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.     

Thermal denaturation of engineered tet repressor proteins and their complexes with tet operator and tetracycline studied by temperature gradient gel electrophoresis

The effects of Trp to Phe exchanges in the Tet repressor on the thermal stability of the proteins and their complexes with operator DNA and inducer have been studied by temperature gradient polyacrylamide gel electrophoresis. The denaturation temperatures obtained by this method are compared with the results from temperature-dependent fluorescence and binding activities of the proteins. It is established that exchanging the interior Trp75 to Phe reduces the thermal stability of the Tet repressor by 8 degrees C while exchanging the exterior Trp43 to Phe has no effect on the stability of the protein. Binding of the inducer tetracycline increases the thermal stability of wild-type and Trp43 to Phe mutant Tet repressors by 5 degrees C, while the ones with the Trp75 to Phe mutation are stabilized by 10 degrees C. The stabilizing effect of operator binding is 20 degrees C in the Trp75 to Phe mutant and only 9 degrees C in the ones with the Trp43 to Phe exchange. In addition to the denaturation temperatures, the gel mobility shifts observed in temperature gradient gel electrophoresis reveal also information about the intermediates of the denaturation reaction. The free proteins and their complexes with the inducer tetracycline exhibit monophasic transitions upon denaturation. The operator complexes of wild-type and Trp75 to Phe mutant repressors denature in more complex reactions. At low temperature they exhibit a stoichiometry of two repressor dimers per tandem tet operator DNA. Upon elevating the temperature they form complexes with only one repressor dimer per DNA fragment. When the temperature is further increased the double-stranded DNA begins to melt from one end resulting in a complex with partially single-stranded DNA which exists only in a narrow temperature range. Finally, the denatured protein and single-stranded DNA are formed at high temperature. The associated mobility shifts are analyzed by changing the ionic strength and characterizing multiphasic melting of a pure DNA fragment by temperature gradient gel electrophoresis.     

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.     

Tet repressor-tet operator contacts probed by operator DNA-modification interference studies

Contacts between tet operator DNA and Tet repressor protein are characterized by modification interference studies. The modified DNA fragments are separated into fractions with high, intermediate and low affinities for Tet repressor by polyacrylamide gel electrophoresis. Ethylation of the phosphates with N-ethylnitrosourea reveals 12 contacts of a repressor dimer to tet operator. Eight of these contacts appear to be important for Tet repressor binding, as judged by the strong interference at these positions, while four contacts are probably less important. All of the phosphate contacts are located on the same side of the B-DNA structure. The sequences of tet operators proposed to interact with the recognition alpha-helix of Tet repressor are TCTATC in three cases and CCTATC in one case. After methylation of N-7 with dimethylsulfate, strong interference is observed at the guanine residues at positions +/- 2. None of the N-7 functions of other guanine residues seems to be involved in tight contacts to Tet repressor. Tet repressor subunits form identical phosphate and guanine N-7 contacts with each half side of the two tet operators indicating twofold dyad symmetry of the complexes. Attempts to analyze the methylation interference at the adenine N-3 sites reveal different results for the operators. Modification of DNA fragments with diethylpyrocarbonate yields hypersensitive sites in the tet operators, indicating different local DNA structures. Carbethoxylation interference studies confirm the contacts at the purines found by methylation interference. All of the sequence-specific protein-DNA contacts detected in this study are centered at the inside four base-pairs in each tet operator half side. The contacts are discussed with respect to the structure of the repressor-operator complex.     

Tn10 tet operator mutations affecting Tet repressor recognition.

The effect of single base pair alterations of the Tn10 encoded tet operator on recognition of Tet repressor was studied in vivo using a repressor titration system and in vitro by dissociation rate determinations of the respective complexes. Both methods reveal that the two operators, O1 and O2, which are in a tandem arrangement in the wild type, are recognized with a two-fold different affinity when separated. Studies on synthetic operator sequences indicate that the Tet repressor binds with higher affinity to the non-palindromic O2 wildtype than to the respective palindromic sequences. The in vivo repressor titration system links the expression of lacZ to the affinity of tet operator to Tet repressor. It was used to isolate tet operator mutations with reduced affinity to the repressor. The in vivo and in vitro obtained results with these mutants agree quantitatively and indicate, that the GC base pairs at positions 2, 6, and 8 are involved in interaction with the Tet repressor. Their importance for recognition decreases in that order. Transitions at position 7 of the tet operator show smaller effects on recognition than transversions.     

