How Cro and lambda-repressor distinguish between operators: the structural basis underlying a genetic switch

Knowledge of the three-dimensional structures of the lambda-Cro and lambda-repressor proteins in complex with DNA has made it possible to evaluate how these proteins discriminate between different operators in phage lambda. As anticipated in previous studies, the helix-turn-helix units of the respective proteins bind in very different alignments. In Cro the recognition helices are 29 A apart and are tilted by 55 degrees with respect to each other, but bind parallel to the major groove of the DNA. In lambda-repressor [Beamer, L. J. & Pabo, C. O. (1992) J. Mol. Biol. 227, 177-196] the helices are 34 A apart and are essentially parallel to each other, but are inclined to the major grooves. The DNA is much more bent when bound by Cro than in the case with lambda-repressor. The first two amino acids of the recognition helices of the two proteins, Gln-27 and Ser-28 in Cro, and Gln-44 and Ser-45 in lambda-repressor, make very similar interactions with the invariant bps 2 and 4. There are also analogous contacts between the thymine of bp 5 and, respectively, the backbone of Ala-29 of Cro and the backbone of Gly-46 of lambda-repressor. Otherwise, however, unrelated parts of the two proteins are used in sequence-specific recognition. It appears that similar contacts to the invariant or almost invariant bps (especially 2 and 4) are used by both Cro and lambda-repressor to differentiate the operator sites as a group from other sites on the DNA. The discrimination of Cro and lambda-repressor between their different operators is more subtle and seems to be achieved primarily through differences in van der Waals contacts at bp 3', together with weaker, less direct effects at bps 5' and 8', all in the nonconsensus half of the operators. The results provide further support for the idea that there is no simple code for DNA-protein recognition.     

Crystal structure of LacI member, PurR, bound to DNA: minor groove binding by alpha helices

The three-dimensional structure of a ternary complex of the purine repressor, PurR, bound to both its corepressor, hypoxanthine, and the 16-base pair purF operator site has been solved at 2.7 A resolution by x-ray crystallography. The bipartite structure of PurR consists of an amino-terminal DNA-binding domain and a larger carboxyl-terminal corepressor binding and dimerization domain that is similar to that of the bacterial periplasmic binding proteins. The DNA-binding domain contains a helix-turn-helix motif that makes base-specific contacts in the major groove of the DNA. Base contacts are also made by residues of symmetry-related alpha helices, the "hinge" helices, which bind deeply in the minor groove. Critical to hinge helix-minor groove binding is the intercalation of the side chains of Leu54 and its symmetry-related mate, Leu54', into the central CpG-base pair step. These residues thereby act as "leucine levers" to pry open the minor groove and kink the purF operator by 45 degrees.     

Conformational changes necessary for gene regulation by Tet repressor assayed by reversible disulfide bond formation

We constructed and characterized four Tet repressor (TetR) variants with engineered cysteine residues which can form disulfide bonds and are located in regions where conformational changes during induction by tetracycline (tc) might occur. All TetR mutants show nearly wild-type activities in vivo, and the reduced proteins also show wild-type activities in vitro. Complete and reversible disulfide bond formation was achieved in vitro for all four mutants. The disulfide bond in NC18RC94 immobilizes the DNA reading head with respect to the protein core and prevents operator binding. Formation of this disulfide bond is possible only in the tc-bound, but not in the operator-bound conformation. Thus, these residues must have different conformations when bound to these ligands. The disulfide bonds in DC106PC159' and EC107NC165' immobilize the variable loop between alpha-helices 8 and 9 located near the tc-binding pocket. A faster rate of disulfide formation in the operator-bound conformation and a lack of induction after disulfide formation show that the variable loop is located closer to the protein core in the operator-bound conformation and that a movement is necessary for induction. The disulfide bond in RC195VC199' connects alpha-helices 10 and 10' of the two subunits in the dimer and is only formed in the tc-bound conformation. The oxidized protein shows reduced operator binding. Thus, this bond prevents formation of the operator-bound conformation. The detection of conformational changes in three different regions is the first biochemical evidence for induction-associated global internal movements in TetR.     

