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.     

Identification of functionally important residues in the DNA binding region of the mnt repressor

Single amino acid substitutions have been introduced throughout the N-terminal DNA binding region of the Mnt repressor, and the operator binding properties of the resulting mutant repressors have been assayed. These studies show that the side chains of Arg2, His6, Asn8, and Arg10 are critical for high affinity binding to operator DNA. Other side chains in the N-terminal region do not appear to play major roles in DNA recognition and binding. Specific alterations in the pattern of methylation protection afforded by the Arg2----Lys mutant protein suggest that Arg2 contacts the N7 groups of guanines 10 and 12 in the operator. In conjunction with previous results, these findings suggest that part of the N-terminal region of Mnt binds as an extended polypeptide strand within the major groove of the mnt operator.     

Biochemical and genetic analysis of operator contacts made by residues within the beta-sheet DNA binding motif of Mnt repressor.

Residues 2, 6, 8 and 10 of Mnt repressor are the major determinants of operator DNA binding and recognition. Here, we investigate the interaction of wild-type Mnt and mutants bearing the Arg2----Lys, His6----Ala, Asn8----Ala and Arg10----Lys mutations with operator DNA modified by methylation or by symmetric base substitutions. The wild-type pattern of methylation interference is altered in specific ways for each of the mutant proteins. In addition, some of the mutant proteins show a 'loss of contact' phenotype with specific mutant operators. Taken together, these and previous results predict the following contacts between side chains in the Mnt tetramer and operator DNA: Arg2 recognizes the guanines at operator positions 10 and 12; His6 contacts the guanines at operator positions 5 and 17; Asn8 contacts operator positions 4, 7, 15 and 18; Arg10 contacts the guanines at operator positions 8 and 14. The proposed contacts can be accommodated in a structural model in which the anti-parallel beta-sheet motifs of Mnt dimers lie in the major grooves of each operator half-site, centered over pseudo-symmetry axes that are 5.5 bp from the central dyad axis of the operator.     

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.     

In vivo selection of randomly mutated retroviral genomes.

Darwinian evolution, that is the outgrowth of the fittest variants in a population, usually applies to living organisms over long periods of time. Recently, in vitro selection/amplification techniques have been developed that allow for the rapid evolution of functionally active nucleic acids from a pool of randomized sequences. We now describe a modification of the nucleic acid-evolution protocol in which selection and amplification take place inside living cells by means of a retroviral-based replication system. We have generated a library of HIV-1 DNA genomes with random sequences in particular domains of the TAR element, which is the binding site for the Tat trans-activator protein. This mixture of HIV genomes was transfected into T cells and outgrowth of the fittest viruses was observed within two weeks of viral replication. The results of this in vivo selection analysis are consistent with the notion that primary sequence elements in both TAR bulge and loop domains are critical for Tat-mediated trans-activation and viral replication.     

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 a3 helix are T, R, E, S respectively. By using a randomized DNA pool containing the central sequence -CATACAAGAAAGNNNNNNTTT-, 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 1×10-12 mol/L-1×10-11mol/L concentration range. Since the affinity of the natural 434 repressor to its natural operator sites is in the 1×10-9 mol/L range, the observed binding affinity increase is remarkable. It was also found that binding affinity was strongly affected by the flanking bases of the optimal tetramer binding sites, especially by the base at the 5′ position. We constructed a new homodimeric single-chain repressor RTRESRTRES and its DNA-binding specificity was tested by using a series of new operators designed according to the recog-nition properties previously determined for the RTRES domain. These operators containing the con-sensus sequence GTAAGAAARNTTACN or GGAAGAAARNTTCCN (R is A or G) were recognized by RTRESRTRES specifically, and with high binding affinity. Thus, by using a combination of random selection and rational design principles, we have discovered novel, high affinity protein-DNA inter-actions with new specificity. This method can potentially be used to obtain new binding specificity for other DNA-binding proteins.     

Analysis of trp repressor-operator interaction by filter binding.

