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Biomedical subjects

O G Berg

Publications and source records attributed to O G Berg.

At least 55 records · Page 3Linked to original sources
PubMed1989

Facilitated target location in biological systems.

In this minireview we have attempted to provide some overall perspective on the question of how various forms of diffusion in reduced dimensions, or diffusion within a nonspecifically bound state, can speed biological interactions beyond the limits normally set by three-dimensional diffusion processes. To this end we began by discussing the rates expected for small molecules engaged in classical elastic collisions. We then proceeded to modify this picture by introducing first the features of inelastic macromolecular collisions in solution, then the effects of specific electrostatic fields set up around macromolecules of known structure at low ionic strengths, and finally the special rate enhancements available to DNA-protein interactions because of the particular geometry of the DNA molecule and the multiple conformations that can be assumed by the protein component. We hope that this exposition will help to clarify the subject for others and also will stimulate more focused examination of this type of problem, both in DNA-protein interaction systems and in other biological systems where such rate facilitation might apply.

DNA↗
PubMed1988

Selection of DNA binding sites by regulatory proteins: the LexA protein and the arginine repressor use different strategies for functional specificity.

The DNA sequences in the operator sites of the arginine regulon and of the SOS regulon have been subject to a statistical analysis. A quantitative correlation is found between the statistics of sequence choice and the activity at individual operator sites in both systems, as expected from theoretical considerations [Berg & von Hippel, J.Mol.Biol. (1987) 193, 723-750]. Based on these correlations it is possible to predict the effect of various sequence mutations. There is a significant difference in the slopes of the correlation lines between sequence and activity for the two systems. From this difference it can be expected that individual point mutations in the ARG boxes will have a much smaller effect on activity than similar changes in the SOS boxes. This difference may be related to a strong cooperative activity at tandem ARG boxes while the binding at SOS boxes appears to be mostly noncooperative.

Arginine↗
PubMed1988

Selection of DNA binding sites by regulatory proteins. II. The binding specificity of cyclic AMP receptor protein to recognition sites.

The statistics of base-pair usage within known recognition sites for a particular DNA-binding protein can be used to estimate the relative protein binding affinities to these sites, as well as to sites containing any other combinations of base-pairs. As has been described elsewhere, the connection between base-pair statistics and binding free energy is made by an equal probability selection assumption; i.e. that all base-pair sequences that provide appropriate binding strength are equally likely to have been chosen as recognition sites in the course of evolution. This is analogous to a statistical-mechanical system where all configurations with the same energy are equally likely to occur. In this communication, we apply the statistical-mechanical selection theory to analyze the base-pair statistics of the known recognition sequences for the cyclic AMP receptor protein (CRP). The theoretical predictions are found to be in reasonable agreement with binding data for those sequences for which experimental binding information is available, thus lending support to the basic assumptions of the selection theory. On the basis of this agreement, we can predict the affinity for CRP binding to any base-pair sequence, albeit with a large statistical uncertainty. When the known recognition sites for CRP are ranked according to predicted binding affinities, we find that the ranking is consistent with the hypothesis that the level of function of these sites parallels their fractional saturation with CRP-cAMP under in-vivo conditions. When applied to the entire genome, the theory predicts the existence of a large number of randomly occurring "pseudosites" with strong binding affinity for CRP. It appears that most CRP molecules are engaged in non-productive binding at non-specific or pseudospecific sites under in-vivo conditions. In this sense, the specificity of the CRP binding site is very low. Relative specificity requirements for polymerases, repressors and activators are compared in light of the results of this and the first paper in this series.

Base Sequence↗
PubMed1988

Selection of DNA binding sites by regulatory proteins. Functional specificity and pseudosite competition.

The frequency of base-pair occurrence in a set of recognition sequences for a particular DNA-binding protein is strongly related to the contributions to the binding free energy from the individual base pairs. Thus from the statistics of base-pair choice, it is possible to estimate the relative binding strengths of any base-pair sequences and to predict the effect of point mutations in specific sites. On the same basis, one can describe the binding properties of random DNA sequences and thereby the expected competitive effects from all the nonspecific DNA sites in the genome of a living cell. The statistical selection theory [Berg & von Hippel.J. Mol. Biol. 193 (1987) 723-750] describing these relations is extended and tested with computer simulations. The theory is shown to hold up well also in the case when base pairs contribute cooperatively to the binding interaction. The simulations also demonstrate the effects of the statistical small-sample uncertainty that appears due to the limited size of all sets of recognition sites identified.

Base Composition↗
PubMed1987

Selection of DNA binding sites by regulatory proteins. Statistical-mechanical theory and application to operators and promoters.

