To analyze the role of amino acids in the steroid receptor DNA binding domain (DBD) recognition helix in binding of the receptor to the estrogen response element (ERE), we adapted the powerful P22 challenge phage selection system for use with a vertebrate protein. We used the progesterone receptor DNA binding domain and selected for mutants that gained the ability to bind to the ERE. We used a mutagenesis protocol based on degenerate oligonucleotides to create a large and diverse pool of mutants in which 10 nonconsensus amino acids in the DNA recognition helix of the progesterone receptor DNA binding domain were randomly mutated. After a single cycle of modified P22 challenge phage selection, 37 mutant proteins were identified, all of which lost the ability to bind to the progesterone response element. In gel mobility shift assays, approximately 70% of the genetically selected mutants bound to the consensus ERE with a >4-fold higher affinity than the naturally occurring estrogen receptor DBD. In the P-box region of the DNA recognition helix, the selected mutants contained the amino acids found in the wildtype estrogen receptor DBD, as well as other amino acid combinations seen in naturally occurring steroid/nuclear receptors that bind the aGGTCA half-site. We also obtained high affinity DBDs with Trp 585 as the first amino acid of the P-box, although this is not found in the known steroid/nuclear receptors. In the linker region between the two zinc fingers, G597R was by far the most common mutation. In transient transfections in mammalian cells using promoter interference assays, the mutants displayed enhanced affinity for the ERE. When linked to an activation domain, the transfected mutants activated transcription from ERE-containing reporter genes.We conclude that the P-box amino acids can display considerable variation and that the little studied linker amino acids play an important role in determining affinity for the ERE. This work also demonstrates that the P22 challenge phage genetic selection system, modified for use with a mammalian protein, provides a novel, single cycle selection for steroid/nuclear receptor DBDs with altered specificity and greatly enhanced affinity for their response elements.Steroid/nuclear receptors (1-4) and many transcription factors belong to protein superfamilies whose members bind to related, but distinct, DNA sequences. Individual proteins within the superfamily must bind to their DNA response elements with high specificity and affinity. The steroid/nuclear receptors bind to a specific DNA sequence, termed a hormone response element (HRE). 1 In general, HREs are composed of two core sequences 5Ј-AGNNCA-3Ј that are separated by a spacer region of 0 -6 nucleotides and are arranged as either a direct repeat or an inverted or everted palindrome.Recognition of HREs by steroid/nuclear receptors is mediated through a DNA binding domain (DBD) of 65-70 amino acids. The core DBD is highly conserved (3). Structural analyses of several DBDs (5-8) showed that they usually contain two ind...