Peter H. von Hippel

Theoretical aspects of DNA-protein interactions: Co-operative and non-co-operative binding of large ligands to a one-dimensional homogeneous lattice☆

Abstract The interaction of proteins binding non-specifically to DNA, as well as the properties of many other interacting ligand-lattice systems important in molecular biology, requires a fundamentally different type of theoretical analysis than that provided by the classical Scatchard independent-binding-site treatment. Exact and relatively simple equations describing the binding of both non-interacting and interacting (co-operative) ligands to a homogeneous one-dimensional lattice are derived in terms of ligand site size, intrinsic binding constant and ligand-ligand co-operativity (equations (10) and (15) in the text). The mathematical approach is based on simple conditional probabilities, and reveals some largely unrecognized characteristics of such lattice binding systems. The results indicate that the binding of any non-interacting ligand covering more than one lattice residue results in non-linear (convex downward) Scatchard plots. The introduction of positive ligand-ligand co-operativity antagonizes this non-linearity, and eventually leads to plots of the opposite curvature. The maxima, limiting slopes, and intercepts of such plots can be used to estimate the required binding parameters. The method can be extended to systems involving heterogeneous ligands, and some types of heterogeneous lattices. Procedures for applying the method to a variety of interacting systems are presented, and a preliminary analysis is carried out for some selected sets of data from the literature.

NEUTRAL SALTS: THE GENERALITY OF THEIR EFFECTS ON THE STABILITY OF MACROMOLECULAR CONFORMATIONS.

The effects of various neutral salts on the temperature of the thermally-induced denaturation of the globular protein ribonuclease are described and compared with the effects of these salts on helix-coil transition temperatures in other macromolecules. These agents affect the stability of the native form of macromolecules as diverse as ribonuclease, collagen, DNA, and myosin in very similar ways; salts such as KSCN and CaCl2 serve as very potent general structural destabilizers or denaturants, while salts such as (NH4)2SO4 and K2HPO4 strongly stabilize the native conformation. The effectiveness of the neutral salts as ribonuclease destabilizers is compared with that of urea and the guanidinium salts.

The structure of collagen and gelatin.

Publisher Summary This chapter reviews that collagen constitutes the major protein component of skin, bone, tendon, and all the other forms of connective tissue. An understanding of collagen seems to the clinician to be a necessary prerequisite to a rational attack on many and diverse connective tissue disorders currently lumped together as “collagen diseases.” The unusual amino acid composition of collagen had also been recognized for some time. One-third of the residues of all collagens seemed to be glycine, while about one-fourth were proline and hydroxyproline. However, the stereochemical consequences of the presence of these residues has only become clear as a result of the detailed studies of synthetic homo- and copolymers of glycine and proline. Consideration of such synthetic polypeptides as simplified models of certain features of collagen and gelatin has been extremely helpful in recent years, and constitutes the rationale for the inclusion of a section dealing specifically with these synthetic polypeptides in this chapter. It also reviews that the collagen ⇆ gelatin transformation in solution has been recognized as a reversible first-order phase transition, subject to the same physical laws which govern the crystalline ⇆ amorphous phase transitions observed in systems of linear polymers. The direct relationship between the transition in solution and the well-known thermal shrinkage phenomenon exhibited by collagen fibers has also been established.

Interactions of bacteriophage T4-coded gene 32 protein with nucleic acids. I. Characterization of the binding interactions.

Abstract In this paper we examine molecular details of the interaction of bacteriophage T4-coded gene 32 protein with oligo- and polynucleotides. It is shown that the binding affinity ( K oligo ) of oligonucleotides of length ( l ) from two to eight nucleotide residues for gene 32 protein is essentially independent of base composition or sugar type. This binding also shows little dependence on salt concentration and on oligonucleotide length; even the expected statistical length factor in K oligo is not observed, suggesting that binding occurs at the end of the oligonucleotide lattice and that the oligonucleotide is not free to move across the binding site. Co-operative (contiguous) or isolated binding of gene 32 protein to polynucleotides is very different; here binding is highly salt dependent ( ∂ log Kω ∂ log [NaCl] ∼- −7 ) and essentially stoichiometric at salt concentrations less than ~0.2 m (for poly(rA)). Binding becomes much weaker and the binding isotherms appear typically co-operative (sigmoid) in protein concentration at higher salt concentrations. We demonstrate, by fitting the co-operative binding isotherms to theoretical plots at various salt concentrations and also by measuring binding at very low protein binding density (ν), that the entire salt dependence of Kω is in the intrinsic binding constant ( K ); the co-operativity parameter (ω) is essentially independent of salt concentration. Furthermore, by determining titration curves in the presence of salts containing a series of different anions and cations, it is shown that the major part of the salt dependence of the gene 32 protein-polynucleotide interaction is due to anion (rather than to cation) displacement effects. Binding parameters of oligonucleotides of length sufficient to bind two or more gene 32 protein monomers show behavior intermediate between the oligonucleotide and the polynucleotide binding modes. These different binding modes probably reflect different conformations of the protein; the results are analyzed to produce a preliminary molecular model of the interactions of gene 32 protein with nucleic acids in its different binding modes.