Saturation mutagenesis of the Tn10-encoded tet operator O1. Identification of base-pairs involved in Tet repressor recognition

Saturation mutagenesis of Tn10-encoded tet operator O1 was performed by chemical synthesis of 30 sequence variants yielding all possible point mutations of an operator half side. Their effect on Tet repressor binding was scored by an in-vivo repressor titration system. Tet repressor affinities of selected operator mutants were further characterized in vitro by dissociation rate measurements. The O1 sequence spans 19 base-pairs. Out of these, all 18 palindromic base-pairs are involved in Tet repressor recognition. The central base-pair does not contribute to sequence-specific binding of Tet repressor. At position 1 a pyrimidine residue is sufficient for maximal affinity to the repressor. At positions 2, 3 and 4, each mutation reduces repressor binding at least tenfold. Mutations at positions 5, 6, 7, 8 and 9 result in less drastic reductions of Tet repressor binding. Differential effects of mutations at a given position are used to deduce the chemical functions contacted by Tet repressor. The T.A to A.T transversion at position 9 increases Tet repressor affinity slightly, while all other mutations decrease repressor binding. The increased affinity of the wild-type tet operator O2 compared to wild-type O1 results from the addition of two favorable transversions at positions +/- 9 and an unfavorable T.A to C.G transition at position -7. Deletion or palindromic doubling of the central base-pair of the O1 palindrome reveals that the wild-type spacing of both operator half sides is crucial for efficient Tet repressor binding.     

Amino acids determining operator binding specificity in the helix-turn-helix motif of Tn10 Tet repressor.

Each of 22 amino acids in the proposed alpha-helix-turn-alpha-helix operator binding motif of the Tn10 encoded Tet repressor was replaced by alanine and one residue was replaced by valine to determine their role in tet operator recognition by a 'loss of contact' analysis with 16 operator variants. One class of amino acids consisting of T27 and R28 in the first alpha-helix and L41, Y42, W43 and H44 in the recognition alpha-helix are quantitatively most important for wild-type operator binding. These residues are probably involved in the structural architecture of the motif. A second class of residues is quantitatively less important for binding, but determines specificity by forming base pair specific contacts to three positions in tet operator. This property is most clearly demonstrated for Q38 and P39 and to a lesser extent for T40 at the N-terminus of the recognition alpha-helix. The contacted operator base pairs indicate that the N-terminus of the recognition alpha-helix is located towards the palindromic center in the repressor-operator complex. Although the orientation of the recognition alpha-helix in the Tet repressor-tet operator complex is inversed as compared with the lambda- and 434 repressor-operator complexes, the reduced operator binding of the TA27 mutation in the first alpha-helix suggests that the hydrogen bonding networks connecting the two alpha-helices may be similar in these proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

How Lac repressor finds lac operator in vitro.

Filter-binding and gel mobility shift assays were used to analyse the kinetics of the interaction of Lac repressor with lac operator. A comparison of the two techniques reveals that filter-binding assays with tetrameric Lac repressor have often been misinterpreted. It has been assumed that all complexes of Lac repressor and lac operator DNA bind with equal affinity to nitrocellulose filters. This assumption is wrong. Sandwich or loop complexes where two lac operators bind to one tetrameric Lac repressor are not or are only badly retained on nitrocellulose filters under normal conditions. Taking this into account, dimeric and tetrameric Lac repressor do not show any DNA-length dependence of their association and dissociation rate constants when they bind to DNA fragments smaller than 2455 base-pairs carrying a single symmetric ideal lac operator. A ninefold increased association rate to ideal lac operator on lambda DNA is observed for tetrameric but not dimeric Lac repressor. It is presumably due to intersegment transfer involving lac operator-like sequences.     

