@article{dean_allen_miller_2005, title={In vitro selection of phage RB69 RegA RNA binding sites yields UAA repeats}, volume={336}, ISSN={["0042-6822"]}, DOI={10.1016/j.virol.2005.03.002}, abstractNote={The SELEX method of in vitro selection was used to isolate RNAs that bind the RB69 RegA translational repressor protein immobilized on Ni-NTA agarose. After five rounds of SELEX, the pool of selected RNA displayed striking sequence uniformity: UAAUAAUAAUAAUA was clearly enriched in the 14 nucleotides that underwent selection. Individual, cloned molecules displayed a repeating (UAA) sequence, with only two RNAs having a 3' AUG. Removing the 3' AUG slightly reduced binding in gel shift assays, moving the AUG 5' proximal of the (UAA) slightly improved binding, but (UAA)4 alone still bound the purified protein. Dissociation constants showed that RNA shortened to (UAA)3 and (UAA)2 also retained binding, whereas cytosine clearly prevented binding by RB69 RegA. Scanning of RB69 gene starts and ends with an RB69 RegA SELEX information weight matrix yielded 21 sequences as potential RegA sites. One site, on the mRNA for the pentameric (4:1) phage gp44/62 DNA polymerase clamp loader complex, has the RB69 gene 44 stop codon and 3'-adjacent gene 62 initiation codon in a sequence (GAAAUAAUAUG) that is similar to in vitro selected RNA and was shown to bind RB69 RegA. Sequences between the Shine-Dalgarno and initiation codon, which frequently contain a UAA stop codon of a 5'-adjacent gene, appear to be preferred RB69 RegA binding sites.}, number={1}, journal={VIROLOGY}, author={Dean, TR and Allen, SV and Miller, ES}, year={2005}, month={May}, pages={26–36} }
 @article{allen_miller_1999, title={RNA-binding properties of in vitro expressed histidine-tagged RB69 RegA translational repressor protein}, volume={269}, ISSN={["1096-0309"]}, DOI={10.1006/abio.1999.4025}, abstractNote={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.}, number={1}, journal={ANALYTICAL BIOCHEMISTRY}, author={Allen, SV and Miller, ES}, year={1999}, month={Apr}, pages={32–37} }