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

ABSTRACT: 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 in vivo 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 micF DNA promoter A-box residue Gua6. The interactions predominantly involved Watson-Crick-like H bonding. The present study focused primarily on the micF 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 micF promoter DNA and the functional consequences resulting from the interaction of micF 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-micF DNA affinity in parallel with increasing antibiotic and Hg2+ resistance, while aromatic amino acid replacements increased the affinity but not the antibiotic or Hg2+ resistance. The demonstration of an increased affinity through strong base stacking interactions was notable.

SUBMITTER: Zhang C

PROVIDER: S-EPMC7301883 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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

Zhang Chao C Chen Shengxi S Dedkova Larisa M LM Hecht Sidney M SM

Biochemistry 20200526 22

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 <i>in vivo</i> and succeeded in demonstrating that an alteration of the protein-DNA affinity can affect specific phenotypes ass ...[more]

PMID: 32412234

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Project description:Time-resolved fluorescence and differential scanning calorimetry (DSC) were used to examine how two amino acids, L-phenylalanine (L-PA) and N-acetyl-DL-tryptophan (NAT), affect the temperature-dependent membrane affinity of two structurally similar coumarin solutes for 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles. The 7-aminocoumarin solutes, coumarin 151 (C151) and coumarin 152 (C152), differ in their substitution at amine position-C151 is a primary amine, and C152 is a tertiary amine-and both solutes show different tendencies to associate with lipid bilayers consistent with differences in their respective log-P-values. Adding L-PA to the DPPC vesicle solution did not change C151's propensity to remain freely solvated in aqueous solution, but C152 showed a greater tendency to partition into the hydrophobic bilayer interior at temperatures below DPPC's gel-liquid crystalline transition temperature (Tgel-lc). This finding is consistent with L-PA's ability to enhance membrane permeability by disrupting chain-chain interactions. Adding NAT to DPPC-vesicle-containing solutions changed C151 and C152 affinity for the DPPC membranes in unexpected ways. DSC data show that NAT interacts strongly with the lipid bilayer, lowering Tgel-lc by up to 2°C at concentrations of 10 mM. These effects disappear when either C151 or C152 is added to solution at concentrations below 10 μM, and Tgel-lc returns to a value consistent with unperturbed DPPC bilayers. Together with DSC results, fluorescence data imply that NAT promotes coumarin adsorption to the vesicle bilayer surface. NAT's effects diminish above Tgel-lc and imply that unlike L-PA, NAT does not penetrate into the bilayer but instead remains adsorbed to the bilayer's exterior. Taken in their entirety, these discoveries suggest that amino acids-and by inference, polypeptides and proteins-change solute affinity for lipid bilayers with specific effects that depend on individualized amino-acid-lipid-bilayer interactions.

Dataset Information

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

Publications

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

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