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.     

RNA polymerase and gal repressor bind simultaneously and with DNA bending to the control region of the Escherichia coli galactose operon.

The Escherichia coli galactose operon contains an unusual array of closely spaced binding sites for proteins governing the expression from the two physically overlapping gal promoters. Based on studies of two gal promoter-up mutants we have previously suggested RNA-polymerase-induced DNA bending of gal promoter DNA. Here we present new evidence confirming and extending this interpretation. It was obtained by the circular permutation assay of gel electrophoretic mobility [Wu and Crothers (1984), Nature, 308, 509-513] applied to three analogous series of circularly permuted fragments derived from wild-type and two promoter-up mutant DNAs. The same circularly permuted DNA fragments have further been used to study the binding of gal repressor to its operator sites by electrophoretic mobility shift and by DNase I footprinting techniques. The main results are: (i) complexes carrying repressor either exclusively at the upstream operator O1 or at the downstream operator O2 exhibit different electrophoretic mobilities; (ii) binding to either one of the operators results in protein-induced DNA bending by the criteria of the circular permutation mobility assay; and (iii) occupation of both gal operators by gal repressor does not prevent cAMP-CRP-independent binding of RNA polymerase to the gal promoters, as judged by DNase I protection and gel retardation assays. The latter finding imposes constraints on any attempt to model the regulation of gal expression by assumed DNA-protein and protein-protein interactions.     

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.     

Bending of synthetic bacteriophage 434 operators by bacteriophage 434 proteins.

The extent of DNA bending induced by 434 repressor, its amino terminal DNA binding domain (R1-69), and 434 Cro was studied by gel shift assay. The results show that 434 repressor and R1-69 bend DNA to the same extent. 434 Cro-induced DNA bends are similar to those seen with the 434 repressor proteins. On approximately 265 base pair fragments, the cyclic AMP receptor protein of Escherichia coli (CRP) produces larger mobility shifts than does 434 repressor. This indicates that the 434 proteins bend DNA to a much smaller extent than does CRP. The effects of central operator sequence on intrinsic and 434 protein-induced DNA bending was also examined by gel shift assay. Two 434 operators having different central sequences and affinities for 434 proteins display no static bending. The amount of gel shift induced by 434 repressor on these operators is identical, showing that the 434 repressor bends operators with different central sequences to the same extent. Hence, mutations in the central region of the operator do not influence the bent structure of the unbound or bound operator.     

Quantitative analysis of Tn10 Tet repressor binding to a complete set of tet operator mutants.

A saturating oligonucleotide-directed mutagenesis of both tet operators in the tet regulatory sequence was performed yielding mutants with four identical base pair exchanges at equivalent positions in the four tet operator half sides. The mutants were cloned between bipolar lacZ and galK indicator genes on a multicopy plasmid allowing the quantitative analysis of their effects in vivo. In the absence of Tet repressor the mutations lead to considerably different expression levels of both genes. They are discussed with respect to the promoter consensus sequences. In particular, the -10 region of the in vivo active tetPR2 promoter is unambiguously defined by these results. In the presence of Tet repressor most of the mutants exhibit a lower affinity for that protein as determined quantitatively by their reduced expression levels. In general, tet operator recognition is most strongly affected by alterations of base pairs near the center of the palindromic sequence. The most important position is the third base pair, followed by base pairs two, four, five and six, the latter showing similar effects as base pair one. At each position, the four possible base pairs show different affinities for Tet repressor. They are discussed according to their exposure of H-bond donors and -acceptors in the major and minor grooves of the B-DNA. The results are in agreement with major groove contacts at positions two, three and five. At position four a low potential correlation of efficiencies with the H-bonding in the minor groove is found, while mutations at position six seem to influence repressor binding by other mechanisms.     

