Mutants in position 69 of the Trp repressor ofEscherichia coli K12 with altered DNA-binding specificity

Structural analysis by X-ray crystallography has indicated that direct contact occurs between Arg69, the second residue of the first helix of the helix-turn-helix (HTH) motif of the Trp repressor, and guanine in position 9 of the α-centred consensustrp operator. We therefore replaced residue 69 of the Trp repressor with Gly, Ile, Leu or Gln and tested the resultant repressor mutants for their binding to synthetic symmetrical α-or β-centredtrp operator variants, in vivo and in vitro. We present genetic and biochemical evidence that Ile in position 69 of the Trp repressor interacts specifically with thymine in position 9 of the α-centredtrp operator. There are also interactions with other bases in positions 8 and 9 of the α-centredtrp operator. In vitro, the Trp repressor of mutant RI69 binds to the consensus α-centredtrp operator and a similartrp operator variant that carries a T in position 9. In vivo analysis of the interactions of Trp repressor mutant RI69 with symmetrical variants of the β-centredtrp operator shows a change in the specificity of binding to a β-centred symmetricaltrp operator variant with a gua-nine to thymine substitution in position 5, which corresponds to position 9 of the α-centredtrp operator.     

The possible roles of residues 79 and 80 of the Trp repressor from Escherichia coli K-12 in trp operator recognition

We constructed mutants of the Trp repressor from Escherichia coli K-12 with all possible single amino acid exchanges at positions 79 and 80 (residues 1 and 2 of the recognition helix). We tested these mutants in vivo by measuring the repression of synthesis of β-galactosidase with symmetric variants of α- and β-centered trp operators, which replace the lac operator in a synthetic lac system. The Trp repressor carrying a substitution of isoleucine 79 by lysine, showed a marked specificity change with respect to base pair 7 of the α-centered trp operator. Gel retardation experiments confirmed this result. Trp repressor mutant IR79 specifically recognizes a trp operator variant with substitutions in positions 7 and 8. Another mutant, with glycine in position 79, exhibited loss of contact at base pair 7. We speculate that the side chain of Ile79 interacts with the AT base pairs 7 and 8 of the α-centered trp operator, possibly with the methyl groups of thymines. Replacement of thymine in position 7 or 8 by uracil confirms the involvement of the methyl group of thymine 8 in repressor binding. Several Trp repressor mutants in position 80 (i.e. AI80, AL80, AM80 and AP80) broaden the specificity of the Trp repressor for α-centered trp operator variants with exchanges in positions 3, 4 and 5.     

Mutants in position 69 of the Trp repressor ofEscherichia coli K12 with altered DNA-binding specificity

Structural analysis by X-ray crystallography has indicated that direct contact occurs between Arg69, the second residue of the first helix of the helix-turn-helix (HTH) motif of the Trp repressor, and guanine in position 9 of the -centred consensustrp operator. We therefore replaced residue 69 of the Trp repressor with Gly, Ile, Leu or Gln and tested the resultant repressor mutants for their binding to synthetic symmetrical -or -centredtrp operator variants, in vivo and in vitro. We present genetic and biochemical evidence that Ile in position 69 of the Trp repressor interacts specifically with thymine in position 9 of the -centredtrp operator. There are also interactions with other bases in positions 8 and 9 of the -centredtrp operator. In vitro, the Trp repressor of mutant RI69 binds to the consensus -centredtrp operator and a similartrp operator variant that carries a T in position 9. In vivo analysis of the interactions of Trp repressor mutant RI69 with symmetrical variants of the -centredtrp operator shows a change in the specificity of binding to a -centred symmetricaltrp operator variant with a gua-nine to thymine substitution in position 5, which corresponds to position 9 of the -centredtrp operator.     

The possible roles of residues 79 and 80 of the Trp repressor from Escherichia coli K-12 in trp operator recognition

We constructed mutants of the Trp repressor from Escherichia coli K-12 with all possible single amino acid exchanges at positions 79 and 80 (residues 1 and 2 of the recognition helix). We tested these mutants in vivo by measuring the repression of synthesis of -galactosidase with symmetric variants of - and -centered trp operators, which replace the lac operator in a synthetic lac system. The Trp repressor carrying a substitution of isoleucine 79 by lysine, showed a marked specificity change with respect to base pair 7 of the -centered trp operator. Gel retardation experiments confirmed this result. Trp repressor mutant IR79 specifically recognizes a trp operator variant with substitutions in positions 7 and 8. Another mutant, with glycine in position 79, exhibited loss of contact at base pair 7. We speculate that the side chain of Ile79 interacts with the AT base pairs 7 and 8 of the -centered trp operator, possibly with the methyl groups of thymines. Replacement of thymine in position 7 or 8 by uracil confirms the involvement of the methyl group of thymine 8 in repressor binding. Several Trp repressor mutants in position 80 (i.e. AI80, AL80, AM80 and AP80) broaden the specificity of the Trp repressor for -centered trp operator variants with exchanges in positions 3, 4 and 5.     

