Genetic selection for mutations that impair the co-operative binding of lambda repressor

Bacteriophage λ repressor binds co-operatively to adjacent pairs of DNA target sites. A novel combination of positive genetic selections, involving two different operon fusions derived from P22 challenge phages, was used to isolate mutant λ repressors that have lost the ability to bind co-operatively to tandem sites yet retain the ability to bind a strong, single site. These cb (co-operative binding) mutations result in 10 different amino acid changes, which define eight residues in the carboxyl-terminus of repressor. Because challenge phage derivatives may be applied to study essentially any specific protein-DNA interaction, analogous combinations of genetic selections may be used to explore the ways that a variety of proteins interact to assemble regulatory complexes.     

Primary structure of the lambda repressor

The complete covalent structure of the bacteriophage lambda repressor has been determined by sequential Edman degradation, gas chromatographic-mass spectrometric peptide sequencing, and DNA sequencing of the repressor gene cI. The repressor is a single-chain, acidic protein containing 236 amino acids. The amino terminal 40 residues are highly polar and basic. Lysines and arginines in the sequence tend to be clustered.     

N-terminal sequence of phage lambda repressor

Summary Lambda repressor was purified from an E. coli strain which produces 150 times more lambda repressor than a single lysogen. The sequence of the fifty N-terminal residues was determined by automated Edman degradation. It contains 43% of all arginine and lysine residues of the chain and constitutes according to the genetic data of Oppenheim et al. (1975) a substantial part of the operator-DNA-binding site of the repressor.     

Sites of contact between lambda operators and lambda repressor.

DNA bearing lambda operator sequences was methylated by dimethyl sulfate (DMS) in the presence or absence of lambda repressor. Under the experimental conditions, DMS methylates only the purine residues. The presence of lambda repressor affects only the methylation of certain G residues in the operators. Repressor blocks the methylation of certain G's and enhances the methylation of other G's. Since the reactive ring-nitrogen of G lies in the major groove of double-stranded DNA, and the reactive ring-nitrogen of A lies in the minor groove, the above results imply that the repressor makes contacts in the major groove of the helix. The repressor effect on G-methylation is sharply confined to the three 17 base pair units within each lambda operator previously proposed as the repressor-binding sites.     

An engineered intersubunit disulfide enhances the stability and DNA binding of the N-terminal domain of lambda repressor

Site-directed mutagenesis has been used to replace Tyr-88 at the dimer interface of the N-terminal domain of lambda repressor with cysteine. Computer model building had suggested that this substitution would allow formation of an intersubunit disulfide without disruption of the dimer structure [Pabo, C. O., & Suchanek, E. G. (1986) Biochemistry (preceding paper in this issue)]. We find that the Cys-88 protein forms a disulfide-bonded dimer that is very stable to reduction by dithiothreitol and has increased operator DNA binding activity. The covalent Cys88-Cys88' dimer is also considerably more stable than the wild-type protein to thermal denaturation or urea denaturation. As a control, Tyr-85 was replaced with cysteine. A Cys85-Cys85' disulfide cannot form without disrupting the wild-type structure, and we find that this disulfide bond reduces the DNA binding activity and stability of the N-terminal domain.     

Comparison of the structures of cro and lambda repressor proteins from bacteriophage lambda

The three-dimensional structures of cro repressor protein and of the amino-terminal domain of lambda repressor protein, both from bacteriophage lambda, are compared. The second and third alpha-helices, alpha 2 and alpha 3, are shown to have essentially identical conformations in the two proteins, confirming the significance of the amino acid sequence homology previously noted between these and other DNA binding proteins in the region corresponding to these helices. The correspondence between the two-helical units in cro and lambda repressor protein is better than the striking agreement noted previously between two-helical units in cro and catabolite gene-activator protein. Parts of the first alpha-helices of repressor and cro show a structural correspondence that suggests a revised sequence homology between the two proteins in their extreme amino-terminal regions. In particular, there is a short loop between the alpha 1 and alpha 2 helices of lambda repressor that is missing from cro. This structural difference may account for the observed differences found with different cros and repressors in the pattern of phosphates whose ethylation prevents the binding of these proteins to their specific recognition sites. Although the two proteins have strikingly similar alpha 2-alpha 3 helical units that are presumed to bind to DNA in an essentially similar manner, stereochemical restrictions prevent the alpha 2-alpha 3 units of the respective proteins aligning on the DNA in exactly the same way.     

