Statistical fluctuations of the level of operator filling by repressor determine the level of noise of reporter gene expression

The regulation of the reporter gene activity in a single bacterial cell by means of lambda-phage C1 repressor has been described by the methods of statistical thermodynamics. The equations for calculation of the mean production rate of the reporter protein and its standard deviation as a function of C1 repressor concentration in the cell have been obtained. The stochastic nature of C1 repressor binding with O_R1 and O_R2 operator sites becomes apparent when both repressor molecules and operators are present in the bacterial cell in a small number of copies. In this case, the number of repressor molecules that bind to O_R1 and O_R2 sites fluctuates considerably. The in vitro binding of C1 repressor to O_R1 and O_R2 sites, their mutant forms, and nonspecific DNA regions has been well studied. Using the binding constants of in vitro binding of C1 repressor to O_R1, O_R2, and nonspecific DNA regions and also the value of the cooperativity parameter for C1 repressor binding to O_R1 and O_R2 sites, we calculated the mean rate of synthesis of the reporter protein and its standard deviation as a function of repressor concentration in the cell. The theoretical relations fit well the experimental results. The results of calculations confirm the assumption that gene expression noise in a single cell at a repressor concentration exceeding 100 nM is related to the stochastic nature of binding of repressor dimers to O_R1 and O_R2 sites. Other mechanisms of the generation of gene expression noise (for example, monomer-dimer balance) make a significant contribution at concentrations less than 100 nM.     

Selection and design of high affinity DNA ligands for mutant single-chain derivatives of the bacteriophage 434 repressor

Single-chain repressor RRTRES is a derivative of bacteriophage 434 repressor, which contains covalently dimerized DNA-binding domains (amino acids 1-69) of the phage 434 repressor. In this single-chain molecule, the wild type domain R is connected to the mutant domain RTRES by a recombinant linker in a head-to-tail arrangement. The DNA-contacting amino acids of RTRES at the -1, 1,2, and 5 positions of the α3 helix are T, R, E, S respectively. By using a randomized DNA pool containing the central sequence -CATACAAGAAAGNNNNNTTT-. a cyclic, in vitro DNA-binding site selection was performed. The selected population was cloned and the individual members were characterized by determining their binding affinities to RRTRES. The results showed that the optimal operators contained the TTAC or TTCC sequences in the underlined positions as above, and that the Kd values were in the 1×10-12mol/L1×10-11mol/L concentration range. Since the affinity of the natural 434 repressor to its natural operator sites is in the     

Kinetics and equilibrium studies of Tet repressor-operator interaction

Binding of a Tet repressor mutant containing a single Trp43 residue in the tet operator recognition -helix leads to the quenching of the protein fluorescence down to about 23% in the case of the tet O₁ operator and to 40% in the case of the tet O₂ operator. We have used fluorescence detection to describe the binding equilibrium and kinetics of the Tet repressor interaction with the 20-bp DNA operators tet O₁ and tet O₂. Stopped-flow measurements in an excess of the tet operators performed in 5 mM NaCl or 150 mM NaCl indicate that the reaction can be described by at least three exponentials characterized by different relaxation times. The mechanism of interaction for both operators as well as for two salt concentrations used can be described as TetR + Operator Complex 1 Complex 2 Complex 3. Only the much faster process can be described as a second-order reaction characterized by a bimolecular rate constant equal to 2.8 × 10⁶ M^–1 sec^–1 for both operators. The medium and slow processes may be described by relaxational times ranging from 50 msec to seconds. The results of the binding equilibrium measurements extrapolated to 1 M NaCl concentration, which reflects the specific nonionic interaction between TetR and tet operators, indicate K _as equal to 3.2 × 10⁴ and 4.0 × 10⁵ M^–1 for tet O₁ and tet O₂, respectively. The number of monovalent ions replaced upon binding can be calculated as about 5 and 3 for tet O₁ and tet O₂, respectively. The binding of Tet repressor to the operators leads to changes in the circular dichroism spectra of the DNA which could indicate transitions of B-DNA into A-like DNA structure.     

Rearrangements between the operators in the bacteriophage lambda

Summary The left operator mutant v2s develops poorly during infection as a result of constitutive expression of the left operon. A revertant of v2s, designated iri, was found to contain an inversion of the cI region with the inversion endpoints to be within the lambda operators o _L and o _R. Formation of the inversion is facilitated by a translocation of right operator o _R ^c mutant sequence to the left operator in v2s. The inversion in iri positions wild-type o _R sequence at o _L returning control of the left operon to repression by the lambda cro repressor.     

