Alteration of Transcriptional Regulator Rob In Vivo: Enhancement of Promoter DNA Binding and Antibiotic Resistance in the Presence of Nucleobase Amino Acids

The identification of proteins that bind selectively to nucleic acid sequences is an ongoing challenge. We previously synthesized nucleobase amino acids designed to replace proteinogenic amino acids; these were incorporated into proteins to bind specific nucleic acids predictably. An early example involved selective cell free binding of the hnRNP LL RRM1 domain to its i-motif DNA target via Watson-Crick-like H-bonding interactions. In this study, we employ the X-ray crystal structure of transcriptional regulator Rob bound to its micF promoter, which occurred without DNA distortion. Rob proteins modified in vivo with nucleobase amino acids at position 40 exhibited altered DNA promoter binding, as predicted on the basis of their Watson-Crick-like H-bonding interactions with promoter DNA A-box residue Gua-6. Rob protein expression ultimately controls phenotypic changes, including resistance to antibiotics. Although Rob proteins with nucleobase amino acids were expressed in Escherichia coli at levels estimated to be only a fraction of that of the wild-type Rob protein, those modified proteins that bound to the micF promoter more avidly than the wild type in vitro also produced greater resistance to macrolide antibiotics roxithromycin and clarithromycin in vivo, as well as the β-lactam antibiotic ampicillin. Also demonstrated is the statistical significance of altered DNA binding and antibiotic resistance for key Rob analogues. These preliminary findings suggest the ultimate utility of nucleobase amino acids in altering and controlling preferred nucleic acid target sequences by proteins, for probing molecular interactions critical to protein function, and for enhancing phenotypic changes in vivo by regulatory protein analogues.