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     

Substituting an alpha-helix switches the sequence-specific DNA interactions of a repressor

It has been suggested that many DNA-binding proteins use an alpha-helix for specific sequence recognition. We have used amino acid sequence homologies to identify the presumptive DNA-recognition helices in two related proteins whose structures are unknown--the repressor and cro protein of bacteriophage 434. The 434 repressor and cro protein each bind to three similar sites in the rightward phage 434 operator, OR, and they make different contacts in each binding site, as revealed by the chemical probe dimethyl sulfate. We substituted the putative recognition alpha-helix of 434 repressor with the putative recognition alpha-helix of 434 cro protein to create a hybrid protein named repressor*. The specific DNA contacts made by repressor* are like those of 434 cro protein.     

TET repressor.tet operator complex formation induces conformational changes in the tet operator DNA.

The structural changes of the tet operator DNA upon binding of the TET repressor protein are examined by circular dichroism. For this purpose a 70 bp DNA fragment was prepared which contains both tet operators. About 67% of the base pairs of this DNA are involved in specific interaction with the TET repressor. A rather large change in the CD of the DNA is induced by binding of the TET repressor. The shape of the CD difference spectrum is similar to the respective difference found for the lac operator DNA upon complex formation with the lac repressor. However, the effect induced by the TET repressor on tet operator DNA seems to comprise both the specific and non-specific effect of the lac repressor on the structure of DNA [Culard, F. and Maurizot, J.C. (1981) Nucl. Acids Res. 9, 5157-5184]. Specificity of binding is confirmed by the lack of any effect of the TET repressor on the CD of a 95 bp lac operator containing DNA fragment, by the reduced mobility of TET repressor.tet operator complexes on polyacrylamide gels under CD conditions, and by a titration experiment of tet operator DNA with TET repressor employing the CD change. The latter experiment reveals a stoichiometry of four TET repressors per tet operon control region.     

lac operator DNA modification in the presence of proteolytic fragments of the repressor protein

Singly end-labeled DNA fragments containing the lactose operator were methylated in the presence of the lactose repressor and homogeneous preparations of its proteolytic fragments. Binding of core protein produced by mild trypsin digestion yielded a methylation perturbation pattern that differed significantly from that elicited by binding to intact repressor, although similarities in the patterns for these related proteins were noted in the central, asymmetric region of the operator. An NH2-terminal peptide (residues 1 to 56) from lac repressor bound operator fragments in a nitrocellulose filter assay, but failed to perturb DNA methylation significantly relative to the pattern in the absence of peptide. Binding of hybrid tetramers of core and intact repressor monomers produced related but unique methylation patterns for the purines on the operator fragment. The general pattern of perturbation observed suggests preferred binding of a single NH2 terminus to the promoter-distal region of the operator and asymmetric interaction of the core region with the operator sequence. Differences in purine methylation patterns produced by the presence of effector complexes of repressor and core protein suggest the possible nature of changes in protein topology that result in the affinity changes accompanying induction.     

Anatomy of specific interactions between lambda repressor and operator DNA

Recognition of specific DNA sequences by proteins is essential for regulation of gene expression. To fully understand the recognition mechanism, it is necessary to understand not only the structure of the specific protein-DNA interactions but also the energetics. We therefore performed a computer analysis in which a phage DNA-binding protein, lambda repressor, was used to examine the changes in binding free energy (DeltaDeltaG) and its energy components caused by single base mutations. We then determined which of the calculated energy components best correlated with the experimental data. The experimental DeltaDeltaG values were well reproduced by the calculations. Component analysis revealed that the electrostatic and hydrogen bond energies were most strongly correlated with the experimental data. Among the 51 single base-substitution mutants examined, positive DeltaDeltaG values, corresponding to weakened binding, were caused by the loss of favorable electrostatic interactions and hydrogen bonds, the introduction of steric collisions and electrostatic repulsion, the loss of favorable interactions with a thymine methyl group, and the increase of unfavorable hydration energy from isolated DNA. This analysis also showed distinct patterns of recognition at A-T and G-C positions, as different combinations of energy components were involved in DeltaDeltaG caused by the two substitution types. We have thus been able to identify the energy components that most strongly correlate with sequence-dependent DeltaDeltaG and determine their contribution to the specificity of DNA sequence recognition by the lambda repressor. Application of this method to other systems should provide additional insight into the molecular mechanism of protein-DNA recognition.     

