Effects of Nucleobase Amino Acids on the Binding of Rob to Its Promoter DNA: Differential Alteration of DNA Affinity and Phenotype

Nucleic acid binding proteins have been studied extensively, but the nature of the interactions that control their affinity, selectivity, and DNA and RNA functions is still not well understood. To understand the nature and functional consequences of such interactions, we introduced nucleobase amino acids at specific positions of the transcriptional regulator Rob protein and succeeded in demonstrating that an alteration of the protein-DNA affinity can affect specific phenotypes associated with Rob protein-DNA interactions. Previously, we inserted different nucleobase amino acids in lieu of Arg40; this residue is known (via X-ray crystallography) to interact with the DNA promoter A-box residue Gua6. The interactions predominantly involved Watson-Crick-like H bonding. The present study focused primarily on the DNA promoter B-box; the crystallographically determined interaction involves H bonding between the agmatine moiety of Arg90 within an HTH motif of Rob and a phosphate oxygen anion to the 5'-side of Thy14. We had two main goals, the first of which was to demonstrate enhanced Rob-binding to the promoter DNA and the functional consequences resulting from the interaction of DNA with Rob analogues containing Arg90 nucleobase mimics. The second was to explore the possible functional consequences of enhancing the protein-DNA affinity with nucleobase replacements, which mechanistically mediate interactions differently than those reported to be operative for specific protein-DNA interactions. Nucleobase replacement at position 90 with Arg isosteres enhanced the Rob protein- DNA affinity in parallel with increasing antibiotic and Hg resistance, while aromatic amino acid replacements increased the affinity but not the antibiotic or Hg resistance. The demonstration of an increased affinity through strong base stacking interactions was notable.