Action at a distance: DNA-looping and initiation of transcription

Effective initiation of transcription, especially in eukaryotes, requires the specific assembly of large protein complexes at promoters. We ask here how activator proteins that are bound hundreds or thousands of base pairs away from the promoter might facilitate this process if protein-protein interactions occur via looping of the intervening DNA. We show that the local concentration at the promoter of activator proteins bound at vicinal DNA sites can be substantially regulated by intrinsic or protein-induced distortion of the regular DNA conformation.

Model nucleoprotein complexes: Studies on the interaction of cationic homopolypeptides with DNA

Abstract Complexes of natiye calf thymus DNA with the cationic polypeptides poly-lornithine, poly-l-lysine, poly-l-arginine and poly-l-homoarginine, have been prepared and their solubility, stoichiometry, absorption spectra and thermal denaturation studied. Increasing the peptide cation/DNA phosphate ratio, up to electrostatic equivalence, yielded progressively more insoluble products and increased the turbidity of the “soluble” fraction. Certain spectral changes were observed which may be largely attributed to anomalous scattering in the absorbing region. Addition of the polypeptides to DNA resulted in a marked stabilization of the helix against thermal denaturation. At peptide cation/DNA phosphate ratios less than electrostatic equivalence, thermal denaturation monitored at 260 and 280 mμ revealed a biphasic transition profile: the first transition had a melting temperature similar to DNA under the same solvent conditions; the second melting temperature was characteristic for the type of polypeptide in the complex. Thermal denaturation monitored at 350 mμ (i.e., turbidity transitions) showed a monophasic profile at the higher melting temperature of the DNA-polypeptide complex. The different polypeptides stabilized DNA against melting to different extents. In order of decreasing degree of stabilization they are: poly-l-ornithine > poly-l-lysine > poly-l-arginine > poly-l-homoarginine. Analysis of the dispersion of hyperchromicity demonstrated that poly-l-ornithine and poly-l-lysine preferentially stabilize A-T rich regions, whereas poly-l-arginine and poly-l-homoarginine appear less discriminating. The soluble DNA-polypeptide complexes could be subfractionated by ultracentrifugation into a fraction which melted like “naked” DNA, and a fraction which melted at the higher temperature characteristic of the complex and showed a peptide cation/DNA phosphate ratio close to electrostatic equivalence. The experimental data imply that, under proper conditions of annealing, the basic polypeptides form definable molecular structures with DNA; the binding reaction is stoichiometric and co-operative. Model-building suggests that the polypeptides could interact with either the large or small groove of DNA.

A General Model for Nucleic Acid Helicases and Their “Coupling” within Macromolecular Machines

In this article we present a general framework that can highly processive, efficient, specific for the type (DNA be used to describe the molecular mechanisms whereby or RNA) of ssNA substrate (lattice) to which they bind, ATP-driven helicases separate and rearrange the com- and directional (59!39 or 39!59) in their movements plementary strands of double helical nucleic acids. This along the target ssNA lattice (for a general review of framework also permits us to consider how these heli- helicase mechanisms see Lohman and Bjornson, 1996). cases might be functionally coupled to other compo- The processivity of a helicase at a given lattice (e.g., nents within the macromolecular machines that carry template) position is defined as the probability that the out physiological processes. We then proceed to define helicase at that position will continue to translocate forparameters that can be used to quantify helicase func- ward by one step along the NA substrate, divided by tion and derive simple thermodynamic equations that the probability that the helicase will dissociate from the describe helicase reactions in isolation and in coupled substrate lattice at that position. The processivity of a systems. helicase is often regulated by additional protein compoAspects of the molecular mechanisms of helicase nents or “coupling” factors, which may interact with the function are then developed using known systems of helicase either directly, or indirectly via the nucleic acid increasing complexity. We begin by considering simple components of the system (see Table 1 and below). DNA “melting proteins” that can—under some condi- Such processivity coupling factors can be operationally tions—open DNA without binding or hydrolyzing ATP. defined as components that interact functionally with We then discuss the cargo-carrying molecular motors the helicase to “trap” intermediate ssNA reaction prodthat, like helicases, use the chemical free energy of ATP ucts of the dsNA opening reaction and facilitate their hydrolysis to translocate directionally along specific cy- subsequent use (e.g., as an ssNA template) by the macromolecular machine within which the helicase operates toplasmic “tracks” but do not, of course, “open” the

