Donald M. Crothers

Studies of the binding of actinomycin and related compounds to DNA

Equilibrium, kinetic and hydrodynamic studies are reported of the binding to DNA of actinomycin, its derivatives, and some simpler analogs. The major conclusions are: (1) The actinomycin chromophore is intercalated between the base pairs in the DNA complex. (2) Binding can occur adjacent to any GC pair, but binding at a given site produces a distortion of the helix that greatly disfavors binding of another actinomycin closer than six base pairs away. (3) The source of the helix distortion is probably a pair of hydrogen bonds formed between the deoxyribose ring oxygens and the -CONH- groups attached to the chromophore. (4) The specificity for guanine among the common bases results from electronic interactions in the π-complex formed in an intercalated structure. Studies of complex formation between actinomycin and simple aromatic systems make this conclusion plausible. (5) Several forms of the complex exist at equilibrium; these result from conformational changes within the cyclic peptide rings of actinomycin. (6) In the most stable form of the complex, the peptide rings have undergone conformational changes which adapt their structures to interact specifically with the DNA backbone, one ring interacting with each strand of the double helix. (7) The physical property which distinguishes actinomycin from the simpler analogs (which lack biological activity) is a very slow dissociation reaction, several orders of magnitude slower than for the non-active analogs. The structural basis for the slow dissociation is the slow reversal of the conformational change of the peptide rings. The peptide ring containing five amino acids is probably selected for this function because of the great steric hindrance to conformational changes inherent in such a tightly packed structure.

Calculation of binding isotherms for heterogeneous polymers

The matrix method of statistical mechanics is used to calculate equilibria for the binding of small molecules to polymers. When there is only one kind of binding site the problem is simple; some examples are given for illustrative purposes. If, however, the binding sites are not all equivalent and the bound molecules interact or interfere with each other, the problem is no longer trivial, being formally analogous with calculation of the helix–coil transition equilibrium in a heterogeneous polypeptide. Particular difficulties arise when the sequence of binding sites is aperiodic; most naturally occurring materials fall in this class. The purpose of this paper is to point out that problems of this type are readily solved with good accuracy by use of random‐number methods on a high‐speed digital computer. One such calculation is presented for illustration. The methods developed are applicable to such systems as the binding of actinomycin, Hg–, and acridine dyes to DNA.

The influence of polyvalency on the binding properties of antibodies.

Abstract We present a general formalism for predicting the relationship between the binding affinities in several kinds of antigen-antibody interactions. The reference binding constant is that of monovalent antibody for monovalent antigen. The equations then allow one to predict the ratio of this constant to that for attaching divalent antibody to divalent antigen. We further develop the theory to consider the binding of multivalent antibodies to a multideterminant antigen particle, in the restricted case of small fractional occupancy of the determinant sites by antibodies. There is good agreement between the calculated results and certain data from the literature. In situations where multisite adherence to a single particle and cross-linking of discrete particles are both possible, the former is predicted to predominate strongly. That the opposite appears sometimes to be experimentally the case means that special features favoring agglutination reactions must be present in these instances. Finally, those factors that potentially make polyvalent γM antibodies more effective agglutinators than bivalent γG antibodies are delineated briefly.

Relaxation studies of the proflavine-DNA complex: The kinetics of an intercalation reaction☆

Abstract We report temperature-jump relaxation kinetic studies of the proflavine-DNA complex. Consideration is restricted to the “strong binding” region commonly thought to represent intercalation of the dye between the base pairs. Two relaxation times are found, indicating that there are two forms of the complex at equilibrium. Evidence is presented for the view that in the less strongly bound form the dye is attached externally to the double helix, and that this outside binding precedes insertion in the intercalation reaction. The relaxation method is able to measure the forward and reverse rates for insertion of proflavine between the base pairs, and is therefore able to determine accurately the equilibrium distribution between intercalated and externally bound states of the dye. External binding is found to contribute only a small percentage of the total binding when the salt concentration is high (about 7% in 0.2 m -Na + ), but is much more important in low salt concentrations. Glucosylation of the DNA, as in T2 bacteriophage, increases the fraction of external binding by a factor of about three. The insertion reaction occurs in roughly the millisecond time range, as a firstorder process from the externally bound state. Lower salt concentrations accelerate intercalation, whereas glucosylation of the DNA slows the process markedly. Two limiting mechanisms for the insertion reaction are proposed, distinguished by whether or not an adjacent base pair must open to allow the dye to enter. The results favor, but do not conclusively prove, a mechanism that proceeds without opening of base pairs.