Single-chain repressors containing engineered DNA-binding domains of the phage 434 repressor recognize symmetric or asymmetric DNA operators

Single-chain (sc) DNA-binding proteins containing covalently dimerized N-terminal domains of the bacteriophage 434 repressor cI have been constructed. The DNA-binding domains (amino acid residues 1 to 69) were connected in a head-to-tail arrangement with a part of the natural linker sequence that connects the N and C-terminal domains of the intact repressor. Compared to the isolated N-terminal DNA-binding domain, the sc molecule showed at least 100-fold higher binding affinity in vitro and a slightly stronger repression in vivo. The recognition of the symmetric O(R)1 operator sequence by this sc homodimer was indistinguishable from that of the naturally dimerized repressor in terms of binding affinity, DNase I protection pattern and in vivo repressor function. Using the new, sc framework, mutant proteins with altered DNA-binding specificity have also been constructed. Substitution of the DNA-contacting amino acid residues of the recognition helix in one of the domains with the corresponding residues of the Salmonella phage P22 repressor c2 resulted in a sc heterodimer of altered specificity. This new heterodimeric molecule recognized an asymmetric, artificial 434-P22 chimeric operator with high affinity. Similar substitutions in both 434 domains have led to a new sc homodimer which showed high affinity binding to a natural, symmetric P22 operator. These findings, supported by both in vitro and in vivo experiments, show that the sc architecture allows for the introduction of independent changes in the binding domains and suggest that this new protein framework could be used to generate new specificities in protein-DNA interaction.     

Sequence context effects on mutational properties of cis-opened benzo[c]phenanthrene diol epoxide-deoxyadenosine adducts in site-specific mutation studies

Diastereomeric N6-substituted dAdo adducts (cis B[c]PhDE-2/1R and cis B[c]PhDE-2/1S) that correspond to cis-opening at C-1 of the enantiomeric benzo[c]phenanthrene 3,4-diol 1,2-epoxides in which the epoxide oxygen and the benzylic hydroxyl group are trans (DE-2) were synthetically incorporated into oligonucleotide 16-mers. Each adduct was placed at the fourth nucleotide from the 5'-end of each of two different oligonucleotide sequences derived from the E. coli supF gene. Each adduct was also placed in two additional oligonucleotide sequences that were constructed by interchanging the adduct site and the immediately adjacent nucleotides between the two original sequences. These oligonucleotides were designed for use in site-specific mutation studies, with a single-stranded bacteriophage M13mp7L2 vector, to determine if the effects of sequence context on types and frequencies of base substitution mutations are attributable only to nucleotides immediately adjacent to these polycyclic aromatic hydrocarbon diol epoxide-dAdo adducts, or whether more distant nucleotide residues also affect the mutagenic response. In SOS-induced Escherichia coli SMH77, total base substitution mutation frequencies for the cis B[c]PhDE-2/1R-dAdo adduct were relatively low (0.62-5.6%) compared with those for the cis B[c]PhDE-2/1S-dAdo adduct (11.9-56.5%). Depending on sequence context, cis B[c]PhDE-2/1R-dAdo gave predominantly A-->T or a more equal distribution of A-->T and A-->G mutations whereas cis B[c]PhDE-2/1S-dAdo gave either predominantly A-->T or predominantly A-->G base substitutions. Our results clearly indicate that nucleotides that are distal as well as those that are proximal to the adduct site are capable of influencing both the mutation frequency and the distribution of base substitution mutations.     