We present a statistical-mechanical selection theory for the sequence analysis of a set of specific DNA regulatory sites that makes it possible to predict the relationship between individual base-pair choices in the site and specific activity (affinity). The theory is based on the assumption that specific DNA sequences have been selected to conform to some requirement for protein binding (or activity), and that all sequences that can fulfil this requirement are equally likely to occur. In most cases, the number of specific DNA sequences that are known for a certain DNA-binding protein is very small, and we discuss in detail the small-sample uncertainties that this leads to. When applied to the binding sites for cro repressor in phage lambda, the theory can predict, from the sequence statistics alone, their rank order binding affinities in reasonable agreement with measured values. However, the statistical uncertainty generated by such a small sample (only 6 sites known) limits the result to order-of-magnitude comparisons. When applied to the much larger sample of Escherichia coli promoter sequences, the theory predicts the correlation between in vitro activity (k2KB values) and homology score (closeness to the consensus sequence) observed by Mulligan et al. (1984). The analysis of base-pair frequencies in the promoter sample is consistent with the assumption that base-pairs at different positions in the sites contribute independently to the specific activity, except in a few marginal cases that are discussed. When the promoter sites are ordered according to predicted activities, they seem to conform to the Gaussian distribution that results from a requirement for maximal sequence variability within the constraint of providing a certain average activity. The theory allows us to compare the number of specific sites with a certain activity to the number that would be expected from random occurrence in the genome. While strong promoters are "overspecified", in the sense that their probability of random occurrence is very low, random sequences with weak promoter-like properties are expected to occur in very large numbers. This leads to the conclusion that functional specificity is based on other properties in addition to primary sequence recognition; some possibilities are discussed. Finally, we show that the sequence information, as defined by Schneider et al. (1986), can be used directly (at least in the case of equilibrium binding sites) to estimate the number of protein molecules that are specifically bound at random "pseudosites" in the genome.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Composition↗
PubMed1986

On the specificity of DNA-protein interactions.

In this paper we summarize the various factors that must be considered in establishing the operational specificity of the binding of a protein regulator of gene expression to a DNA target site. We consider informational (combinatorial) aspects of binding-site specification, actual recognition mechanisms, and the thermodynamics of target-site selection against a background of competing pseudospecific and non-(sequence)-specific DNA binding sites. The results provide insight into the design, specification, and possibly the evolution of regulatory proteins and their chromosomal binding targets, as well as into practical aspects of the design of regulatory-protein isolation schemes and physicochemical regulatory considerations in vivo.

Binding Sites↗
PubMed1985

Orientation constraints in diffusion-limited macromolecular association. The role of surface diffusion as a rate-enhancing mechanism.

Ligand association to a reactive site on a macromolecular surface could be very slow if the site is small. The effective capture radius of the reactive site can be significantly increased if the ligand can bind weakly to the nonspecific surface around the site and then slide in a two-dimensional diffusion along the surface. In this model, the diffusion along the surface has to be properly coupled with the free diffusion in solution and the effective bimolecular association rate constant to the reactive site can be calculated as a function of the nonspecific affinity. This is carried out both for a plane and spherical surface, modeling the association to a membrane receptor or to the catalytic site on an enzyme. The result of these calculations can be used to assign reasonable values to the parameters in the quasichemical approximation of K. Solc and W. H. Stockmayer (1973, Int. J. Chem. Kinet., 5:733-752). In this way a simple analytical expression can be derived for the diffusion-limited association rate constant of two asymmetrically reactive molecules, with or without surface diffusion contributing.

Binding Sites↗
PubMed1983

Microscopic diffusion-reaction coupling in steady-state enzyme kinetics.

The theory of diffusion-controlled processes is applied to describe the steady state of a reversible enzymatic reaction with special emphasis on the effects of enzyme saturation. A standard macroscopic steady-state treatment requires only that the average diffusional influx of substrate equals the net reaction flux as well as the average diffusional efflux of product. In contrast, the microscopic diffusion-reaction coupling used here takes properly into account the conditional concentration distributions of substrate and product: Only when the enzyme is unoccupied will there be a diffusional association flux; when the enzyme is occupied, the concentration distributions will relax towards their homogeneous bulk values. In this way the relaxation effects of the non-steady state will be constantly reoccurring as the enzyme shifts between unoccupied and occupied states. Thus, one is forced to describe the steady state as the weighted sum of properly time-averaged non-stationary conditional distributions. The consequences of the theory for an appropriate assessment of the parameters obtained in Lineweaver-Burk plots are discussed. In general, our results serve to justify the simpler macroscopic coupling scheme. However, considerable deviations between the standard treatment and our analysis can occur for fast enzymes with an essentially irreversible product release.

