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