Specificity of the interaction between lambda cro repressor protein and operator DNA fragments

Cro repressor protein is known to interact with specific sites in the operator DNA. The cro protein of lambda phage was isolated and the mode of its interaction with three different DNA fragment, lambda-OR3 17mer, phi 80-OR2 19mer and CAP binding site 22-mer, were examined by the use of proton NMR. Some of the imino proton resonances of lambda-OR3 shifted and were broadened remarkably on addition of lambda-cro protein, which indicated the induction of conformational change with complexation. In the spectrum of phi 80-OR2 which has a six base pair sequence common to lambda-OR3 the signals of the common base pairs revealed slight shifts on addition of lambda-cro protein. The imino proton signals of the CAP site DNA, however, did not show any change at all on mixing with lambda-cro. Combining the data of photo CIDNP of lambda-cro, we could postulate the mode of interaction between lambda-cro repressor and operator DNA.     

Photoaffinity polyamines: sequence-specific interactions with DNA.

ANB-spermine is a photoaffinity analog of the naturally-occurring polyamine, acetylspermine. ANB-spermine was used to determine its binding sites on naked double stranded DNA, at the nucleotide level, using a modification of the primer extension technique. A total of 1,275 nucleotides was examined in 5 sequences of DNA from Saccharomyces cerevisiae. Binding sites were non-random. The primary determinant of binding was the presence of a thymidine residue. Secondary determinants appeared to depend on the secondary structure of the DNA, with runs of thymidines providing unusually poor binding sites while TA and, especially, TATA providing the strongest binding sites. The 'TATA element' upstream of the URA3 gene from S. cerevisiae was the strongest binding site. The data indicate that ANB-spermine binding to DNA is a probe for DNA secondary structure and suggest a role for polyamines in regulating the structure of chromatin in vivo.     

Interaction of the Mnt repressor with 37-base pair synthetic operator DNA fragments. Importance of symmetric GC pairs

Mnt repressor is indirectly responsible for the maintenance of lysogeny of the phage P22. This repressor interacts with a 21-base pair operator DNA constituting within it a 17-base pair perfect 2-fold symmetric sequence whose bases make a direct contact with the protein. We have synthesized six 37-base pair DNAs consisting of 21 base pair natural operator and its modifications in which certain symmetrically situated GC base pairs were replaced systematically with ATs to understand their importance. The binding interaction studies of Mnt repressor to such natural and modified operator DNAs reported here indicate that the GCs close to the center of symmetry make major contacts with the protein whereas, GCs nearer to the periphery form weak contacts. Methylation protection experiments indicated that when the GCs near the center of symmetry were replaced with AT, the central GC became more accessible for dimethyl sulfate methylation with possible conformational change in DNA. The circular dichroism studies indicated that upon repressor binding conformational changes in DNA takes place with a possible increase in helicity of the repressor protein.     

Escherichia coli integration host factor stabilizes bacteriophage Mu repressor interactions with operator DNA in vitro

Using gel retardation and DNase I protection techniques, we have demonstrated that the Escherichia coli integration host factor (IHF) stabilizes the interaction between Mu repressor and its cognate operator-binding sites in vitro. These results are discussed in terms of a model in which IHF may commit the phage to the lytic or lysogenic pathway depending on the occupancy of the operator sites by the repressor.     

Effect of sequence-specific interactions on the stability of helical conformations in polypeptides

A modification of the Zimm–Bragg two-state model for the helix–coil transition in polypeptides, which considers the effect of charge–dipole, charge–charge, and other specific interactions on helix stability, is presented. The new model introduces a series of adjustable parameters whose values are estimated by fitting to recent spectroscopic results on medium-sized peptides. This formalism, based on traditional two-state helix–coil transition models, provides a framework in which data on the helix contents of peptides of specific sequence can be rationalized by a statistical mechanical theory.     