How Trp repressor binds to its operator.

We propose that the generally accepted model of a single Trp repressor dimer binding to a center of symmetry in the natural trp operator (Otwinowski et al., 1988) is wrong. We show here that the Trp repressor binds to a sequence whose center is located four base pairs either to the right or to the left of the central axis of symmetry that was previously identified. We show that: (i) the oligonucleotide used by Otwinowski et al. is not retarded by the Trp repressor in a mobility shift assay under conditions wherein a shorter oligonucleotide carrying our consensus sequence is retarded, (ii) that methylation protection experiments on the full natural operator sequence and the short oligonucleotide protect similar patterns and (iii) that by varying every base in the shorter oligonucleotide, we can demonstrate an optimal sequence for Trp repressor binding.     

Computational design of a substrate specificity mutant of a protein

The wild-type trp repressor of E. coli bound 5-methoxytryptophan, a Trp analogue, less tightly than Trp. A mutant repressor (Val⁵⁸→Ala) that should bind 5-methoxytryptophan preferentially to Trp was computationally designed by free-energy calculations accompanied by free-energy decomposition. The designed mutant was demonstrated by experiments to bind 5-methoxytryptophan more tightly than Trp, consistent with the computational prediction. This success indicates the usefulness of free energy decomposition in protein design. Proteins 26:459–464 © 1996 Wiley-Liss, Inc.     

Interaction of the trp repressor with trp operator DNA fragments

The interaction of the trp repressor with several trp operator DNA fragments has been examined by DNA gel retardation assays and by circular dichroism, in the absence and presence of the corepressor l-tryptophan. The holorepressor binds stoichiometrically to both the trpO and aroH operators, forming 1:1 complexes. In the presence of excess protein, additional complexes are formed with these operator fragments. The relative electrophoretic mobilities of the 1:1 complexes differ significantly for trp and aroH operators, indicating that they differ substantially in gross structure. A mutant trp operator, trpO ^c, has low affinity for the holorepressor, and forms only complexes with stoichiometries of 2:1 (repressor: DNA) or higher, which have a very low electrophoretic mobility. Specific binding is also accompanied by a large increase in the intensity of the near ultraviolet circular dichroism, with only a small blue shift, which is consistent with significant changes in the conformation of the DNA. Large changes in the chemical shifts of three resonances in the ³¹P NMR spectrum of both the trp operator and the aroH operator occur on adding repressor only in the presence of L-tryptophan, consistent with localised changes in the backbone conformation of the DNA.Abbreviations CD circular dichroism - trpO, trpR aroH trp operator fragments - trpO ^c trpMH mutant trp operator fragments     

Studies of the Escherichia coli Trp repressor binding to its five operators and to variant operator sequences.

The Escherichia coli Trp repressor binds to promoters of very different sequence and intrinsic activity. Its mode of binding to trp operator DNA has been studied extensively yet remains highly controversial. In order to examine the selectivity of the protein for DNA, we have used electromobility shift assays (EMSAs) to study its binding to synthetic DNA containing the core sequences of each of its five operators and of operator variants. Our results for DNA containing sequences of two of the operators, trpEDCBA and aroH are similar to those of previous studies. Up to three bands of lower mobility than the free DNA are obtained which are assigned to complexes of stoichiometry 1 : 1, 2 : 1 and 3 : 1 Trp repressor dimer to DNA. The mtr and aroL operators have not been studied previously in vitro. For DNA containing these sequences, we observe predominantly one retarded band in EMSA with mobility corresponding to 2 : 1 complexes. We have also obtained retardation of DNA containing the trpR operator sequence, which has only been previously obtained with super-repressor Trp mutants. This gives bands with mobilities corresponding to 1 : 1 and 2 : 1 complexes. In contrast, DNA containing containing a symmetrized trpR operator sequence, trpRs, gives a single retarded band with mobility corresponding solely to a 1 : 1 protein dimer-DNA complex. Using trpR operator variants, we show that a change in a single base pair in the core 20 base pairs can alter the number of retarded DNA bands in EMSA and the length of the DNase I footprint observed. This shows that the binding of the second dimer is sequence selective. We propose that the broad selectivity of Trp repressor coupled to tandem 2 : 1 binding, which we have observed with all five operator sequences, enables the Trp repressor to bind to a limited number of sites with diverse sequences. This allows it to co-ordinately control promoters of different intrinsic strength. This mechanism may be of importance in a number of promoters that bind multiple effector molecules.     

