Several lines of evidence indicate that the phage X repressor recognizes its operator by using, in part, an a helix (the "recognition helix"), which it inserts into the major groove of DNA. In addition to its recognition helix, X repressor has an "arm," consisting of the first six amino acids, that wraps around the DNA helix. We constructed plasmids that, in Escherichia coli, direct the expression of derivatives of A repressor that lack the NH2-terminal one, three, six, or seven amino acids. We studied these modified proteins in vivo and in vitro, and from our results we argue that the arm: (i) contributes a large portion of the binding energy; (ii) helps to determine sequence specificity of binding and, in particular, the relative affinities for two wild-type binding sites; (iii) determines entirely repressor's response to one operator mutation (a "back-side" mutation); (iv) magnifies repressor's response to other operator mutations ("front-side" mutations); and (v) increases the sensitivity of repressor binding to salt concentration and temperature.Models based upon x-ray crystallographic studies (1-3) and upon sequence homologies (4,5) suggest that many prokaryotic regulatory proteins recognize their binding sites by a common mechanism. According to these models (6-8), such proteins make sequence-specific contacts by inserting an a helix, the "recognition helix," into the major groove of DNA. In one case it has been shown that replacing the recognition helix from one protein with the recognition helix of another alters the binding specificity (9). Phage X repressor is unusual because, in addition to a recognition helix, it also bears a flexible NH2-terminal "arm" that consists of the first six amino acids and evidently wraps around the DNA (10). According to the current picture (6), each monomer of a repressor dimer inserts a recognition helix into the major groove on one face of the DNA helix, the "front side." The two arms of the dimer wrap around the DNA, making specific contacts in the major groove on the opposite face, the "back side," and also making nonspecific contacts to the DNA phosphates. Phage X Cro protein, which lacks an arm, binds to the same operator sites as does repressor. Cro also binds as a dimer, inserting a recognition helix from each monomer into the major groove on the front side of the DNA helix, but does not contact the back side of the operator (7).Previous experiments have shown that phage X repressor derivatives with defects in the arm have a reduced affinity for operator. A point mutation changing Lys-4 to Gln-4 greatly reduced repressor binding (11). Proteolytic removal of the NH2-terminal three amino acid residues decreased the affinity of a repressor fragment [the NH2-terminal domain (12)] for an operator site and altered the ability of the same repressor fragment to protect guanines in the operator from methylation by dimethyl sulfate (10). Since removal of the first three amino acids does not change the global conformation of the protein (13), alterations made to the...

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Flexible regions of proteins play an important role in catalysis, ligand binding, and macromolecular interactions. Because of its enhanced sensitivity to motional narrowing, two-dimensional coupling constant J-correlated 1H NMR may be used to observe these regions selectively. Dynamic filtering is an intrinsic feature of this experiment because cross-peak amplitude decays rapidly as linewidths approach the coupling constant. We demonstrate here the flexibility of the NH2-terminal arm of phage A, repressor, which is thought to wrap around the double helix in the repressor-operator complex. The assignment of arm resonances is made possible by the construction of mutant repressor genes containing successive NH2-terminal deletions.Phage X repressor regulates gene expression by binding to specific operator sequences in the phage genome (1). The determination of the crystal structure of its operator-binding domain has focused attention upon the molecular details of protein-DNA recognition (2)(3)(4). This domain, consisting of the first 92 residues, contains five a helices and an extended NH2-terminal arm. The former fold into a globular domain whose second and third helices are thought to interact with the operator. Homologous helices are observed also in a number of other DNA-binding proteins and may provide a general scaffold for operator recognition (5-10). The NH2- terminal arm (NH2-Ser-Thr-Lys-Lys-Lys-Pro-) is thought to wrap around the double helix to contact the back of the operator site (11) as illustrated in Fig. 1. Flexibility of the arm is an essential feature of this model, since otherwise there would be large kinetic barriers to operator binding and release.We report here a new application of two-dimensional NMR that directly demonstrates the flexibility of the NH2-terminal arm of intact phage X repressor. The coupling constant J-correlated pulse sequence (12, 13, §) is shown to be a dynamic filter, enabling the selective observation of mobile regions of macromolecules. By a coincidence of time scales, this filter attenuates signals from spins whose transverse relaxation is dominated by macromolecular rotation. Accordingly, the correlated spectrum of X repressor contains only a few spin systems. These must belong to residues that are reorienting more quickly than the rate of overall rotation. Most of these resonances are rigorously assigned to the NH2-terminal arm through the study of mutant repressors containing successive NH2-terminal deletions. MATERIALS AND METHODSWild-type phage X repressor was purified as described (14, 15 (11). in this model, the NH2-terminal arms (thick lines) of the protein (hatched dimer) wrap around the double helix (cylinder) to contact the back of the operator site. An essential feature of this model is the flexibility of the arm. In crystallographic studies of the isolated operator-binding domain (residues 1-92), no electron density was observed for serine-1, threonine-2, and lysine-3. In addition, residues 4-6 extended away from the rest of the domain (2).in the l...