Deuteriation of sugar protons simplify NMR assignments and structure determination of large oligonucleotide by the 1H-NMR window approach

The concept of the 1H-NMR window has been developed and examined through a comparative study of NOESY spectra of a self-complementary Dickerson's dodecamer (I) [5'd(5C6G7C8G9A10A11T12T13C-14G15C16G)2(3')], a self-complementary 20-mer (II) [(5'd(1C2G3C4G5C6G7C8G9A10A11T12T13C14G15C16G17C18G19C20G)2(3')] in which the core part consists of the same Dickerson's dodecamer sequence with the flanking CGCG residues at both 3' and 5'-ends, and the partly-deuteriated (shown by underlined CGCG residues at both 3' and 5'-ends) analogous duplex (III) [5'd(1C2G3C4G5C6G7C8G9A10A11T12T13C14G15C16G17C18G19C20G)2(3')] in which the core 5C to 16G part (i.e. 1H-NMR window) consists of the natural Dickerson's dodecamer sequence. A comparison of their NOESY spectra clearly demonstrates that the severe overlap of proton resonances in the larger DNA duplex (II) has been successfully reduced in the partly-deuterated duplex (III) as a result of specific incorporations of the sugar-deuteriated nucleotide residues in the latter [stereospecific > 97 atom % 2H enrichment at H2', H2' and H3' sites, approximately 85 atom % 2H enrichment at H4' and approximately 20 atom % 2H enrichment at H1' (see refs. 10 and 11) in the 20-mer duplex (III)]. These simplifications of the resonance overlap by the deuteriation approach have enabled unequivocal chemical shift assignments and extraction of the quantitative NOE data in the 1H-NMR window part of duplex (III). A comparison of the 12-nucleotide long 1H-NMR window in (III) with that of the 12-mer duplex (I) also shows the scope of studying the changes in conformation and dynamics of the core 12-mer region in (III) which result from the increase of molecular weight due to the DNA chain extension. It is noteworthy that such a study is clearly impossible for the natural 20-mer (II) because of the inherent problem of the overlap of resonances.     

Critical base pairs and amino acid residues for protein-DNA interaction between the TyrR protein and tyrP operator of Escherichia coli

In Escherichia coli K-12, the repression of tyrP requires the binding of the TyrR protein to the operator in the presence of coeffectors, tyrosine and ATP. This operator contains two 22-bp palindromic sequences which are termed TyrR boxes. Methylation, uracil, and ethylation interference experiments were used to identify the important sites in the TyrR boxes that make contacts with the TyrR protein. Methylation interference studies demonstrated that guanines at positions +8, -5, and -8 of the strong TyrR box and positions +8, -4, and -8 of the weak box are close to the TyrR protein. Uracil interference revealed that strong van der Waals contacts are made by the thymines at position -7 and +5 of the top strands of both strong and weak boxes and that weaker contacts are made by the thymines at positions +7 (strong box) and -5 and +7 (weak box) of the bottom strand. In addition, ethylation interference suggested that the phosphate backbone contacts are located at the end and central regions of the palindrome. These findings are supported by our results derived from studies of symmetrical mutations of the tyrP strong box. Overall, the results confirm the critical importance of the invariant (G x C)(C x G)8 base pairs for TyrR recognition and also indicate that interactions with (T x A)(A x T)7 are of major importance. In contrast, mutations in other positions result in weaker effects on the binding affinity of TyrR protein, indicating that these positions play a lesser role in TyrR protein recognition. Alanine scanning of both helices of the putative helix-turn-helix DNA-binding motif of TyrR protein has identified those amino acids whose side chains play an essential role in protein structure and DNA binding.     

