverleaf formation is biologically significant for regulating viral gene expression.