In vivoandin vitroStudies of TrpR-DNA Interactions

The binding of tryptophan repressor (TrpR) to its operators was examined quantitatively usingin vitroandin vivomethods. DNA sequence requirements for 1:1 and tandem 2 :1 (TrpR : DNA) binding in various sequence contexts were studied. The results indicate that the optimal half-site sequence for recognition by one helix-turn-helix motif of one TrpR dimer is_{3′ CNTGA 5′}^{5′ GNACT 3′}, consistent with contacts observed by X-ray diffraction analysis of cocrystalline 1:1 and 2 :1 complexes. Half-sites can be paired to form a palindrome either by direct abutment, forming the nucleation site for a tandem 2 :1 complex, or with an 8-base-pair spacer, forming a 1:1 target. Dimethylsulfate (DMS) methylation-protection footprintingin vitroof 1:1 and 2 :1 complexes formed sequentially on the two unequal half-site pairs of thetrpEDCBA operator fromSerratia marcescensindicated an obligate hierarchy of site occupancy, with one half-site pair serving as the nucleation site for tandem binding. DMS footprinting ofEscherichia colioperatorsin vivoshowed that, over a wide range of intracellular TrpR concentration, thetrpEDCBA operator is occupied by three repressor dimers,aroH is occupied by two dimers, and the 1:1 binding mode is used on thetrpR operator. The coexistence of these distinct occupancy states implies that changes in protein concentration affect only the fractional occupancy of each operator rather than the binding mode, which is determined by the number of half-site sequences present in the operator region. Cooperativity of tandem complex formation measured by gel retardation using a symmetrized synthetic operator containing identical, optimal sites spaced as in natural operators was found to be modest, implying a maximum coupling free energy of ∼−2 kcal/mol. On other sequences the apparent degree of cooperativity, as well as the apparent affinity, varied with sequence and sequence context in a manner consistent with the structural models and which suggests compensation between affinity and cooperativity as a mechanism that allows tolerance of operator sequence variation.     

DNA-induced conformational changes in bacteriophage 434 repressor

Although bacteriophage 434 repressor binds to its specific DNA sites only as a dimer, formation of the dimers in solution occurs at concentrations three orders of magnitude higher than those needed to bind the 434 operator DNA. Our results suggest that both specific and non-specific DNA induce conformational changes in repressor that lead to formation of repressor dimers. The repressor conformational changes induced by DNA occur at concentrations much lower than those needed for binding of repressor, suggesting that the alternative conformations of repressor persist even if the protein is not in direct contact with DNA. Hence, DNA acts in a "catalytic" fashion to induce a steady-state amount of an alternative repressor conformation that has an enhanced affinity for its specific binding site. These findings suggest that the repressor conformer induced by non-specific DNA is the form of the repressor that is optimized for searching for DNA binding sites along non-specific DNA. Upon finding a binding site, the repressor protein undergoes an additional conformational change that allows it to "lock-on" to its specific site.     

Radiation-induced tetramer-to-dimer transition of Escherichia coli lactose repressor

The wild type lactose repressor of Escherichia coli is a tetrameric protein formed by two identical dimers. They are associated via a C-terminal 4-helix bundle (called tetramerization domain) whose stability is ensured by the interaction of leucine zipper motifs. Upon in vitro γ-irradiation the repressor losses its ability to bind the operator DNA sequence due to damage of its DNA-binding domains. Using an engineered dimeric repressor for comparison, we show here that irradiation induces also the change of repressor oligomerisation state from tetramer to dimer. The splitting of the tetramer into dimers can result from the oxidation of the leucine residues of the tetramerization domain.     