A filter binding assay was developed that allows measurement of specific binding of trp repressor to operator DNA. The most important feature of this procedure is the concentration and type of salt present in the binding buffer. Using this assay the dissociation constant of the repressor-operator complex was determined to be 2.6 X 10(-9) M, and 1.34 repressor dimers were found to be bound to each operator-containing DNA molecule. These values agree with those obtained by more complex methods. The dissociation constant of the repressor for the corepressor L-tryptophan in the presence of operator DNA was shown to be 2.5 X 10(-5) M. A synthetic 48 bp operator fragment was used to determine the repressor-operator dissociation constant in the presence of tryptophan or tryptophan analogs which have higher or lower affinities for aporepressor. The rate of dissociation of repressor from operator DNA also was determined. Our findings indicate that dissociation is influenced by the concentration of tryptophan or tryptophan analogs and suggest that release of the corepressor may be the first step in dissociation of the repressor-operator complex.     

The LexA repressor and its isolated amino-terminal domain interact cooperatively with poly[d(A-T)], a contiguous pseudo-operator, but not with random DNA: a circular dichroism study

The interaction of the entire LexA repressor and its amino-terminal DNA binding domain with poly[d(A-T)] and random DNA has been studied by circular dichroism. Binding of both protein species induces an about 2-fold increase of the positive circular dichroism band at about 270 nm of both polynucleotides, allowing a precise determination of the principal parameters as a function of mono- and divalent salt concentration and pH. Both proteins interact much more strongly (about 2000-fold) with poly[d(A-T)] than with random DNA as expected from the homology with the specific consensus binding site of LexA (CTGTATATATATACAG). For both LexA and its DNA binding domain we find that the interaction with poly[d(A-T)] is cooperative with a cooperativity factor omega of about 50-70 for both proteins over a wide range of solvent conditions, suggesting that the carboxy-terminal domain of LexA is not involved in this type of cooperativity. On the contrary, no cooperativity could be detected for the interaction of the LexA DNA binding domain with a random DNA fragment. The overall binding constant K omega (or simply K in the case of random DNA) depends strongly on the salt concentration as observed for most protein-DNA interactions, but the behavior of LexA is unusual in that the steepness of this salt dependence (delta log K omega/delta log [NaCl]) is much more pronounced at slightly acidic pH values as compared to that at neutral or slightly alkaline pH. The behavior is not easily understood in terms of the current theories on the electrostatic contribution to protein-DNA interactions on the basis of polyelectrolyte theory. A comparison of the overall binding constant K omega of the entire LexA repressor and its DNA binding domain reveals that LexA binds only 20-50-fold stronger under a wide variety of salt and pH conditions. This result tends to demonstrate further that the additional energy due to the dimerization of LexA via the carboxy-terminal domain should be rather weak as expected from the small dimerization constant of LexA (2 X 10(-4) M-1).     

Dynamics of repressor-operator recognition: the Tn10-encoded tetracycline resistance control

Binding of the Tet repressor to nonspecific and specific DNA leads to quenching of the Tet fluorescence by approximately 22% and approximately 35%, respectively. This effect is used for a direct, quantitative characterization of the binding equilibria and dynamics involved in the recognition of the operator by its repressor. From the dependence of the nonspecific binding constant on the ion concentration, it is concluded that nonspecific binding is almost completely driven by the entropy change resulting from the release of three to four Na+ ions from the double helix upon protein binding. Formation of the specific complex is driven by a higher entropy term resulting from the release of seven to eight Na+ ions and in addition by a free energy term of -33 kJ/mol from nonelectrostatic interactions, which are attributed to the specific contacts. The dynamics of the repressor-operator recognition are resolved by stopped-flow measurements at various salt concentrations and for different DNA chain lengths into two separate steps. The first step follows a second-order mechanism and results in an intermediate complex associated with formation of about three to four electrostatic contacts between protein and DNA; apparently, this complex is equivalent to the nonspecific complex. The existence of an intermediate is also indicated by experiments in mixed Na+-Mg2+ buffers, which can be described with high accuracy by competition of Mg2+ and protein. The intermediate complex is formed at a rate of 3 X 10(8) M-1 s-1 and is converted in the second reaction step to the specific complex with a rate constant of 6 X 10(4) s-1, which is almost independent of the salt concentration. Our interpretation and the parameters obtained from our model are confirmed by competition of nonspecific DNA with operator DNA for repressor binding. The observed maximal rate constant of 3 X 10(8) M-1 s-1 is very close to theoretical predictions for the association without a sliding mechanism. The very small dependence of the observed rate constants on the chain length shows that the Tet repressor is not able to slide over any substantial distance even at low salt concentrations. The question of a potential contribution from sliding under our experimental conditions is critically discussed. The absence of sliding in the case of the Tet repressor under physiological conditions is compared with the high sliding efficiency of the lac repressor and is discussed with respect to possible molecular mechanisms of sliding in relation to biological function.     

