DNA-binding proteins. The physiological role of DNA-binding proteins is determined by the affinity and specificity of the DNA-protein interaction. These properties depend upon the precise interactions between amino acids in the DNA-binding protein and nucleotides in the DNA-binding site. Protein-protein interactions are required for efficient DNA-protein interactions as well. DNA-binding sites. Gene expression is often regulated by proteins that activate or repress transcription by binding to short, specific DNA sequences. Such cis-acting sites are usually located close to the promoter (RNA polymerase binding site) for the regulated gene. Sometimes DNA-binding proteins bring together two distant DNA-binding sites and loop out the intervening DNA. When positioned close enough to the promoter, almost any specific DNA binding protein may act as a repressor. Repression may be caused by competition with RNA polymerase binding to the promoter, blocking the isomerization of RNA polymerase required for initiation of transcription, bending the DNA, or inhibiting the elongation of RNA polymerase after transcription has initiated. Repression due to binding to a single site is usually strongest when the binding site is close to the promoter (usually within about 20 bp).
How can features of a DNA-binding site or DNA-binding protein which determine DNA-protein interactions be identified? Many DNA-binding proteins have common structural motifs involved in DNA-protein interactions, but it is not possible to identify the amino acids involved in DNA-binding by simple sequence gazing. For example, even though a protein may have an amino acid sequence with all the features of a consensus DNA-binding motif, that amino acid sequence may not be involved in DNA-binding. Furthermore, different DNA-binding proteins that share common motifs may recognize DNA differently. Therefore, the critical nucleotides in a DNA-binding site and the interacting amino acids in DNA-binding protein must be determined empirically. The DNA-protein interaction can be dissected using in vitro biochemical approaches or using in vivo genetic approaches. Even when DNA-protein interactions have been characterized in vitro, genetic analysis of the interaction is needed to confirm that the specific DNA-protein contacts identified in vitro are necessary and sufficient for DNA-binding under physiological conditions.
Any genetic approach for characterizing DNA-protein interactions that regulate gene expression requires the isolation of mutants that affect regulation. A general approach for isolating regulatory mutations is to place the expression of a selectable reporter gene under the control of the specific DNA-protein interaction. Transcription of the reporter gene is then regulated by binding of the protein to a specific DNA sequence upstream of the reporter gene. This approach can be applied to many different types of DNA binding proteins.
Maloy, S., V. Stewart, and R. Taylor. 1996. Genetic Analysis of Pathogenic Bacteria. Cold Spring Harbor Laboratory Press, NY.
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Last modified July 15, 2002