title: RNA-binding properties of in vitro expressed histidine-tagged RB69 RegA translational repressor protein.
abstract: To facilitate RNA-binding studies of the phage RB69 RegA translational repressor protein, regA was configured to add six histidines to the carboxyl end of the protein. In vitro transcription-translation from the T7 promoter on plasmid pSA1 yielded a RegA69-His6 protein that binds nickel-Sepharose and elutes with 0.5 M imidazole. The system was further modified to avoid cloning and the toxic effects of RegA on Escherichia coli by the polymerase chain reaction (PCR), producing linear templates with the configuration T7 promoter-TIR-regA-His6. A translation initiation region was used that conforms to consensus E. coli and eukaryotic initiation sites and eliminates the target for RegA autogenous repression. RegA69-His6 synthesized in E. coli S30 or wheat germ extracts displayed RNA-binding properties similar to wild-type RB69 RegA. Specificity of RNA binding was demonstrated by in vitro repression of T4 gp44 and gp45 but not beta-lactamase, by differential binding to poly(U)- and poly(C)-agarose, and by site-specific binding to a 23-base gene 44 target RNA but not to mutant 44 RNA. Therefore, addition of the His6 tag to the C-terminus of RB69 RegA does not dramatically alter RNA binding, indicating that this region is not directly involved in site recognition. With access to several T4-like phage genomes and regA mutant sequences, in vitro synthesis of His-tagged proteins directly from linear PCR products provides a convenient and efficient system to study RegA and other interesting RNA-binding proteins.
authors: Department of Microbiology, North Carolina State University, Raleigh, North Carolina 27696-7615, USA.
authors: S V
authors: SV
authors: Allen
authors: E S
authors: ES
authors: Miller
mesh_terms: D017122:Bacteriophage T4 / Q000235:genetics; D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D009532:Nickel / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D013698:Templates, Genetic; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
chemical_list: D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; C074698:regA protein, bacteriophage RB69; D006639:Histidine; D012313:RNA; D009532:Nickel