The role of DNA shape in protein-DNA recognition is a key factor in how proteins bind to specific DNA sequences. This study shows that proteins often recognize DNA through interactions with the minor groove, particularly in narrow regions. Narrow minor grooves enhance the negative electrostatic potential of DNA, which is exploited by proteins like the Hox protein Sex combs reduced (Scr) to distinguish between similar sequences. The ability to detect these shape and electrostatic variations allows proteins to recognize DNA with high specificity, even when the minor groove offers few opportunities for base-specific hydrogen bonds. Arginines are significantly enriched in narrow minor grooves, especially those with widths less than 5.0 Å. This is because arginines can recognize the enhanced electrostatic potential in these narrow regions. The study also shows that AT-rich sequences tend to narrow the minor groove, which is often associated with A-tracts—sequences of four or more As or Ts that exclude the flexible TpA step. Even shorter A-tracts, as short as three base pairs, can narrow the minor groove, contributing to DNA binding specificity. The study further demonstrates that arginines recognize the enhanced electrostatic potential in narrow minor grooves, which is crucial for DNA recognition. For example, in the Hox protein Ultrabithorax (Ubx) complex, arginine residues insert into narrow regions formed by A-tracts. Similarly, in the MogR repressor complex, arginines bind to narrow regions in AT-rich DNA, contributing to the specificity of DNA binding. In the nucleosome, arginines are found in narrow minor groove regions, which are associated with A-tracts. These regions are important for DNA bending around the histone core. The periodicity of A-tracts in nucleosomal DNA is also significant, as they help facilitate DNA bending and contribute to the recognition of DNA sequences by proteins. The study also highlights the physical mechanisms underlying the electrostatic potential in narrow minor grooves. These mechanisms are influenced by the dielectric properties of the surrounding environment, which affect the distribution of electrostatic potentials. The results suggest that the shape and electrostatic properties of DNA play a critical role in protein-DNA recognition, and these factors must be considered when predicting transcription factor binding sites in genomes. Overall, the study provides new insights into the structural and energetic origins of protein-DNA binding specificity, emphasizing the importance of DNA shape in the recognition of specific DNA sequences by proteins.The role of DNA shape in protein-DNA recognition is a key factor in how proteins bind to specific DNA sequences. This study shows that proteins often recognize DNA through interactions with the minor groove, particularly in narrow regions. Narrow minor grooves enhance the negative electrostatic potential of DNA, which is exploited by proteins like the Hox protein Sex combs reduced (Scr) to distinguish between similar sequences. The ability to detect these shape and electrostatic variations allows proteins to recognize DNA with high specificity, even when the minor groove offers few opportunities for base-specific hydrogen bonds. Arginines are significantly enriched in narrow minor grooves, especially those with widths less than 5.0 Å. This is because arginines can recognize the enhanced electrostatic potential in these narrow regions. The study also shows that AT-rich sequences tend to narrow the minor groove, which is often associated with A-tracts—sequences of four or more As or Ts that exclude the flexible TpA step. Even shorter A-tracts, as short as three base pairs, can narrow the minor groove, contributing to DNA binding specificity. The study further demonstrates that arginines recognize the enhanced electrostatic potential in narrow minor grooves, which is crucial for DNA recognition. For example, in the Hox protein Ultrabithorax (Ubx) complex, arginine residues insert into narrow regions formed by A-tracts. Similarly, in the MogR repressor complex, arginines bind to narrow regions in AT-rich DNA, contributing to the specificity of DNA binding. In the nucleosome, arginines are found in narrow minor groove regions, which are associated with A-tracts. These regions are important for DNA bending around the histone core. The periodicity of A-tracts in nucleosomal DNA is also significant, as they help facilitate DNA bending and contribute to the recognition of DNA sequences by proteins. The study also highlights the physical mechanisms underlying the electrostatic potential in narrow minor grooves. These mechanisms are influenced by the dielectric properties of the surrounding environment, which affect the distribution of electrostatic potentials. The results suggest that the shape and electrostatic properties of DNA play a critical role in protein-DNA recognition, and these factors must be considered when predicting transcription factor binding sites in genomes. Overall, the study provides new insights into the structural and energetic origins of protein-DNA binding specificity, emphasizing the importance of DNA shape in the recognition of specific DNA sequences by proteins.