Kinetics and mechanism in the reaction of gene regulatory proteins with DNA

We have measured the kinetic properties of the Escherichia coli cAMP receptor protein (CAP) and lac repressor interacting with lac promoter restriction fragments. Under our reaction conditions (10 mM-Tris X HCl (pH 8.0 at 21 degrees C), 1 mM-EDTA, 10 microM-cAMP, 50 micrograms bovine serum albumin/ml, 5% glycerol), the association of CAP is at least a two-step process, with an initial, unstable complex formed with rate constant kappa a = 5(+/- 2.5) X 10(7) M-1 s-1. Subsequent formation of a stable complex occurs with an apparent bimolecular rate constant kappa a = 6.7 X 10(6) M-1 s-1. At low total DNA concentration, the dissociation rate constant for the specific CAP-DNA complex is 1.2 X 10(-4) s-1. The ratio of formation and dissociation rate constants yields an estimate of the equilibrium constant, Keq = 5 X 10(10) M-1, in good agreement with static results. We observed that the dissociation rate constant of both CAP-DNA and repressor-DNA complexes is increased by adding non-specific "catalytic" DNA to the reaction mixture. CAP dissociation by the concentration-dependent pathway is second-order in added non-specific DNA, consistent with either the simultaneous or the sequential participation of two DNA molecules in the reaction mechanism. The results imply a role for distal DNA in assembly-disassembly of specific CAP-DNA complexes, and are consistent with a model in which the subunits in the CAP dimer separate in the assembly-disassembly process. The dissociation of lac repressor-operator complexes was found to be DNA concentration-dependent as well, although in contrast to CAP, the reaction is first-order in catalytic DNA. Added excess operator-rich DNA gave more rapid dissociation than equivalent concentrations of non-specific DNA, indicating that the sequence content of the competing DNA influences the rate of repressor dissociation. The simplest interpretation of these observations is that lac repressor can be transferred directly from one DNA molecule to another. A comparison of the translocation rates calculated for direct transfer with those predicted by the one-dimensional sliding model indicates that direct transfer may play a role in the binding site search of lac repressor.     

Dissociation of the lactose repressor-operator DNA complex: effects of size and sequence context of operator-containing DNA

The dissociation kinetics for repressor-32P-labeled operator DNA have been examined by adding unlabeled operator DNA to trap released repressor or by adding a small volume of concentrated salt solution to shift the Kd of repressor-operator interaction. The dissociation rate constant for pLA 322-8, an operator-containing derivative of pBR 322, was 2.4 X 10(-3) s-1 in 0.15 M KCl. The dissociation rate constant at 0.15 M KCl for both lambda plac and pIQ, each of which contain two pseudooperator sequences, was approximately 6 X 10(-4) s-1. Elimination of flanking nonspecific DNA sequences by use of a 40 base pair operator-containing DNA fragment yielded a dissociation rate constant of 9.3 X 10(-3) s-1. The size and salt dependences of the rate constants suggest that dissociation occurs as a multistep process. The data for all the DNAs examined are consistent with a sliding mechanism of facilitated diffusion to/from the operator site. The ability to form a ternary complex of two operators per repressor, determined by stoichiometry measurements, and the diminished dissociation rates in the presence of intramolecular nonspecific and pseudooperator DNA sites suggest the formation of an intramolecular ternary complex. The salt dependence of the dissociation rate constant for pLA 322-8 at high salt concentrations converges with that for a 40 base pair operator. The similarity in dissociation rate constants for pLA 322-8 and a 40 base pair operator fragment under these conditions indicates a common dissociation mechanism from a primary operator site on the repressor.     

Changing Folding and Binding Stability in a Viral Coat Protein: A Comparison between Substitutions Accessible through Mutation and Those Fixed by Natural Selection

