Direct genetic selection of two classes of R17/MS2 coat proteins with altered capsid assembly properties and expanded RNA-binding activities.
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.
Wang
S
S
Department 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.
True
H L
HL
Seitz
E M
EM
Bennett
K A
KA
Fouts
D E
DE
Gardner
J F
JF
Celander
D W
DW
D000595:Amino Acid Sequence; D017100:Bacteriophage P22 / Q000235:genetics*; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics*; D036022:Capsid Proteins; D003062:Codon; D017931:DNA Primers; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism; D005814:Genes, Viral; D007700:Kinetics; D008242:Lysogeny; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D016601:RNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D011995:Recombination, Genetic; D012486:Salmonella typhimurium / Q000821:virology; D012641:Selection, Genetic; D014158:Transcription, Genetic; D015678:Viral Structural Proteins / Q000235:genetics
D036022:Capsid Proteins; D003062:Codon; D017931:DNA Primers; D004279:DNA, Viral; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D015678:Viral Structural Proteins