Translational control by a long range RNA-RNA interaction; a basepair substitution analysis

Area of Science:

• Molecular Biology
• Genetics
• Virology

Background:

• Translational coupling is a key regulatory mechanism for gene expression in single-stranded RNA coliphages.
• This process ensures that the replicase cistron is only translated when the upstream coat cistron is actively being translated.
• Previous studies identified a regulatory region within the coat gene influencing this coupling.

Purpose of the Study:

• To identify the molecular basis of translational polarity in RNA coliphage gene expression.
• To elucidate the specific RNA-RNA interactions responsible for translational coupling between the coat and replicase cistrons.

Main Methods:

• Genetic analysis to identify regulatory regions.
• Deletion studies to narrow down the critical sequence.
• RNA-RNA interaction mapping using base-pairing complementarity.
• Site-directed mutagenesis to disrupt and restore RNA interactions.

Main Results:

• A long-distance RNA-RNA interaction of 6 base pairs was identified as the structural basis for translational coupling.
• The interaction involves complementary sequences located in the coat-replicase intercistronic region and the coat protein gene (encoding amino acids 31-32).
• Disrupting mutations abolished translational coupling, while compensatory mutations restoring base-pairing re-established it.

Conclusions:

• Long-range RNA-RNA base-pairing is the fundamental mechanism driving translational coupling in this system.
• This interaction acts as a crucial regulatory element for replicase gene expression in RNA coliphages.
• The findings provide a detailed molecular explanation for translational polarity in viral RNA genomes.