'Muprints' of the lac operon demonstrate physiological control over the randomness of in vivo transposition

A method called Muprinting has been developed that uses PCR to generate a detailed picture of the bacteriophage Mu transposition sites in chosen domains of the bacterial chromosome. Muprinting experiments In Escherichia coli show that the frequency of phage integration changes dramatically near two repressor binding sites in the lac operon. When the lac operon was repressed, hotspots for Mu transposition were found near the O₁ and O₂ operators that are proposed to make a repression loop. When cells were grown in lactose, Mu transposition near these operators was greatly diminished. Striking changes In transposition frequencies were limited to the control region and were not found in a region of the lacZ gene lying beyond the O₂ operator. Muprints of the bgl operon showed a different pattern; hotspots for Mu transposition detected in sequences upstream of the bglC promoter when the operon was silenced changed when the operon became activated by mutation. By targeting transposition to the regulatory regions around non-expressed genes, Mu may demonstrate a self-restraint mechanism that allows the virus to move through its host genome without disrupting the functions that contribute to a healthy cell physiology.     

Fluorescence and Phosphorescence Study of Tet Repressor–Operator Interaction

Fluorescence and phosphorescence measurements have been carried out on single-p tryptophan (Trp 43 or Trp 75)-containing mutants of Tet repressor (Tet R). Tet R containing Trp 43, the residue localized in the DNA recognition helix of the repressor, has been used to observe the binding of Tet R to two 20-bp DNA sequences of tet O₁ and tet O₂ operators. Binding of Tet R to tet O₁ operator leads to a 78% decrease of the repressor fluorescence intensity, with an accompanying 20-nm blue shift of its fluorescence emission maximum to 330 nm. Upon binding of Tet R to tet O₂ operator, the Trp 43 fluorescence intensity is quenched by 60%, and a 10-nm shift of its emission maximum to 340 nm occurs. Solute fluorescence quenching studies, using acrylamide, performed at low ionic strength indicate that in both the complex of Tet R with the O₁ and that with the O₂ operator, Trp 43 is moderately buried, as indicated by a bimolecular rate quenching constant of about 1.8 × 10⁹ M^–1 sec^–1. In contrast to the Tet R–tet O₂ complex, the Stern–Volmer acrylamide quenching constant K _sv of the complex with tet O₁ operator changes from 7.5 M^–1 at 5 mM NaCl to 22 M^–1 at 200 mM NaCl, indicating different exposures of Trp 43 in the two complexes in solutions of higher ionic strength. Phosphorescence studies showed a 0–0 vibronic transition at 408 and 403 nm for Trp 43 and Trp 75, respectively. Upon binding of Tet R to the tet operators, we observed red shifts of 0–0 vibronic bands of Trp 43 to 413 and 412 nm for tet O₁ and tet O₂ operator, respectively, and the phosphorescence triplet lifetime of Trp 43 at 75 K was quenched from 6.0–5.5 to 3.5–3.3 sec. The thermal phosphorescence quenching profile ranged from –200°C to –20°C, and differed drastically for the two complexes, suggesting different dynamics of the microenvironment of the Trp 43 residue. The luminescence data for Trp 43 of Tet R suggest that the recognition helix of Tet R interacts in different fashions with the tet O₁ and tet O₂ operators.     

Kinetics and Equilibrium Studies of Tet Repressor–Operator Interaction

Binding of a Tet repressor mutant containing a single Trp43 residue in the tet operator recognition α-helix leads to the quenching of the protein fluorescence down to about 23% in the case of the tet O₁ operator and to 40% in the case of the tet O₂ operator. We have used fluorescence detection to describe the binding equilibrium and kinetics of the Tet repressor interaction with the 20-bp DNA operators tet O₁ and tet O₂. Stopped-flow measurements in an excess of the tet operators performed in 5 mM NaCl or 150 mM NaCl indicate that the reaction can be described by at least three exponentials characterized by different relaxation times. The mechanism of interaction for both operators as well as for two salt concentrations used can be described as TetR + Operator ? Complex 1 ? Complex 2 ? Complex 3. Only the much faster process can be described as a second-order reaction characterized by a bimolecular rate constant equal to 2.8 × 10⁶ M^?1 sec^?1 for both operators. The medium and slow processes may be described by relaxational times ranging from 50 msec to seconds. The results of the binding equilibrium measurements extrapolated to 1 M NaCl concentration, which reflects the specific nonionic interaction between TetR and tet operators, indicate K _as equal to 3.2 × 10⁴ and 4.0 × 10⁵ M^?1 for tet O₁ and tet O₂, respectively. The number of monovalent ions replaced upon binding can be calculated as about 5 and 3 for tet O₁ and tet O₂, respectively. The binding of Tet repressor to the operators leads to changes in the circular dichroism spectra of the DNA which could indicate transitions of B-DNA into A-like DNA structure.     