Non-additivity of sequence-specific enzyme-DNA interactions in the EcoRI DNA methyltransferase.

We describe a novel strategy to characterize protein-DNA interactions involving monomeric enzymes such as DNA methyltransferases (Mtases). This strategy is applied to our investigation of the EcoRI DNA Mtase, which binds its double stranded recognition site 5'-G-AATTC-3' and methylates the central adenosine of each strand using S-adenosyl-L-methionine as the methyl donor. We show that prior methylation of adenosine in either strand does not perturb catalysis. In contrast, substrates substituted with deoxyinosine at either guanosine position (T-BMI5 and TI5-BM) show the minor groove residing N2 amino group of both guanosines contribute to DNA recognition since specificity constants for the modified substrates are reduced 13 and 39 fold. Similar analysis of a substrate containing deoxyinosine at both positions (TI5-BMI5) clearly shows that some communication occurs between the sites. To determine the extent to which structural changes in the DNA alone contribute to this lack of additivity, we performed DNA melting analysis of the singly and doubly substituted substrates, and also found non-additivity. Although our functional and structural analyses suggest that deoxyinosine incorporation causes long range conformational effects, the similarity of KmAdoMet for all substrates suggests that no large-scale structural changes occur in the Mtase-DNA-AdoMet complex. Our results support the following conclusions: 1) The non-additivity shown in this system contrasts with the widespread demonstration of additivity involving repressors [Lehming et al., 1990; Takeda et al., 1989; Ebright et al., 1987], suggesting that sequence discrimination by enzymes may involve more complex mechanisms. Further, this non-additivity precludes quantitative assignment of individual interactions and we suggest that future analyses of this and related enzyme systems with base analogs include detailed information about the long range structural consequences of individual substitutions. 2) Although TI5-BM and T-BMI5 are shown to be radically different by thermodynamic analysis, the similar specificity constants with the Mtase suggest that the underlying structural differences (e.g., altered helical parameters of the DNA) are not critical for sequence-recognition. 3) The significance of minor groove Mtase-DNA interactions to specificity is confirmed.     

The absence of non-local conformational changes in OR3 operator DNA on complexing with the cro repressor

The Interaction of the cro protein of lambda phage with a synthetic OR3 operator having 17 base pairs in length and with its 9 bp fragment has been studied using the circular dichroism (CD) method. In both cases, a considerable change in the CD of the samples was found in the region 260-300 nm upon the addition of the cro protein. The stoichiometry obtained by the CD titration was identical for OR3 and its 9 bp fragment: one duplex per dimeric cro. NaCl addition makes the complexes dissociate so that the 9 bp fragment becomes free at [NaCl] greater than 0.2 M while the whole OR3 becomes free at [NaCl] greater than 0.5 M. The CD spectra of both the free duplexes show a typical B-form conservative pattern with a positive CD band (270 nm) and a negative one (250 nm). The specific complexing of both the duplexes results in a substantial CD depression in the positive band. The most pronounced effect occurs at 280 nm. This spectral change is quite distinct from those in the B to A transition and in the non-cooperative winding of the DNA within the B-family of forms. The interaction of the cro protein with the non-operator DNAs, calf thymus DNA and a synthetic 10 bp duplex, reveals no visible CD changes at all. An inference is drawn that the CD change in the specific complexes is mainly due to the induced CD in tyr-26 upon its interaction with a specific base pair in the operator or its fragment, the operator DNA conformation being conserved in a B-like form as a whole.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tryptophan in alpha-helix 3 of Tet repressor forms a sequence-specific contact with tet operator in solution