Use of inducible feedback-resistant N-acetylglutamate synthetase (argA) genes for enhanced arginine biosynthesis by genetically engineered Escherichia coli K-12 strains

The goal of this work was to construct Escherichia coli strains capable of enhanced arginine production. The arginine biosynthetic capacity of previously engineered E. coli strains with a derepressed arginine regulon was limited by the availability of endogenous ornithine (M. Tuchman, B. S. Rajagopal, M. T. McCann, and M. H. Malamy, Appl. Environ. Microbiol. 63:33-38, 1997). Ornithine biosynthesis is limited due to feedback inhibition by arginine of N-acetylglutamate synthetase (NAGS), the product of the argA gene and the first enzyme in the pathway of arginine biosynthesis in E. coli. To circumvent this inhibition, the argA genes from E. coli mutants with feedback-resistant (fbr) NAGS were cloned into plasmids that contain "arg boxes," which titrate the ArgR repressor protein, with or without the E. coli carAB genes encoding carbamyl phosphate synthetase and the argI gene for ornithine transcarbamylase. The free arginine production rates of "arg-derepressed" E. coli cells overexpressing plasmid-encoded carAB, argI, and fbr argA genes were 3- to 15-fold higher than that of an equivalent system overexpressing feedback-sensitive wild-type (wt) argA. The expression system with fbr argA produced 7- to 35-fold more arginine than a system overexpressing carAB and argI genes on a plasmid in a strain with a wt argA gene on the chromosome. The arginine biosynthetic capacity of arg-derepressed DH5 alpha strains with plasmids containing only the fbr argA gene was similar to that of cells with plasmids also containing the carAB and argI genes. Plasmids containing wt or fbr argA were stably maintained under normal growth conditions for at least 18 generations. DNA sequencing identified different point mutations in each of the fbr argA mutants, specifically H15Y, Y19C, S54N, R58H, G287S, and Q432R.     

Carboxyl-terminal domain dimer interface mutant 434 repressors have altered dimerization and DNA binding specificities

Strong dimerization of the repressor, mediated by the carboxyl (C)-terminal domain, is a prerequisite for forming a specific complex with DNA and cooperative DNA binding to form tetramers. We have generated a computer model of the C-terminal domain of the 434 repressor based on the crystal structure of the homologous UmuD' protein. This model predicts that residues in the primary sequence between 93 and 168 contribute to the dimer interface. We changed several amino acid residues located in this region. Gel filtration and crosslinking assays were used to characterize the strength and specificity of dimerization of the purified repressor C-terminal domain dimer interface mutants. These results indicate that amino acid residues K121, H139, D161 and N163 contribute to the strength and/or specificity of dimerization. The relative affinity of the bacteriophage 434 repressor for 434 operators is determined, in part, by the repressor's ability to detect sequence-dependent structural alterations in the non-contacted region at the center of an operator site. We find that the relative ability of C-terminal domain dimer interface mutant repressors to dimerize does not necessarily predict their relative abilities to bind DNA, and that these proteins are deficient in detecting non-contacted base-dependent differences in operator strength. Our results show that the structure of the DNA in complex with these mutant proteins differs from that found in wild-type repressor-operator complexes, even though the sites of these mutations lie in a separate domain from that which contacts the DNA. These observations demonstrate that the structural integrity of the C-terminal domain dimer interface is required to appropriately orient the DNA binding information contained within the DNA-contacting N-terminal domain.     

Solution structure of the d(T-C-G-A) duplex at acidic pH. A parallel-stranded helix containing C+ .C, G.G and A.A pairs

The solution structure of the d(T-C-G-A) sequence at acidic pH has been determined by a combination of NMR and molecular dynamics calculations including NOE intensity based refinements. This sequence forms a right-handed parallel-stranded duplex with C+ .C (three hydrogen bonds along Watson-Crick edge), G.G (two symmetry related N2-H.. N3 hydrogen bonds) and A.A (two symmetry related N6-H..N7 hydrogen bonds) homo base-pair formation at acidic pH. The duplex is stabilized by intra-strand base stacking at the C2-G3 step and cross-strand base stacking at the G3-A4 step. The thymine residues on partner strands are directed towards each other and are positioned over the C+ .C base-pair. All four residues adopt anti glycosidic torsion angles and C2'-endo type sugar conformations in the parallel-stranded d(T-C-G-A) duplex which exhibits large changes in twist angles between adjacent steps along the duplex. This study rules out previously proposed models for the structure of the d(T-C-G-A) duplex at acidic pH and supports earlier structural contributions, which established that d(C-G) and d(C-G-A) containing sequences at acidic pH pair through parallel-stranded alignment. We have also monitored hydration patterns in the symmetry related grooves of the parallel-stranded d(T-C-G-A) duplex.     

