Analysis in vivo of factors affecting the control of transcription initiation at promoters containing target sites for trp repressor

An investigation of repression in the trp system of Escherichia coli was undertaken using operon fusions and plasmids constructed via recombinant DNA technology. The promoters of the trp operon and the trpR gene were fused to lacZ, enabling the activity of these promoters to be evaluated under various conditions through measurements of beta-galactosidase production. In confirmation of earlier studies, the trpR gene was shown to be regulated autogenously. This control feature of the trp system was found to maintain intracellular Trp repressor protein at essentially invariant levels under most conditions studied. Increasing the trpR+ gene dosage did not significantly elevate Trp repressor protein levels, nor did the introduction of additional operator "sinks" result in significantly decreased levels of Trp repressor protein. Definite alterations in intracellular Trp repressor protein levels were achieved only by subverting the normal trpR regulatory elements. The placement of the lacUV5 or the lambda PL promoters upstream of the trpR gene resulted in significant increases in repression of the trp system. Substituting the primary trp promoter/operator for the native trpR promoter/operator resulted in an altered regulatory response of the trp system to tryptophan limitation or excess. The regulation of the trpR gene effectively imparts a broad range of expression to the trp operon in a manner finely attuned to fluctuations in intracellular tryptophan levels.     

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.     

The influence of tryptophan on mobility of residues in the trp repressor of Escherichia coli

The relative mobility of residues in the trp repressor of Escherichia coli has been examined in the absence and presence of the corepressor L-tryptophan by one- and two-dimensional 1H NMR. A comparison of relative intensities of cross peaks in NOESY and COSY spectra allowed a rigid Tyr and a mobile Tyr residue, three mobile Ser residues and three mobile Lys residues to be detected. The two Tyr residues were assigned by selective nitration with tetranitromethane. The singly nitrated molecule (on Tyr7) binds the trp operator with an affinity close to that of the unmodified repressor. Measurements of the intraring cross-relaxation rate constant as a function of temperature for Tyr7 shows the presence of considerable internal motion on the subnanosecond time scale in the flexible N-terminal arm. The order parameter, S2, characterising the motion is 0.35, which increases to about 0.5 in the presence of Trp. Trp decreases both the amplitude of the motion and the rate of the motion. At least three of the six Ser residues of the trp repressor have greater mobility than expected for a rigid body, and two of the Ser residues are sensitive to the presence of Trp. The more mobile Ser residues are probably those on the N-terminal arm and the C-terminal sequence. These results complement the single-crystal X-ray diffraction studies for which the electron density of the first ten and last three amino acid residues is weak. The solution data are consistent with proposals that the flexible N-terminal arm of the trp repressor makes important contacts with the DNA.     

Trp scanning analysis of Tet repressor reveals conformational changes associated with operator and anhydrotetracycline binding.

We analysed the conformational states of free, tet operator-bound and anhydrotetracycline-bound Tet repressor employing a Trp-scanning approach. The two wild-type Trp residues in Tet repressor were replaced by Tyr or Phe and single Trp residues were introduced at each of the positions 162-173, representing part of an unstructured loop and the N-terminal six residues of alpha-helix 9. All mutants retained in vivo inducibility, but anhydrotetracycline-binding constants were decreased up to 7.5-fold when Trp was in positions 169, 170 and 173. Helical positions (168-173) differed from those in the loop (162-167) in terms of their fluorescence emission maxima, quenching rate constants with acrylamide and anisotropies in the free and tet operator-complexed proteins. Trp fluorescence emission decreased drastically upon atc binding, mainly due to energy transfer. For all proteins, either free, tet operator bound or anhydrtetracycline-bound, mean fluorescence lifetimes were determined to derive quenching rate constants. Solvent-accessible surfaces of the respective Trp side chains were calculated and compared with the quenching rate constants in the anhydrotetracycline-bound complexes. The results support a model, in which residues in the loop become more exposed, whereas residues in alpha-helix 9 become more buried upon the induction of TetR by anhydrotetracycline.     

The Mnt repressor of bacteriophage P22: role of C-terminal residues in operator binding and tetramer formation

A set of C-terminal deletion mutants of the Mnt repressor of bacteriophage P22 has been constructed, and the corresponding truncated proteins have been purified. A truncated protein lacking the three C-terminal residues, Lys80-Thr81-Thr82, binds operator DNA with an affinity near wild type and has a normal tetrameric structure. Loss of the next residue, Lys79, causes a 600-fold decrease in operator affinity, but the truncated protein is still tetrameric. Further sequential deletions of Tyr78 and Leu77 cause modest decreases in operator affinity, but the truncated proteins are now dimeric. These results indicate that Lys79 is an important determinant of the high affinity of Mnt repressor for operator DNA and that Tyr78 is an important determinant of tetramer formation by Mnt repressor.     

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.     

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.     

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.     

Intracellular Trp repressor levels in Escherichia coli.