Structure and function of the repressor of bacteriophage lambda

Summary The high-affinity mutant cI gene of cIha (Nag et al. 1984) was cloned in the multicopy plasmid pBR322. In the resulting plasmid, pMD 102, a lacUV5 promoter was inserted giving the lacUV5-cIha fusion plasmid pMD 205. Bacteria carrying pMD 102 and pMD 205 contain 2.5 and 15 times, respectively, the level of repressor in a monolysogen of cIha. Results of the study of certain properties of the bacteria carrying these plasmids suggest that the ha repressor also has a higher affinity for the virulent mutant operators as well as the prm promoter of .     

Amino acid changes in the repressor of bacteriophage lambda due to temperature-sensitive mutations in its cI gene and the structure of a highly temperature-sensitive mutant repressor

The mutant cIts genes from seven different lambdacIts phages carrying tsU50, tsU9, tsU46, ts1, tsU51, tsI-22 and ts2 mutations were cloned in plasmid. The positions of these mutations and the resulting changes of amino acids in the repressor were determined by DNA sequencing. The first four mutations mapping in the N-terminal domain show the following changes: I21S, G53S, A62T and V73A, respectively. Of the three remaining mutations mapping in the C-terminal domain, cItsI-22 and cIts2 show N207T and K224E substitutions respectively, while the mutant cItsU51 gene carries F141I and P153L substitutions. Among these ts repressors, CIts2 having the charge-reversal change K224E was overexpressed from tac promoter in a plasmid and purified, and its structure and function were studied. Operator-binding studies suggest that the ts2 repressor is somewhat defective in monomer-dimer equilibrium and/or cooperativity even at permissive temperatures and loses its operator-binding ability very rapidly above 25 degrees C. Comparative studies of fluorescence and CD spectra, sulfhydryl group reactivity and elution behaviour in size-exclusion HPLC of both wild-type and ts2-mutant repressors at permissive and non-permissive temperatures suggest that the C-terminal domain of the ts2 repressor carrying a K224E substitution has a structure that does not favor tetramer formation at non-permissive temperatures.     

Increasing and decreasing protein stability: effects of revertant substitutions on the thermal denaturation of phage lambda repressor

The thermal denaturations of five revertant lambda repressors containing single amino acid substitutions in their N-terminal domains have been studied by differential scanning calorimetry. Two substitutions slightly decrease stability, and the remaining three render the protein more stable than wild type. The Gly48----Asn and Gly48----Ser proteins are 4 degrees C more stable than wild type. These two substitutions replace an alpha helical residue, and in each case a poor helix forming residue, glycine, is replaced by a residue with a higher helical propensity. We also present data showing that one revertant, Tyr22----Phe, has reduced operator DNA binding affinity despite its enhanced stability.     

Inactivation of prophage lambda repressor in vivo.

Jacob &; Monod (1961) postulated that prophage A induction results from the inactivation of the λ repressor by a cellular inducer. Although it has been shown that the phage A repressor is inactivated by the recA gene product in vitro (Roberts et al., 1978), we wanted to determine the action of the “cellular inducer” in vivo. Our results have led to a new model, which defines the relationship between the “cellular inducer” and the recA gene product.In order to quantitate the action of the cellular inducer on the λ repressor, we made use of bacteria with elevated cellular levels of the λ repressor (hyperimmune lysogens). We determined the kinetics of repressor inactivation promoted by three representative inducing treatments: ultraviolet light irradiation, thymine deprivation and temperature shift-up of tif-1 mutants.The kinetics of repressor decay in wild-type monolysogens indicate that repressor inactivation is a relatively slow cellular process that takes a generation time to reach completion. Incomplete inactivation of the repressor without subsequent prophage development may occur in a cell. We call this phenomenon detected at the biochemical level “subinduction”. In hyperimmune lysogens. subinduction is always the case.A high cellular level of A repressor that prevents prophage λ induction does not prevent induction of a heteroimmune prophage such as 434 or 80. Although the cellular inducer does not seem specific for any inducible prophage, it does not inactivate two prophage repressors present in a cell in a random manner. We have called this finding “preferential repressor inactivation”. Preferential repressor inactivation may be accounted for by considering that the intracellular concentration of a repressor determines its susceptibility to the action of the inducer.In bacteria with varying repressor levels, a fixed amount of repressor molecules is inactivated per unit of time irrespective of the initial repressor concentration. The rate of repressor inactivation depends on the catalytic capacity of the cellular inducer that behaves as a saturated enzyme. In wild-type bacteria the cellular inducer seems to be produced in a limited amount, to have a weak catalytic capacity and a relatively short half-life. The amount of the inducer formed after tif-1 expression is increased in STS bacteria overproducing a tif-1-modified RecA protein. This result is an indication that a modified form of the RecA protein causes repressor inactivation in vivo.From the results obtained we propose a model concerning the formation of the cellular inducer. We postulate that the cellular inducer is formed in a two-step reaction. The is model visualises how the RecA protein can be induced to high cellular concentrations, even though the RecAp protease molecules remain at a low concentration. The latter accounts for the limited proteolytic activity found in vivo.     