Free energy of imperfect nucleic acid helices. II. Small hairpin loops.

Abstract Physical studies of enzymically synthesized oligonucleotides of defined sequence are used to evaluate quantitatively the stability of small RNA hairpin loops and helices. The series (Ap) 4 G(pC) N (pU) 4 , N = 4, 5 or 6, exists as monomolecular hairpin helices when N ≥ 5, and as imperfect dimer helices when N ≤ 4. In this size range, hairpin loops become more favorable (less destabilizing thermodynamically) as they increase in size from 3 to 4 to 5 unbonded nucleotides. Very small hairpin loops are particularly destabilizing; molecules whose base sequence would imply a hairpin loop of three nucleotides will generally exist with a loop of five, including a broken terminal base pair. Thermodynamic parameters for base pair and loop formation are calculated by a method which makes unnecessary the use of measured enthalpies of polynucleotide melting. Literature data on oligonucleotide double helices yield estimates of the free energy contribution from each of the six types of stacking interactions between three possible neighboring base pairs. The advantage of this approach is that the properties of oligonucleotides are used in predicting the stability of small RNA helices, avoiding the long extrapolation from the properties of high polymers. We provide Tables of temperature-dependent free energies that allow one to predict the stability and thermal transition temperature of many simple RNA secondary structures (applicable to ~1 m -Na + concentration). As an example, we apply the rules to an isolated fragment of tRNA Ser (yeast) (Coutts, 1971), whose properties were not used in calculating the free-energy parameters. The experimental melting temperature of 88 °C is predicted with an error margin of 5 deg. C.

The molecular mechanism of thermal unfolding of Escherichia coli formylmethionine transfer RNA

Abstract The molecular mechanism of thermal unfolding of Escherichia coli tRNA fMet (in 0.17 m -NaCl without Mg 2+ ) has been elucidated by a combination of relaxation kinetics and proton nuclear magnetic resonance spectroscopy. We measured the n.m.r. ‡ spectrum of the hydrogen-bonded ring NH protons at different temperatures and found that the resonances assigned to each arm of the cloverleaf broaden and disappear together, yielding four distinct n.m.r. “melting” transitions. Temperature-jump measurements in the same solvent showed five co-operative melting transitions, varying in relaxation time from a few microseconds to ten milliseconds. The relaxation and n.m.r. measurements were correlated by the following model. When the lifetime of a hydrogen-bonded proton in a helix is five milliseconds, its n.m.r. line will be broadened to approximately twice its intrinsic low-temperature width and appear to “melt”. The helix dissociation time constants of the relaxation effects were extrapolated by the Arrhenius equation to lower temperatures where their values were five milliseconds. The correlation of extrapolated dissociation time constants with n.m.r. melting of specific helices allowed assignments of the structural basis for each relaxation effect. The results show that the principal path for the reversible thermal unfolding of tRNA 1 fMet under these solution conditions is first, transient opening of the dihydrouridine helix, followed by simultaneous melting of the dihydrouridine helix and a “tertiary” interaction, which does not correspond to a cloverleaf helix. The tertiary interaction is much less stable in tRNA 3 fMet , with T m lowered by 16 °C from tRNA 1 fMet . The sequence of melting steps at higher temperatures is the same in the two isoacceptors: first the TΨC helix melts, followed by the anticodon helix and finally the acceptor stem helix. Thermodynamic and kinetic parameters are reported for these steps. The method of sequential melting, combining n.m.r. and relaxation kinetic techniques, is a powerful procedure for elucidating RNA secondary structure. In addition, this method allows assignment of many hydrogen-bonded ring NH proton resonances that are unresolved in the low-temperature spectrum.

Free energy of imperfect nucleic acid helices: III. Small internal loops resulting from mismatches☆☆☆