Trapping DNA-protein binding reactions with neutral osmolytes for the analysis by gel mobility shift and self-cleavage assays

We take advantage of our previous observation that neutral osmolytes can strongly slow down the rate of DNA–protein complex dissociation to develop a method that uses osmotic stress to ‘freeze’ mixtures of DNA–protein complexes and prevent further reaction enabling analysis of the products. We apply this approach to the gel mobility shift assay and use it to modify a self-cleavage assay that uses the nuclease activity of the restriction endonucleases to measure sensitively their specific binding to DNA. At sufficiently high concentrations of neutral osmolytes the cleavage reaction can be triggered at only those DNA fragments with initially bound enzyme. The self-cleavage assay allows measurement of binding equilibrium and kinetics directly in solution avoiding the intrinsic problems of gel mobility shift and filter binding assays while providing the same sensitivity level. Here we compare the self-cleavage and gel mobility shift assays applied to the DNA binding of EcoRI and BamHI restriction endonucleases. Initial results indicate that BamHI dissociation from its specific DNA sequence is strongly linked to water activity with the half-life time of the specific complex increasing ~20-fold from 0 to 1 osmolal betaine.     

Coupling of protein assembly and DNA binding: biotin repressor dimerization precedes biotin operator binding

The kinetics of coupling of protein dimerization and DNA binding have been investigated in the biotin repressor system. Two repressor monomers bind to the 40 base-pair biotin operator sequence. In previous analyses of equilibrium-binding data the weak dimerization of the repressor has justified using a model in which two protein monomers bind cooperatively to the operator site. Here, rapid kinetic methods have been used to directly determine the binding mechanism. Results of rapid-mixing DNaseI footprinting measurements of association of the repressor with operator indicate that the binding process involves at least two steps. Results of measurements of the unimolecular dissociation of the complex reveal a half-life of approximately 400 seconds. Analysis of the data using a combination of simulation and global non-linear least-squares analysis provides support for a binding model in which a preformed repressor dimer associates with the biotin operator. This kinetic model is consistent with the previously proposed model for regulation of the functional switch in the repressor from enzyme to site-specific DNA-binding protein.     

The role of the N terminus in Tet repressor for tet operator binding determined by a mutational analysis.

The N-terminal residues preceding the alpha-helix-turn-alpha-helix motif on the Tn10 Tet repressor protein were probed by oligonucleotide-directed deletion mutagenesis for their role in protein activity. All deletion mutants showed decreased repression in vivo, emphasizing the importance of the N terminus for tet operator binding. Only two of the mutants, TetR delta 2-23 and TetR delta 3-8 displayed a reduced intracellular protein level. The remaining deletion mutants showed either reduced binding to tet operator and inducibility by tetracycline or transdominance. We conclude that these deletions do not affect stability and overall protein structure. DNA binding activities of residue-wise increasing deletions, TetR delta 9 through TetR delta 9-13, reveal a pattern consistent with an alpha-helical structure of the affected residues. This conclusion is supported by the helical wheel projection and the hydrophobic moment profile calculated for the protein segment ranging from residues S7-V20. We propose that these residues form an amphipathic alpha-helix which packs closely against the alpha-helix-turn-alpha-helix motif and is essential for Tet repressor activity. The residues preceding this putative alpha-helix contribute to DNA binding, but no direct interactions with base pairs of tet operator were revealed in a loss of contact analysis. Individual mutation of the 4 charged residues to alanine at the N terminus shows that no single residue can account for the reduction in repression observed for the deletion mutants.     

Specific binding of lac repressor to linear versus circular polyoperator molecules

Gel shift assays were used to examine the binding of the lactose (lac) repressor to polyoperator DNA molecules. Specific binding was differentiated from nonspecific DNA association by (i) equilibrating repressor-operator complexes below the nonspecific association constant and (ii) demonstrating the effects of the inducer isopropyl beta-D-thiogalactoside (IPTG) on the formation of repressor-operator complexes. With the linear polyoperator molecules, all eight operator sites could be simultaneously bound by distinct repressors. However, with circular molecules, the eight operator sites were saturable by repressor only in the nicked circular state and not in the covalently closed circular form. Under the experimental conditions used, there was no evidence of bifunctional repressor binding or loop formation. The results suggest that the conformational perturbation of DNA that occurs upon specific repressor binding was retained in topologically closed molecules and could modify other operator sites so as to make them unavailable for specific binding.