RNA-binding properties of in vitro expressed histidine-tagged RB69 RegA translational repressor protein

To facilitate RNA-binding studies of the phage RB69 RegA translational repr essor protein, regA was configured to add six histidines to the carboxyl en d of the protein. In vitro transcription-translation from the T7 promoter o n plasmid pSA1 yielded a RegA69-His(6) protein that binds nickel-Sepharose and elutes with 0.5 M imidazole. The system was further modified to avoid c loning and the toxic effects of RegA on Escherichia coil by the polymerase chain reaction (PCR), producing linear templates with the configuration T7 promoter-TIR-regA-His(6). A translation initiation region was used that con forms to consensus E. coli and eukaryotic initiation sites and eliminates t he target for RegA autogenous repression. RegA69-His(6) synthesized in E. c oli S30 or wheat germ extracts displayed RNA- binding properties similar to wild-type RB69 RegA Specificity of RNA binding was demonstrated by in vitr o repression of T4 gp44 and gp45 but not beta-lactamase, by differential bi nding 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 t he His, tag to the C-terminus of RB69 RegA does not dramatically alter RNA binding, indicating that this region is not directly involved in site recog nition. With access to several T4-like phage genomes and regA mutant sequen ces, in vitro synthesis of His-tagged proteins directly from Linear PCR pro ducts provides a convenient and efficient system to study RegA and other in teresting RNA-binding proteins, (C) 1999 Academic Press.