Abstract Physical studies of enzymioally synthesized oligoribonucleotides of defined sequence are used to evaluate quantitatively the destabilizing influence of mismatched bases in a double helix. The series (A-) 4 G(-C) n (-U) 4 , N = 1 to 6, exist as imperfect dimer helices when N is equal to or less than 4, and as monomolecular hairpin helices when N is 5 and 6. Internal loops become progressively more destabilizing as their size increases from 2 to 4 to 6 nucleotides resulting from 1, 2 and 3 consecutive mismatched base pairs. However, the stability of a helix will generally be greater if a given number of mismatched pairs occur consecutively rather than in isolation from one another. These data may be used for improved calculations of stability of RNA secondary structure, to estimate the frequency of structural fluctuations in a double helix and to assess the stability of modified polynucleotide helices. An unmodified double helix of one million randomly arranged base pairs should contain on the time average approximately 10 G.C and 500 A.U pairs in non-hydrogen bonded, unstacked conformations at 25 °C. Our estimate of the effect of mismatching on T m values of high polymers is less precise because of the long temperature extrapolation required. However, we estimate that DNA or RNA treated with mutagens which interrupt up to 20% of the nucleotide pairs will show a drop of about 1.2 deg. C in melting temperature with each unit per cent of modification.

DNA-ethidium reaction kinetics: Demonstration of direct ligand transfer between DNA binding sites

Abstract Study of the relaxation kinetics of the interaction of ethidium and DNA reveals a novel and potentially important general binding mechanism, namely direct transfer of the ligand between DNA binding sites without requiring dissociation to free ligand. The measurable relaxation spectrum shows three relaxation times, indicating that three bound dye species are present at equilibrium; about 80% of the dye is in the major intercalated form. For each relaxation the reciprocal relaxation time varies linearly with concentration up to very high DNA concentrations. The failure of the longer relaxation times to plateau at high concentration can be accounted for by including a bimolecular pathway for conversion from one complex form to another. This we envisage as direct transfer of an ethidium molecule, bound to one DNA molecule, to an empty binding site on another DNA molecule. Additional evidence for this direct transfer mechanism was obtained from an experiment showing that DNA (which binds ethidium relatively rapidly) accelerates the binding of ethidium to poly(rA) · poly(rU), presumably by first forming a DNA-ethidium complex and then transferring the ethidium to RNA. The bimolecular rate constant for transfer is found to be about four times larger than the constant for intercalating the free dye. The transfer pathway thus provides a highly efficient means for the ligand to equilibrate over its DNA binding sites, especially at high polymer concentration. The potential importance of direct transfer for DNA-binding regulatory proteins is emphasized.

The DNA binding domain and bending angle of E. coli CAP protein

We use a new gel electrophoretic analysis to map the thermodynamically defined DNA binding domain of Escherichia coli CAP protein in the lac promoter. Strong binding interactions span a 28-30 bp duplex DNA region, substantially larger than that found for typical repressors. Sequence changes outside the central 28 bp of the binding site are found to affect the electrophoretically observed extent of bending. We also report a study of the DNA bending induced at a symmetrized CAP binding site, compared with the wild-type site; binding and bending are stronger at the upstream than at the downstream half of the wild-type site. Bends of the estimated 90 degrees - 180 degrees magnitude could play a vital regulatory role by producing tertiary structure in a local DNA domain, and by storing elastic energy for subsequent use in transcription or replication.

Studies of the complex between transfer RNAs with complementary anticodons: I. Origins of enhanced affinity between complementary triplets☆

Abstract We used the temperature-jump method to study the complex between yeast t RNAPheand Escherichia coli tRNAGlu, which have the complementary anticodons GmAA and s2UUC, respectively. The binding constant (3.6 × 105 m −1 at 25 °C) is about six orders of magnitude larger than expected for two complementary trinucleotides. The association rate constant (3 × 106 m −1 at 25 °C) is similar to typical values observed for oligonucleotides, so the enhanced affinity in the tRNA · tRNA complex is due entirely to a much slower dissociation than expected for a three base-pair helix. We found an association enthalpy of −25 kcal/mol, nearly twice as large as expected for two stacking interactions in a three base-pair helix. The association entropy (−58 cal/deg per mol) is close to the expected value. The reaction occurs with a single relaxation, and therefore does not involve any slow reorganization of the tRNA molecule. We studied structural variations to investigate the origin of affinity enhancement. The following general factors are important. (1) The “loop constraint”, or closure of the two anticodon sequences into hairpin loops, accounts for about a factor 50 in the affinity. (2) “Dangling ends”, or non-complementary nucleotides at the end of the double helix contribute strongly to the affinity. (3) Modified nucleotides, like the Y base, in the dangling ends can contribute a special stabilization of up to a factor seven. These observations can be understood in terms of a model in which the short three base-pair helix is sandwiched between stacked bases and hence stabilized. The potential importance of loop-loop interactions and stacking effects for codon-anticodon bonding is emphasized. The results suggest a possible simple physical basis for the evolutionary choice of a triplet coding system.