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Updated: Aug 8, 2026
Novel RNA-Binding Proteins Isolation by the RaPID Methodology
Published on: September 30, 2016
Direct genetic selection of two classes of R17/MS2 coat proteins with altered capsid assembly properties and expanded
1Department of Microbiology and College of Medicine, University of Illinois at Urbana-Champaign, B103 Chemical and Life Sciences Laboratory, 601 South Goodwin Avenue, Urbana, IL 61801, USA.
Abstract:
RNA challenge phages are derivatives of bacteriophage P22 that enable direct genetic selection for a specific RNA-protein interaction. The bacteriophage P22 R17 encodes a wild-type R17 operator site and undergoes lysogenic development following infection of susceptible bacterial strains that express the R17/MS2 coat protein. A P22 R17 derivative with an OcRNA site (P22 R17 [A(-10)U]) develops lytically following infection of these strains. RNA challenge phages can be used to isolate second-site coat protein suppressors that recognize an OcRNA sequence by selecting for lysogens with a P22 R17 [Oc] phage derivative. The bacteriophage derivative P22 R17 [A(-10)U] was used in one such scheme to isolate two classes of genes that encode R17 coat proteins with altered capsid assembly properties and expanded RNA-binding characteristics. These mutations map outside the RNA-binding surface and include amino acid substitutions that interfere with interactions between coat protein dimers in the formation of the stable phage capsid. One class of mutants encodes substitutions at the highly conserved first and second positions of the mature coat protein. N-terminal sequence analysis of these mutants reveals that coat proteins with substitutions only at position 1 are defective in post-translational processing of the initiator methionine. All selected proteins possess expanded RNA-binding properties since they direct efficient lysogen formation for P22 R17 and P22 R17 [A(-10)U]; however, bacterial strains that express the protein mutants remain sensitive to lytic infection by other P22 R17 [Oc] bacteriophages. The described selection strategy provides a novel genetic approach to dissecting protein structure within RNA-binding proteins.
Insights
RNA challenge phages offer a new way to study RNA-protein interactions. This research identified mutations in coat proteins affecting capsid assembly and RNA binding, advancing our understanding of protein structure.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Bacteriophage P22 derivatives, known as RNA challenge phages, facilitate genetic selection for RNA-protein interactions.
- The R17 operator site in bacteriophage P22 P22 R17 normally supports lysogenic development in bacteria expressing the R17/MS2 coat protein.
- A modified P22 R17 derivative with an OcRNA site (P22 R17 [A(-10)U]) exhibits lytic development in these strains.
Purpose of the Study:
- To isolate and characterize second-site mutations in coat proteins that alter RNA-binding specificity and capsid assembly.
- To investigate the structural basis of RNA-protein interactions using a novel genetic selection strategy.
Main Methods:
- Utilized RNA challenge phages, specifically P22 R17 [A(-10)U], to select for bacterial lysogens expressing mutant R17 coat proteins.
- Employed N-terminal sequence analysis to identify amino acid substitutions and assess their impact on protein processing.
- Evaluated RNA-binding properties by testing lysogen formation with different phage derivatives.
Main Results:
- Isolated two classes of R17 coat protein mutants with altered capsid assembly and expanded RNA-binding characteristics.
- Identified mutations mapping outside the RNA-binding surface, affecting coat protein dimer interactions.
- Discovered that substitutions at the N-terminus can impair post-translational processing of the initiator methionine.
- Observed that all isolated mutants exhibit enhanced RNA-binding, supporting lysogen formation for both wild-type and OcRNA phages.
Conclusions:
- The RNA challenge phage system provides a powerful genetic tool for dissecting the structure-function relationships of RNA-binding proteins.
- Mutations affecting coat protein dimer interactions and N-terminal processing influence both capsid formation and RNA recognition.
- This approach enables the identification of novel protein adaptations and provides insights into the evolution of RNA-binding specificity.
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