The Tn10-encoded Tet repressor contains two tryptophan residues at positions 43 and 75. The typical tryptophan fluorescence is decreased upon binding of tet operator. The Tet repressor gene was engineered to replace either or both of the Trp codons by Phe codons. The resulting single tryptophan mutants are called F43 and F75 and the double mutant F43F75. The mutant proteins were purified to homogeneity. They recognize tet operator DNA only in the absence of the inducer tetracycline, indicating an intact tertiary structure of the engineered proteins. F75 and wild-type bind tet operator with the same association constant. The association constants of F43 and F43F75 with tet operator are about 3 orders of magnitude smaller. This indicates that Trp43 is important for tet operator recognition. Trp43 fluorescence is completely quenched in the complex with tet operator DNA while Trp75 remains unaffected. Binding to nonspecific DNA leads only to a 40% decrease of Trp43 fluorescence. This is interpreted as the contribution of the changed environment while the complete quench reflects a tight sequence-specific contact of tryptophan 43 to tet operator DNA. Trp43 is solvent-exposed, while Trp75 is buried in the hydrophobic interior of the protein. These results are discussed in light of the alpha-helix turn-alpha-helix DNA binding motif deduced from homology to other repressor proteins.     

In vivo interaction of Escherichia coli lac repressor N-terminal fragments with the lac operator

Escherichia coli lac repressor is a tetrameric protein composed of 360 amino acid subunits. Considerable attention has focused on its N-terminal region which is isolated by cleavage with proteases yielding N-terminal fragments of 51 to 59 amino acid residues. Because these short peptide fragments bind operator DNA, they have been extensively examined in nuclear magnetic resonance structural studies. Longer N-terminal peptide fragments that bind DNA cannot be obtained enzymatically. To extend structural studies and simultaneously verify proper folding in vivo, the DNA sequence encoding longer N-terminal fragments were cloned into a vector system with the coliphage T7 RNA polymerase/promoter. In addition to the wild-type lacI gene sequence, single amino acid substitutions were generated at positions 3 (Pro3----Tyr) and 61 (Ser61----Leu) as well as the double substitution in a 64 amino acid N-terminal fragment. These mutations were chosen because they increase the DNA binding affinity of the intact lac repressor by a factor of 10(2) to 10(4). The expression of these lac repressor fragments in the cell was verified by radioimmunoassays. Both wild-type and mutant lac repressor N termini bound operator DNA as judged by reduced beta-galactosidase synthesis and methylation protection in vivo. These observations also resolve a contradiction in the literature as to the location of the operator-specific, inducer-dependent DNA binding domain.     

Interaction of the bacteriophage P22 Arc repressor with operator DNA

Are repressor binds to a single, partially symmetric, 21 base-pair operator site that is centered between the -10 and -35 regions of the Pant promoter. Protection and interference experiments show that Arc makes contacts with the operator on one side of the DNA helix. Although Arc is a small protein (53 residues/subunit), it makes contacts that are farther from the center of the operator than those made by many larger repressors. These extended contacts include the phosphate groups at the ends of the 21 base-pair site. Under standard conditions (pH 7.5, 100 mM-KCl, 3 mM-MgCl2, 22 degrees C) half-maximal operator binding is observed at an Arc concentration of 2.5 X 10(-9) M and the protein-DNA complex is very stable (t1/2 approximately equal to 80 min).     

Tet repressor binding induced curvature of tet operator DNA.

Tet repressor dimer binds to two tet operator sites spaced by 30 bp in the Tn10 encoded tet regulatory DNA. The effect of repressor binding on the gel mobility of circular permutated DNA fragments containing either one or both operator sequences is reported. The EcoRI induced bending of DNA is used to compare the results with other protein binding induced structural perturbations of DNA. Tet repressor bends a DNA fragment with a single tet operator to an angle of 42 degrees +/- 7 degrees. The apparent bend angle of DNA fragments containing the tandem tet operator arrangement occupied by two Tet repressor dimers turns out to be 52 degrees +/- 9 degrees. These results are interpreted with respect to the end to end distances of the bent DNA fragments. They indicate that either the intervening tet regulatory DNA between the operators or the bound operator sequences themselves contain additional perturbations from the canonical B-DNA structure. This finding is discussed in the light of previously obtained results from CD, neutron scattering, and electrooptical studies.     

Tryptic core protein of lactose repressor binds operator DNA.

