Genomic DNA contains all the information necessary for the survival and functioning of a cell. The functions of a cell are mainly carried out by proteins, the primary sequences of which are coded as a sequence of nucleotides in the genome. The decoding process starts with high-fidelity copying of the DNA sequence to mRNA (transcription). The mRNA provides a template for synthesizing the corresponding protein chain (translation) from amino acids. The processes of transcription and translation form the backbone of the process of gene expression. There are thousands of protein-coding genes in a genome, but they cover only a minor fraction of the whole genome in higher eukaryotes. The other part, the non-protein-coding part, of the genome plays a crucial role in many functions, particularly in regulating the expression of the genes. The physical and chemical properties of the DNA double helix are pivotal in regulating gene expression. This chapter recapitulates some of the properties of the DNA double helix that play important roles in the gene regulatory process. Apart from the basic structure of the double helix, we have discussed the origin of the conformational heterogeneity of the DNA as well as the flexibility and dynamics of the double helix. DNA-binding proteins play crucial roles in regulating the flow of information. We have described the structure of different classes of DNA-binding proteins and how these proteins recognize specific nucleotide sequences in the DNA.