Previous studies have shown that most random amino acid substitutions destabilize protein folding (i.e. increase the folding free energy). No analogous studies have been carried out for protein-protein binding. Here we use a structure-based model of the major coat protein in a simple virus, bacteriophage φX174, to estimate the free energy of folding of a single coat protein and binding of five coat proteins within a pentameric unit. We confirm and extend previous work in finding that most accessible substitutions destabilize both protein folding and protein-protein binding. We compare the pool of accessible substitutions with those observed among the φX174-like wild phage and in experimental evolution with φX174. We find that observed substitutions have smaller effects on stability than expected by chance. An analysis of adaptations at high temperatures suggests that selection favors either substitutions with no effect on stability or those that simultaneously stabilize protein folding and slightly destabilize protein binding. We speculate that these mutations might involve adjusting the rate of capsid assembly. At normal laboratory temperature there is little evidence of directional selection. Finally, we show that cumulative changes in stability are highly variable; sometimes they are well beyond the bounds of single substitution changes and sometimes they are not. The variation leads us to conclude that phenotype selection acts on more than just stability. Instances of larger cumulative stability change (never via a single substitution despite their availability) lead us to conclude that selection views stability at a local, not a global, level.     

Statistical fluctuations in processes of gene expression regulation: consideration of the problem from point of view of statistical mechanics

The regulation of gene expression is a basic problem of biology. In some cases, the gene activity is regulated by specific binding of regulatory proteins to DNA. In terms of statistical mechanics, this binding is described as the process of adsorption of ligands on the one-dimensional lattice and has a probability nature. As a random physical process, the adsorption of regulatory proteins on DNA introduces a noise to the regulation of gene activity. We derived equations, which make it possible to estimate this noise in the case of the binding of the lac repressor to the operator and showed that these estimates correspond to experimental data. Many ligands are able to bind nonspecifically to DNA. Nonspecific binding is characterized by a lesser equilibrium constant but a greater number of binding sites on the DNA, as compared with specific binding. Relations are presented, which enable one to estimate the probability of the binding of a ligand on a specific site and on nonspecific sites on DNA. The competition between specific and nonspecific binding of regulatory proteins plays a great role in the regulation of gene activity. Similar to the one-dimensional "lattice gas" of particles, ligands adsorbed on DNA produce "one-dimensional" pressure on proteins located at the termini of free regions of DNA. This pressure, an analog of osmotic pressure, may be of importance in processes leading to changes in chromatin structure and activation of gene expression.     

Transition between Stochastic Evolution and Deterministic Evolution in the Presence of Selection: General Theory and Application to Virology

We present here a self-contained analytic review of the role of stochastic factors acting on a virus population. We develop a simple one-locus, two-allele model of a haploid population of constant size including the factors of random drift, purifying selection, and random mutation. We consider different virological experiments: accumulation and reversion of deleterious mutations, competition between mutant and wild-type viruses, gene fixation, mutation frequencies at the steady state, divergence of two populations split from one population, and genetic turnover within a single population. In the first part of the review, we present all principal results in qualitative terms and illustrate them with examples obtained by computer simulation. In the second part, we derive the results formally from a diffusion equation of the Wright-Fisher type and boundary conditions, all derived from the first principles for the virus population model. We show that the leading factors and observable behavior of evolution differ significantly in three broad intervals of population size, N. The “neutral limit” is reached when N is smaller than the inverse selection coefficient. When N is larger than the inverse mutation rate per base, selection dominates and evolution is “almost” deterministic. If the selection coefficient is much larger than the mutation rate, there exists a broad interval of population sizes, in which weakly diverse populations are almost neutral while highly diverse populations are controlled by selection pressure. We discuss in detail the application of our results to human immunodeficiency virus population in vivo, sampling effects, and limitations of the model.     

Interactions of Arg2 in the Mnt N-terminal arm with the central and flanking regions of the Mnt operator

Arg2, in the N-terminal arm of the Mnt repressor, plays an important role in determining operator-binding specificity. In the complex of the Mnt tetramer with the 21 base-pair mnt operator, there are four potential sites for Arg2 interactions, two in the central region of the operator and two on the outer flanks of the operator. Single-chain variants of the dimeric N-terminal domain of Mnt containing one Arg2 residue and one Lys2 or Met2 residue were constructed and interactions with operator DNA were probed using Fe. EDTA affinity cleavage. The results of these orientation studies show that the majority of the energetically significant interactions mediated by Arg2 occur in the central region of the mnt operator. The RK2, RA2, and RM2 mutations reduce the free energy of operator binding by 1.7 kcal/mol, 3.3 kcal/mol, and 4.9 kcal/mol, respectively. Double-mutant thermodynamic cycle analyses using the RA2, RM2, and operator variants also reveal interaction free energies between Arg2 and operator base-pairs 9, 10, 11, 12 and 13, which in aggregate account for most of the Arg2 contribution to operator binding.