The RES domain toxins of RES‐Xre toxin‐antitoxin modules induce cell stasis by degrading NAD+

Type II toxin‐antitoxin (TA) modules, which are important cellular regulators in prokaryotes, usually encode two proteins, a toxin that inhibits cell growth and a nontoxic and labile inhibitor (antitoxin) that binds to and neutralizes the toxin. Here, we demonstrate that the res‐xre locus from Photorhabdus luminescens and other bacterial species function as bona fide TA modules in Escherichia coli. The 2.2 Å crystal structure of the intact Pseudomonas putida RES‐Xre TA complex reveals an unusual 2:4 stoichiometry in which a central RES toxin dimer binds two Xre antitoxin dimers. The antitoxin dimers each expose two helix‐turn‐helix DNA‐binding domains of the Cro repressor type, suggesting the TA complex is capable of binding the upstream promoter sequence on DNA. The toxin core domain shows structural similarity to ADP‐ribosylating enzymes such as diphtheria toxin but has an atypical NAD⁺‐binding pocket suggesting an alternative function. We show that activation of the toxin in vivo causes a depletion of intracellular NAD⁺ levels eventually leading to inhibition of cell growth in E. coli and inhibition of global macromolecular biosynthesis. Both structure and activity are unprecedented among bacterial TA systems, suggesting the functional scope of bacterial TA toxins is much wider than previously appreciated.     

Immunity repressor of bacteriophage P2: Identification and DNA-binding activity

The product of gene C of the temperate bacteriophage P2, the immunity repressor, can be detected as a unique band eluting from phosphocellulose columns at 0.12 m-potassium phosphate when differentially labelled with a radioactive amino acid: the band is absent when phages that either have lost gene C through deletion or carry a suppressor-sensitive mutation in the gene are used. The repressor in its monomeric form is about 11,000 in molecular weight. At near physiological salt concentrations, the form predominantly recovered is the dimer.In filter-binding assays, the partially purified repressor binds wild-type P2 DNA strongly. It does not bind DNA of P2 vir94, a deletion that removes all the genetic elements involved in the regulation of lysogeny; it also does not bind, or binds inefficiently, DNA of P2 vir3, a mutation in the operator that controls the early replicative functions of P2. At the concentrations employed, the dimer is the active form in binding.The P2 repressor clearly differs in several features from the well-studied immunity repressor of bacteriophage lambda.     

Complex structure of the membrane nuclease of Streptococcus pneumoniae revealed by two-dimensional electrophoresis

Close contacts between Escherichia coli RNA polymerase and specific purine residues in the tryptophan (trp) operon promoter of Salmonella typhimurium were revealed using the methylating agent dimethyl sulfate. RNA polymerase bound to trp promoter DNA caused alterations in the rate of methylation at seven specific sites; in the anti-sense strand, guanine residues at positions ?37, ?34 and ?2 showed enhanced methylation, while those at positions ?14, ?6 and +3 showed reduced methylation. In the sense strand, only the guanine residue at ?32 showed reduced methylation. No RNA polymerase contacts with adenine residues were observed. Using the same method, close interactions between E. coli trp repressor and purine residues in the trp operator of S. typhimurium were examined. Bound trp repressor alters the methylation rates of both guanine and adenine residues from positions ?25 to +3. The points of contact are distributed rather symmetrically on both DNA strands. Three points of close contact are shared by RNA polymerase and trp repressor, supporting previous models of trp repressor action.     

Conformational prediction and circular dichroism studies on the lac repressor

Using the protein predictive model of Chou & Fasman (1974b), the secondary structure of the lac repressor has been elucidated from its amino acid sequence of 347 residues. The conformation is predicted to contain 37% α-helix and 35% β-sheet for the repressor, and 29% helix and 41% β-sheet for the trypsin-resistant core (residues 60 to 327). Circular dichroism studies indicate that native lac repressor contains 40% helix and 42% β-sheet, while the core has 16% helix and 54% β-sheet, in general agreement with the predicted conformation. The sharp reduction in helicity for the trypsinized lac repressor could be due to the loss of two long helical regions, 26–45 and 328–344, predicted at both terminals. There are extensive β-sheets predicted in the 215–324 region, which may be responsible for tetrameric stabilization found in both the lac repressor and the core. Residues 17 to 33 were previously predicted by Adler et al. (1972) to be helical and were proposed to bind in the major groove of DNA. However, the present analysis shows that there are two anti-parallel β-sheet regions: 4–7 and 17–24 at the N-terminal as well as 315–318 and 321–324 at the C-terminal of the lac repressor. These β-sheet pairs may assume the twisted “polypeptide double helix” conformation (Carter & Kraut, 1974) and bind to complementary regions in the major groove of DNA. The OH groups of Tyr at the N-terminal and those of Thr and Ser side chains, in both β-sheets at the N and C-terminal ends, could form hydrogen bonds to specific sites on the lac operator. There are 23 reverse β-turns predicted that may control the tertiary folding of the lac repressor, which is essential for operator binding. The behavior of several lac repressor mutants can be satisfactorily explained in terms of polar to non-polar group replacements as well as conformational changes in light of the present predicted model.