NH2-terminal arm of phage lambda repressor contributes energy and specificity to repressor binding and determines the effects of operator mutations.

Abstract

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...

Recognition of DNA by the Helix-Turn-Helix Global Regulatory Protein Lrp Is Modulated by the Amino Terminus

“…The Nterminal 6-amino-acid arm of CI is required for proper DNA binding by this HTH protein; deleting the arm reduces operator binding by ϳ8,000-fold, and there is structural evidence for its role (Fig. 1C) (5,23,36,47). We report here that, as previously demonstrated for phage CI, the N-terminal arm of Lrp also affects its specificity and function.…”

“…The alignment also shows the similarity to the N terminus of the CI activator/repressor from phage. The "actives" are substitutions in this region of CI that retained the ability to maintain lysogeny (18,23,36). Letters in red are Lrp residues differing from EcoLrp.…”

Identification of the DNA binding domain of the phage lambda cII transcriptional activator and the direct correlation of cII protein stability with its oligomeric forms.

“…These results strongly suggest that the amino-terminal region of the cII protein has an unusual flexibility in its specific amino acid sequence. This amino-terminal flexibility is different from that found in the amino-terminal region of the k repressor (cI), which is known to be very important for DNA binding (Eliason et al 1985). It is of interest to note that cII binds selectively to DNA with a natural affinity that is approximately two orders of magnitude lower than that of the k repressor (Johnson et al 1979).…”

Isolation of Highly Persistent Mutants of Salmonella enterica Serovar Typhimurium Reveals a New Toxin-Antitoxin Module

“…Consistent with other antitoxins, the carboxy terminus is typically unstructured and can serve as a substrate for Lon protease or is important for binding the toxin (57,74). Many DNA binding proteins also have unstructured amino-or carboxy-terminal tails rich in lysine and arginine residues that assist the folded domain of the protein in contacting DNA (75)(76)(77). We propose that the product of shpB1, with its altered carboxy terminus, has reduced affinity for ShpA and, consequently, is not able to fully subdue toxin activity.…”