Thinking quantitatively about transcriptional regulation

By thinking about the chemical and physical mechanisms that are involved in the stepwise elongation of RNA transcripts, we can begin to understand the way that these mechanisms are controlled within the cell to reflect the different requirements for transcription that are posed by various metabolic, developmental and disease states. Here, we focus on the mechanistic details of the single-nucleotide addition (or excision) cycle in the transcription process, as this is the level at which many regulatory mechanisms function and can be explained in quantitative terms.

Kinetics of protein-nucleic acid interactions: use of salt effects to probe mechanisms of interaction

The kinetics of protein-nucleic acid interactions are discussed with particular emphasis on the effects of salt concentration and valence on the observed rate constants. A general review is given of the use of experimentally determined salt dependences of observed kinetic parameters as a tool to probe the mechanism of interaction. Quantitative analysis of these salt dependences, through the application of polyelectrolyte theory, can be used to distinguish reactions which occur in a single step from those reactions which involve distinct intermediates. For those rate constants which display a large salt dependence, in either the association or dissociation reaction, this is due to the high concentration of counterions (e.g., Na+) in the vicinity of the nucleic acid which are subsequently released (or bound in the case of dissociation) at some point before the rate limiting step of the reaction. A general discussion of other features which affect protein-nucleic acid kinetics, such as nucleic acid length and the ratio of nonspecific to specific DNA binding sites (in the case of sequence specific binding proteins), is also given. The available data on the nucleic acid binding kinetics of small ligands (ions, dyes, oligopeptides), nonspecific binding proteins (T4 gene 32 protein, fd gene 5 and Escherichia coli SSB), and sequence specific binding proteins (lac repressor, RNA polymerase, Eco RI restriction endonuclease) are discussed with emphasis on the interpretation of the experimentally determined salt dependences.

Interactions of bacteriophage T4-coded gene 32 protein with nucleic acids: II. Specificity of binding to DNA and RNA☆

In this paper we examine the specificity of the co-operative binding (in the polynucleotide mode) of bacteriophage T4-coded gene 32 protein to synthetic and natural single-stranded nucleic acids differing in base composition and sugar type. It is shown by competition experiments in a tight-binding (low salt) environment that there is a high degree of binding specificity under these (protein-limiting) conditions, with one type of nucleic acid lattice binding gene 32 protein to saturation before any binding to the competing lattice takes place; it is also shown that the same differential specificities apply at high salt concentrations. Procedures developed in the preceding paper (Kowalczykowski et al., 1980) are used to measure the net binding affinities (Kω) of gene 32 protein to a variety of polynucleotides, as well as to determine individual values of K and ω for some systems. For all polynucleotides, virtually the entire specificity and salt dependence of binding of Kω appears to be in K. In ~0.2 m-NaCl, the net binding affinities (Kω) range from ~106 to ~1011 m−1; in order of increasing affinities we find: poly(rC) < poly(rU) < poly(rA) < poly(dA) < poly(dC) < poly(dU) < poly(rI) < poly(dI) < poly-(dT). In general, Kω for a particular homopolyribonucleotide at constant salt concentration is 101 to 104 smaller than Kω for the corresponding homopoly-deoxyribopolynucleotide. Values of Kω for randomly copolymerized polynucleotides and for natural DNA fall at the compositionally weighted average of the values for the individual homopolynucleotides (except for poly(dT), which appears to bind somewhat tighter), indicating that the net affinity represents the sum of the binding free energy contributions of the individual nucleotides. It is shown that these results, on a competition basis under physiological salt conditions, can account quantitatively for the autogenous regulation of the synthesis of gene 32 protein at the translational level (Russel et al., 1976; Lemaire et al., 1978). In addition, these results suggest possible mechanisms by which gene 32 messenger RNA might be specifically recognized (by gene 32 protein) and functionally discriminated from the other mRNAs of phage T4.