Expression of pheA gene using PR-PL tandem promoter of bacteriophage lambda

Summary The genepheA ⁺ coding chorismate niutase P-prephenate dehydratase, one of the regulatory enzymes of phenylalanine biosynthesis, was cloned into the down-stream of P_R-P_L tandem promoter. In this construction, both the native promoter-operator region and the attenuator region ofpheA ⁺ operon were eliminated so as to avoid the repression and attenuation ofpheA ⁺ gene expression. The expression ofpheA ⁺ gene was directed by P_R-P_L tandem promoter of bacteriophage lambda and controlled by a temperature sensitive repressor, cI₈₅₇.It was shown that the expression as well as phenylalanine production was regulated by temperature. Maximum production of phenylalanine, 170 mg/l, was obtained at 40°C. The host strain, MC1065, produced a trace (4 mg/l) of phenylalanine at the same temperature.     

1H NMR study of the interaction of bacteriophage λ Cro protein with the O R 3 operator

The 17 base pair operator O _R ³ oligonucleotide, which is the preferential binding site for the Cro repressor of phage , was studied by two-dimensional NMR spectroscopy. A sequential assignment procedure based on two-dimensional Nuclear Overhauser Effect (NOESY) and scalar coupling correlated (COSY) NMR spectroscopy, together with the knowledge of the oligodesoxynucleotide sequence, made it possible to assign the non-exhangeable base protons and the H1 and the H2-H2 sugar protons of the O _R ³ operator DNA. The pattern of the observed NOE connectivities is consistent with a right-handed helical DNA structure. The base and sugar proton assignments provide the necessary information for further studies of the O _R ³ operator — Cro repressor interaction.Abbreviations COSY correlated spectroscopy - FID free induction decay - NOE nuclear Overhauser effect - NOESY nuclear Overhauser effect spectroscopy - RD relaxation delay - TSP sodium 3-trimethylsilyl-(2,2,3,3-²H₄)propionate - EDTA sodium ethylendiamine tetraacetate     

Role of cis-Acting Sites in Stimulation of the Phage λ PRM Promoter by CI-Mediated Looping

The lysogenic state of phage λ is maintained by the CI repressor. CI binds to three operators each in the right operator (O_R) and left operator (O_L) regions, which lie 2.4 kb apart. At moderate CI levels, the predominant binding pattern is two dimers of CI bound cooperatively at each regulatory region. The resulting tetramers can then interact, forming an octamer and a loop of the intervening DNA. CI is expressed from the P_RM promoter, which lies in the O_R region and is subjected to multiple regulatory controls. Of these, the most recently discovered is stimulation by loop formation. In this work, we have investigated the mechanism by which looping stimulates P_RM. We find that two cis-acting sites lying in the O_L region are involved. One site, an UP element, is required for stimulation. Based on the behavior of other promoters with UP elements located upstream of the −35 region, we suggest that a subunit of RNA polymerase (RNAP) bound at P_RM binds to the UP element located in the O_L region. In addition, adjacent to the UP element lies a binding site for integration host factor (IHF); this site plays a less critical role but is required for stimulation of the weak prm240 allele. A loop with CI at the O_L2 and O_L3 operators does not stimulate P_RM, while one with CI only at O_L2 provides some stimulation. We discuss possible mechanisms for stimulation.     