High prevalence of mitochondrial diabetes mellitus in Japanese patients with major risk factors

To identify diabetes mellitus caused by the mitochondrial gene substitution at genomic nucleotide pair 3243 (M3243A-->G) we selected 87 diabetic patients with high risk factors such as maternal inheritance and hearing loss. Total DNA was extracted from peripheral leukocytes, and mitochondrial DNA fragments containing M3243A-->G were amplified by polymerase chain reaction (PCR). The amplified fragments were digested with a restriction endonuclease Apa1 and analyzed by agarose gel electrophoresis. The incidence of the M3243A-->G mutation was 4.6% (four of 87) in diabetic patients with maternal inheritance and/or hearing loss. In a subgroup with both maternal inheritance and hearing loss, the incidence of the mutation was as high as 21.4% (three of 14). Cardiac disorders were also present in all four diabetic patients with the mutation. This study suggests that maternal inheritance and hearing loss are useful clinical findings to identify diabetic patients with the mutation, and that cardiac involvement is a high risk factor for the M3243A-->G mutation.     

Spectroscopic investigation of an intramolecular DNA triplex containing both G.G:C and T.A:T triads and its complex with netropsin

The triple helix formation by the oligonucleotide 5'd(G4T4G4-[T4]-G4A4G4-[T4]-C4T4C4) ([T4] represents a stretch of 4 thymine residues) has been investigated by UV absorption spectroscopy and circular dichroism. In a 10 mM sodium cacodylate, 0.2 mM disodium EDTA (pH 7) buffer, we show the following significant results: i) In the absence of MgCl2, the oligonucleotide adopts a hairpin duplex structure with the dangling tail 5'd(G4T4G4-[T4]). This 5' extremity, which contains separated runs of four guanine residues, does not assume the expected tetraplex conformation observed when this sequence is free. ii) In the presence of MgCl2, the oligonucleotide folds back on itself twice to give a triple helix via a double hairpin formation, with [T4] single-strand loops. iii) The addition of high concentration of KCl to the preformed triplex does not disrupt the structure. Nevertheless, if the oligonucleotide is allowed to fold back in the presence of K+, triplex formation is inhibited. Circular dichroism studies demonstrate that the oligonucleotide adopts a dimeric conformation, resulting from the association of two hairpin duplexes, via the formation of an antiparallel G-quadruplex by the telomeric 5'd(G4T4G4-[T4]) extremities. iv) Under the experimental conditions used in this report, the triplex melts in a monophasic manner. v) Netropsin, a DNA minor groove ligand, binds to the central site A4/T4 of the duplex and to that of the triplex in an equimolar stoichiometry. In contrast with previous studies concerning pyr.pur:pyr triplexes, thermal denaturation experiments demonstrate that the netropsin binding stabilizes the intramolecular triplex.     

Megavoltage imaging with a large-area, flat-panel, amorphous silicon imager

Mutations in an N-terminal 70-amino acid domain of bacteriophage Mu's repressor cause temperature-sensitive DNA-binding activity. Surprisingly, amber mutations can conditionally correct the heat-sensitive defect in three mutant forms of the repressor gene, cts25 (D43-G), cts62 (R47-Q) and cts71 (M28-I), and in the appropriate bacterial host produce a heat-stable Sts phenotype (for survival of temperature shifts). Sts repressor mutants are heat sensitive when in supE or supF hosts and heat resistant when in Sup degrees hosts. Mutants with an Sts phenotype have amber mutations at one of three codons, Q179, Q187, or Q190. The Sts phenotype relates to the repressor size: in Sup degrees hosts sts repressors are shorter by seven, 10, or 18 amino acids compared to repressors in supE or supF hosts. The truncated form of the sts62-1 repressor, which lacks 18 residues (Q179-V196), binds Mu operator DNA more stably at 42 degrees in vitro compared to its full-length counterpart (cts62 repressor). In addition to influencing temperature sensitivity, the C-terminus appears to control the susceptibility to in vivo Clp proteolysis by influencing the multimeric structure of repressor.     