A radioimmunoassay for the Trp repressor protein of Escherichia coli was developed with antisera raised against purified Trp repressor protein. This assay was used to directly measure the intracellular Trp repressor content in several E. coli K-12 and B/r strains. Repressor levels varied from 2.5- to 3-fold in response to L-tryptophan concentration in the growth medium (15 to 44 ng of repressor per mg of protein). Neither cell growth rate nor culture age had a significant effect on repressor concentrations within the cell. Addition of L-tryptophan to the growth medium resulted in lowered intracellular levels of Trp repressor. The absolute amounts of native Trp repressor molecules per cell varied between 120 and 375 dimers in the presence and absence of L-tryptophan in the culture medium, respectively. Assuming an intracellular volume of 7.3 microliters/10(10) E. coli cells, the Trp repressor concentration varied from 270 to 850 nM in response to extracellular tryptophan levels. These findings represent the first direct measurements of Trp repressor levels in E. coli and confirm the autoregulatory nature of the trpR gene.     

Co-operative binding of two Trp repressor dimers to α- or β-centred trp operators

The α-centred trp operator binds one dimer of the Trp repressor, whereas the β-centred trp operator binds two dimers of the Trp repressor (Carey et al., 1991; Haran et al., 1992). The Trp repressor with a Tyr-Gly-7 substitution binds almost as well as the wild-type Trp repressor to the α-centred trp operator, but it does not bind to the β-centred trp operator. This confirms that Tyr-7 is involved in the interaction between Trp repressor dimers, as seen in the crystal structure (Lawson and Carey, 1993). Further experiments with a-centred trp operator variants showed that positions 1 of the a-centred trp operators play a crucial role in tetramerisation. The two innermost base pairs of the α-centred trp operator are not involved in contacts with the dimer of the Trp repressor binding to it. However, substitutions in these positions (T-A to G-T) effectively transform the α-centred trp operator into a β-centred trp operator, and thus encourage the binding of two Trp repressor dimers to this operator. Finally, we demonstrate, with suitable heterodimers, that one subunit of each dimer suffices to bind to a β-centred trp operator.     

Ligand-mediated conformational changes in Trp repressor protein of Escherichia coli probed through limited proteolysis and the use of specific antibodies

Trp repressor of Escherichia coli K-12 is a dimeric protein (monomer size, 108 amino acids) that acquires high affinity for certain operator targets in double-stranded DNA upon interaction with L-tryptophan. High titer antiserum directed against E. coli Trp repressor protein, elicited in rabbits, was monospecific toward native or denatured Trp repressor. Using an enzyme-linked immunosorbent assay to measure antigen-antibody reaction, we found that the binding of L-tryptophan to Trp repressor was associated with a marked decrease in antibody reactivity that presumably accompanied a conformational change in this protein to a state with strong affinity for trp operator-bearing DNA. We analyzed the pattern of cleavage of Trp repressor by chymotrypsin and trypsin and the effect of L-tryptophan on such hydrolytic cleavages. Chymotrypsin cleaved Trp repressor mainly between residues 71 and 72. In the presence of L-tryptophan this cleavage was slowed. The first-order rate constants for chymotryptic digestion of Trp repressor were 7.6 X 10(-2) and 4.6 X 10(-2) min-1 in the absence and presence of L-tryptophan, respectively. Tryptic digestion was more complex. Initial cleavage of Trp repressor occurred with approximately equal facility between residues 69-70 or 84-85. Subsequent tryptic hydrolyses led eventually to a major core fragment containing the first 54 amino acids of Trp repressor plus four other fragments from the carboxyl-terminal half of the protein. In the presence of L-tryptophan, cleavage by trypsin between residues 54-55 and 84-85 was retarded, even when a previous hydrolytic event elsewhere in the protein had occurred. Tryptophan had essentially no effect on the tryptic hydrolysis of peptide bond 97-98, but accelerated cleavage at peptide bond 69-70. The first-order rate constants for the first tryptic cleavage of Trp receptor were 1.55 X 10(-1) and 1.33 X 10(-1) min-1 in the absence and presence of ligand, respectively. Our results are compatible with a structural model wherein certain amino acid side chains and peptide bonds of Trp repressor (specifically, those of residues 69-85) lie on or near the surface of the protein. This region of Trp repressor has been predicted to contain the operator recognition site. The susceptibility to proteolytic attack of at least four peptide bonds in this area changes when the protein interacts with L-tryptophan.     

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.     

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.     

nalD encodes a second repressor of the mexAB-oprM multidrug efflux operon of Pseudomonas aeruginosa

The Pseudomonas aeruginosa nalD gene encodes a TetR family repressor with homology to the SmeT and TtgR repressors of the smeDEF and ttgABC multidrug efflux systems of Stenotrophomonas maltophilia and Pseudomonas putida, respectively. A sequence upstream of mexAB-oprM and overlapping a second promoter for this efflux system was very similar to the SmeT and TtgR operator sequences, and NalD binding to this region was, in fact, demonstrated. Moreover, increased expression from this promoter was seen in a nalD mutant, consistent with NalD directly controlling mexAB-oprM expression from a second promoter.