The core protein produced by mild proteolytic digestion of lactose repressor protein has been purified from native repressor by chromatography on phosphocellulose. The core protein isolated in this manner binds to operator DNA with an apparent dissociation constant of 10(-7) M, and the observed binding is decreased by the presence of inducer. Competition studies with nonspecific DNA indicate that the binding species in the core protein preparations is neither intact lactose repressor nor mixed tetramers containing varying numbers of intact NH2-terminal regions. This conclusion is supported by experiments designed to measure the rate of dissociation of the core protein from the operator DNA. Calculations based on the assumption that the isolated core protein binds similarly to the corresponding region in intact repressor protein indicate that the core region contributes approximately 40 to 50% of the energy of binding to operator DNA. Furthermore, the change in operator affinity upon inducer binding to core accounts for a minimum of 60% of the free energy change in binding to operator observed for the native protein. The demonstration that core protein binds to operator DNA requires a re-evaluation of the various models for repressor binding to DNA. A possible model based on the available information is presented.     

Characterization of the arginine repressor from Salmonella typhimurium and its interactions with the carAB operator.

Primer extension experiments showed that the argR gene, encoding the arginine repressor in Salmonella typhimurium, is transcribed from a single promoter that is negatively regulated by arginine. A repressor overproducing strain was constructed and the repressor was purified to homogeneity. Gel filtration, sedimentation and cross-linking studies established that the native repressor is a hexamer of identical 17,000 Mr subunits. Gel retardation experiments indicate that the apparent dissociation constant for repressor/carAB operator is 6 x 10(-12) M. These experiments showed that arginine is essential for binding of the repressor to the DNA and that pyrimidine nucleotides have no significant effect on this binding. These results indicate that the effect of pyrimidines on expression of the arginine sensitive "downstream" carAB promoter is not directly mediated by the arginine repressor. These experiments also suggest that a single hexamer binds to the carAB operator, which carries two previously defined "ARG box" sequences that characterize operators for arg genes. Gel retardation experiments with DNA fragments carrying the individual ARG boxes showed that both boxes are required for effective binding of the hexameric repressor to the operator, indicating that the ARG boxes comprise a single binding site for the repressor. Analysis of the potential secondary structure of the arginine repressor does not reveal any of the recognizable structural motifs common to a number of DNA-binding proteins. A combination of DNase I, premethylation interference, depurination and hydroxyl radical footprinting techniques were employed to characterize the interactions of the repressor with the carAB operator, with the results suggesting that the repressor predominantly interacts with A.T residues in this region. Comparative DNA sequence analysis of the known arginine operators of enteric bacteria further indicates that the specificity of interaction may be based more on the precise distance between two defined A.T-rich regions rather than on the specific nucleotide sequence.     

A sequence-specific single-strand DNA binding protein that contacts repressor sequences in the human GM-CSF promoter.

NF-GMb is a nuclear factor that binds to the proximal promoter of the human granulocyte-macrophage colony stimulating factor (GM-CSF) gene. NF-GMb has a subunit molecular weight of 22 kDa, is constitutively expressed in embryonic fibroblasts and binds to sequences within the adjacent CK-1 and CK-2 elements (CK-1/CK-2 region), located at approximately -100 in the GM-CSF gene promoter. These elements are conserved in haemopoietic growth factor (HGF) genes. NF-GMb binding requires the presence of repeated 5'CAGG3' sequences that overlap the binding sites for positive activators. Surprisingly, NF-GMb was found to bind solely to single-strand DNA, namely the non-coding strand of the GM-CSF CK-1/CK-2 region. NF-GMb may belong to a family of single-strand DNA binding (ssdb) proteins that have 5'CAGG3' sequences within their binding sites. Functional analysis of the proximal GM-CSF promoter revealed that sequences in the -114 to -79 region of the promoter containing the NF-GMb binding sites had no intrinsic activity in fibroblasts but could, however, repress tumour necrosis factor-alpha (TNF-alpha) inducible expression directed by downstream promoter sequences (-65 to -31). Subsequent mutation analysis showed that sequences involved in repression correlated with those required for NF-GMb binding.