Escherichia coli purine repressor: key residues for the allosteric transition between active and inactive conformations and for interdomain signaling

The Escherichia coli purine repressor, PurR, exists in an equilibrium between open and closed conformations. Binding of a corepressor, hypoxanthine or guanine, shifts the allosteric equilibrium in favor of the closed conformation and increases the operator DNA binding affinity by 40-fold compared to aporepressor. Glu70 and Trp147 PurR mutations were isolated which perturb the allosteric equilibrium. Three lines of evidence indicate that the allosteric equilibrium of E70A and W147A aporepressors was shifted toward the closed conformation. First, compared to wild-type PurR, these mutant repressors had a 10-30-fold higher corepressor binding affinity. Second, the mutant aporepressors bound to operator DNA with an affinity that is characteristic of the wild-type PurR holorepressor. Third, binding of guanine to wild-type PurR resulted in a near-UV circular dichroism spectral change at 297-305 nm that is attributed to the closed conformation. The circular dichroism spectrum of the E70A aporepressor at 297-305 nm was that expected for the closed conformation, and it was not appreciably altered by corepressor binding. Mutational analysis was used to identify an Arg115-Ser46' interdomain intersubunit hydrogen bond that is necessary for transmitting the allosteric transition in the corepressor binding domain to the DNA binding domain. R115A and S46G PurR mutants were defective in DNA binding in vitro and repressor function in vivo although corepressor binding was identical to the wild type. These results establish that the hydrogen bond between the side chain NH2 of Arg115 and the main chain CO of Ser46' plays a critical role in interdomain signaling.     

Characteristics of insulin receptor binding to various rat tissues

The solution secondary structure of the Oxytricha nova telomeric 3' overhang, d(T4G4)2, has been investigated by Raman spectroscopy, hydrogen-deuterium exchange kinetics and gel electrophoresis. The electrophoretic mobility of d(T4G4)2 in non-denaturing gels indicates a highly compact conformation, consistent with a hairpin secondary structure. Raman markers show that the d(T4G4)2 hairpin contains equal numbers of C2'-endo/syn and C2'-endo/anti deoxyguanosine conformers, as well as G.G base-pairs of the Hoogsteen type. The hydrogen-deuterium exchange kinetics of d(T4G4)2, monitored by time-resolved Raman spectroscopy, reveal two kinetically distinct classes of guanine imino (N1H) protons. The more slowly exchanging fraction (kN1H(1)=4.6x10(-3) min-1), which represents 50% of N1H groups, is attributed to Hoogsteen-paired residues. The more rapidly exchanging fraction (kN1H(2)>/=0.3 min-1) is attributable to solvent-exposed residues. Raman dynamic probe of the kinetics of guanine C8H-->C8(2)H exchange in d(T4G4)2 reveals modest retardation vis-à-vis dGMP, which rules out quadruplex formation by the telomeric repeat and confirms an ordered secondary structure consistent with a Hoogsteen-paired hairpin. Similar Raman, hydrogen-isotope exchange and electrophoretic mobility experiments on the related telomeric model, dT6(T4G4)2, also reveal a hairpin stabilized by Hoogsteen G.G pairs. Presence of the 5' thymidine tail preceding the Oxytricha telomeric repeat has no apparent effect on the hairpin secondary structure. We propose a molecular model for the hairpin conformation of the Oxytricha nova telomeric repeat and consider its possible roles in mechanisms of telomeric DNA interaction in vitro and telomere function in vivo.