Direct demonstration and quantification of long-range DNA looping by the λ bacteriophage repressor

Recently, it was proposed that DNA looping by the λ repressor (CI protein) strengthens repression of lytic genes during lysogeny and simultaneously ensures efficient switching to lysis. To investigate this hypothesis, tethered particle motion experiments were performed and dynamic CI-mediated looping of single DNA molecules containing the λ repressor binding sites separated by 2317 bp (the wild-type distance) was quantitatively analyzed. DNA containing all three intact operators or with mutated o3 operators were compared. Modeling the thermodynamic data established the free energy of CI octamer-mediated loop formation as 1.7 kcal/mol, which decreased to –0.7 kcal/mol when supplemented by a tetramer (octamer+tetramer-mediated loop). These results support the idea that loops secured by an octamer of CI bound at oL1, oL2, oR1 and oR2 operators must be augmented by a tetramer of CI bound at the oL3 and oR3 to be spontaneous and stable. Thus the o3 sites are critical for loops secured by the CI protein that attenuate cI expression.     

Molecular analysis of two ScrR repressors and of a ScrR–FruR hybrid repressor for sucrose and D-fructose specific regulons from enteric bacteria

The scr regulon of pUR400 and the chromosomally encoded scr regulon of Klebsiella pneumoniae KAY2026 are both negatively controlled by a specific repressor (ScrR). As deduced from the nucleotide sequences, both scrR genes encode polypeptides of 334 residues (85.5% identical base pairs, 91.3% identical amino acids), containing an N-terminal helix-turn-helix motif. Comparison with other regulatory proteins revealed 30.6% identical amino acids to FruR, 27.0% to Lacl and 28.1% to GaIR. Six scrR^s super-repressor mutations define the inducer-binding domain. The scr operator sequences were identified by in vivo titration tests of the sucrose repressor and by in vitro electrophoretic mobility shift assays. D-fructose, an intracellular product of sucrose transport and hydrolysis, and D-fructose 1-phosphate were shown to be molecular inducers of both scr regulons. An active ScrR–FruR hybrid repressor protein was constructed with the N-terminal part of the sucrose repressor of K. pneumoniae and the C-terminal part of the fructose repressor of Salmonella typhimurium, LT2. Gel retardation assays showed that the hybrid protein bound to scr-specific operators, and that D-fructose 1-phosphate, the inducer for FruR, was the only inducer. In vivo, neither the operators of the fru operon nor of the pps, operon, the natural targets for FruR, were recognized, but the scr operators were. These data and the data obtained from the super-repressor alleles confirm previous models on the binding of repressors of the Lacl family to their operators.     

Ultrafast electron transfer in the recognition of different DNA sequences by a DNA-binding protein with different dynamical conformations

Ultrafast electron transfer (ET) phenomenon in protein and protein–DNA complex is very much crucial and often leads to the regulation of various kinds of redox reactions in biological system. Although, the conformation of the protein in protein–DNA complex is concluded to play the key role in the ET process, till date very little evidences exist in the literature. λ-repressor–operator DNA interaction, particularly O_R1 and O_R2, is a key component of the λ-genetic switch and is a model system for understanding the chemical principles of the conformation-dependent ET reaction, governed by differential protein dynamics upon binding with different DNA target sequences. Here, we have explored the photoinduced electron transfer from the tryptophan moieties of the protein λ-repressor to two operators DNA of different sequences (O_R1 and O_R2) using picosecond-resolved fluorescence spectroscopy. The enhanced flexibility and different conformation of the C-terminal domain of the repressor upon complexation with O_R1 DNA compared to O_R2 DNA are found to have pronounced effect on the rate of ET. We have also observed the ET phenomenon from a dansyl chromophore, bound to the lysine residue, distal from the DNA-binding domain of the protein to the operator DNA with a specific excitation at 299?nm wavelength. The altered ET dynamics as a consequence of differential protein conformation upon specific DNA sequence recognition may have tremendous biological implications.     

Comparative oxygen affinity of fish and mammalian myoglobins

Summary Myoglobins from rat, coho salmon (Oncorhynchus kisutch), buffalo sculpin (Enophrys bison) hearts, and yellowfin tuna (Thunnus albacares) red skeletal muscle were partially purified and their O₂ binding affinities determined. Commercially prepared sperm whale myoglobin was employed as an internal standard. Tested at 20°C, myoglobins from salmon and sculpin bound O₂ with lower affinity than myoglobins from the rat or sperm whale. Oxygen binding studies at 12°C and 37°C suggest that this difference is adaptive, permitting myoglobins from cold-adapted fish to function at physiologically relevant temperatures. Taken together, purification and O₂ binding data obtained in this study reveal a previously unrecognized diversity of myoglobin structure and function.