Dimerization of the operator binding domain of phage lambda repressor

Dimerization of lambda repressor is required for its binding to operator DNA. As part of a continuing study of the structural basis of the coupling between dimer formation and operator binding, we have undertaken 1H NMR and gel filtration studies of the dimerization of the N-terminal domain of lambda repressor. Five protein fragments have been studied: three are wild-type fragments of different length (1-102, 1-92, and 1-90), and two are fragments bearing single amino acid substitutions in residues involved in the dimer interface (1-102, Tyr-88----Cys; 1-92, Ile-84----Ser). The tertiary structure of each species is essentially the same, as monitored by the 1H NMR resonances of internal aromatic groups. However, significant differences are observed in their dimerization properties. 1H NMR resonances of aromatic residues that are involved in the dimer contact allow the monomer-dimer equilibrium to be monitored in solution. The structure of the wild-type dimer contact appears to be similar to that deduced from X-ray crystallography and involves the hydrophobic packing of symmetry-related helices (helix 5) from each monomer. Removal of two contact residues, Val-91 and Ser-92, by limited proteolysis disrupts this interaction and also prevents crystallization. The Ile-84----Ser substitution also disrupts this interaction, which accounts for the severely reduced operator affinity of this mutant protein.     

lambda Phage cro repressor interaction with DNA

We present here the complete identification of the resonances from the aromatic region of the 1H NMR spectrum of the cro repressor of the Escherichia coli lysogenic phage lambda. This was accomplished by the use of two-dimensional NMR analysis as well as specifically deuterated tyrosines. Not surprisingly, it shows that the published resonance assignment approached by more conventional methods by others includes substantial errors. The effect of complex formation with DNA was examined in the 1H NMR spectrum as well as in 19F NMR spectra from 3-fluorophenylalanine- or 3-fluorotyrosine-substituted cro repressors. The fluoro analogues show the practicality of using a structural gene cloned into an inducible system as a starting point to obtain both material and specific nuclear spin incorporation for NMR spectroscopy. The NMR data offer direct support for the speculative models of cro repressor-DNA complexes proposed from x-ray structure analysis on the protein alone.     

Multiphasic denaturation of the lambda repressor by urea and its implications for the repressor structure.

Urea denaturation of the lambda repressor has been studied by fluorescence and circular dichroic spectroscopies. Three phases of denaturation could be detected which we have assigned to part of the C-terminal domain, N-terminal domain and subunit dissociation coupled with further denaturation of the rest of the C-terminal domain at increasing urea concentrations. Acrylamide quenching suggests that at least one of the three tryptophan residues of the lambda repressor is in a different environment and its emission maximum is considerably blue-shifted. The transition in low urea concentration (midpoint approximately 2 M) affects the environment of this tryptophan residue, which is located in the C-terminal domain. Removal of the hinge and the N-terminal domain shifts this transition towards even lower urea concentrations, indicating the presence of interaction between hinge on N-terminal and C-terminal domains in the intact repressor.     

Bacteriophage lambda repressor allelic modulation of the Rex exclusion phenotype

The sensitivity of delta red-gam delta ren mutants of bacteriophage lambda to Rex exclusion by lambda rexA+ rexB+ lysogens is modulated by the prophage cI repressor allele. We show the following: (i) lambda spi156 delta nin5 forms plaques on a cI+-rexA+-rexB+ lysogen with 10(5)-fold higher efficiency than on cI[Ts]-rexA+-rexB+ derivatives. (ii) The cI[Ts]857 allele augmentation of Rex exclusion is recessive to cI+. (iii) The cI857-mediated increase in Rex exclusion activity involves the participation of a genetic element mapping outside of cI-rexA-rexB.     

A fine structure map of spontaneous and induced mutations in the lambda repressor gene, including insertions of IS elements

Summary Mutations at over 70 sites in the cI gene have been mapped by 4-factor crosses and assigned precise or approximate positions in the DNA sequence. 16 of 25 spontaneous mutations were insertions of IS1, IS3 or IS5 into AT-rich regions of cI. The 5-methylcytosine in the sequence Cm⁵CAGG is a hot spot for spontaneous cI amber mutations. Recombination frequencies between mutations were proportional to distance with the exception of amber mutations at 4 sites, including the host spot for spontaneous mutations. Mutations with a given phenotype are clustered on the genetic map. No missense mutations affecting repressor activity were found in the central one-third of cI, but 5 of 6 ind^- mutations were located in this region. The aminoterminal third of the gene contains the sites of most trans-dominant cI^- mutations, and of all ts mutations that result in repressors that are reversibly inactivated at high temperatures.