Diffusion↗
PubMed1982

Association kinetics with coupled three- and one-dimensional diffusion. Chain-length dependence of the association rate of specific DNA sites.

The effective target size of a specific binding site on a long polymeric chain can be considerably enhanced if the associating molecules slide along the chain in a one-dimensional diffusion. Such a mechanism has been proposed to explain the "faster than diffusion controlled" association of the lac repressor protein with the operator site on a long DNA chain. An important experimental tool for investigating the properties of rate-enhancing mechanisms of this type is now becoming available. In these experiments the DNA chain length is varied and the association rate of the investigated protein is measured. Previous attempts to describe theoretically how such one-dimensional sliding processes behave under those conditions were either oversimplified or connected with fundamental errors. We present here a theory for the association rate of proteins with a specific site on DNA molecules with variable lengths. We use a steady-state approach as do Schranner and Richter (Biophys. Chem. 8 (1978)47) but, in contrast, use boundary conditions which appear to be physically more reasonable.

Chemical Phenomena↗
PubMed1981

Diffusion-driven mechanisms of protein translocation on nucleic acids. 1. Models and theory.

Genome regulatory proteins (e.g., repressors or polymerases) that function by binding to specific chromosomal target base pair sequences (e.g., operators or promoters) can appear to arrive at their targets at faster than diffusion-controlled rates. These proteins also exhibit appreciable affinity for nonspecific DNA, and thus this apparently facilitated binding rate must be interpreted in terms of a two-step binding mechanism. The first step involves free diffusion to any nonspecific binding site on the DNA, and the second step comprises a series of protein translocation events that are also driven by thermal fluctuations. Because of nonspecific binding, the search process in the second step is of reduced dimensionality (or volume); this results in an accelerated apparent rate of target location. In this paper we define four types of processes that may be involved in these protein translocation events between DNA sites. These are (i) "macroscopic" dissociation--reassociation processes within the domain of the DNA molecule, (ii) "microscopic" dissociation--reassociation events between closely spaced sites in the DNA molecule, (iii) "intersegment transfer" (via "ring-closure") processes between different segments of the DNA molecule, and (iv) "sliding" along the DNA molecule. We present mathematical and physical descriptions of each of these processes, and the consequences of each for the overall rate of target location are worked out as a function of both the nonspecific binding affinity between protein and DNA and the length of the DNA molecule containing the target sequence. The theory is developed in terms of the Escherichia coli lac repressor--operator interaction since data for testing these approaches are available for this system [Barkley, M. (1981) Biochemistry 20, 3833; Winter, R. B., & von Hippel, P. H. (1981) Biochemistry (second paper of three in this issue); Winter, R. B., Berg, O. G., & von Hippel, P. H. (1981) Biochemistry (third paper of three in this issue)]. However, we emphasize that this approach is general for the analysis of mechanisms of biological target location involving facilitated transfer processes via nonspecific binding to the general system of which the target forms a small part.

Binding Sites↗
PubMed1981

Diffusion-driven mechanisms of protein translocation on nucleic acids. 3. The Escherichia coli lac repressor--operator interaction: kinetic measurements and conclusions.

The association and dissociation kinetics of the Escherichia coli lac repressor--operator (RO) complex have been examined as a function of monovalent ion concentration and operator-containing DNA fragment length in order to investigate the mechanisms used by repressor in locating (and dissociating from) the operator site. Association rate constants (ka) measured with an 80- or a 203-base-pair lac operator containing DNA fragment are 3--5-fold smaller than those determined with a 6700-base-pair operator fragment or with intact lambda plac5 DNA (50000 base pairs) at all salt concentrations tested. At salt concentrations less than approximately 0.1 M KCl, association rate constants to all operator-containing DNA fragments (except lambda plac5 DNA) are insensitive to variations in salt concentration, but the limiting low salt value of ka appears to depend upon operator-containing DNA length. The value of ka for lambda plac5 DNA decreases significantly from the approximately 0.1 M KCl maximum at low salt. Above approximately 0.1 M KCl, repressor--operator association rate constants for all operator-containing DNA substrates tested show a similar decrease with increasing salt concentration, which does not appear to depend upon the length of the DNA molecule (except for the very small DNA fragments). In contrast to the association reaction, kd, the dissociation rate constant, decreases linearly (on a log kd vs. log [KCl] plot) with decreasing salt concentration over virtually the entire salt concentration range studied (0.05--0.2 M KCl). These results are consistent with the explanation of the unusually fast association kinetics for this system in terms of a two-step model in which repressor initially diffuses to a nonoperator DNA binding site (forming an RD complex) and then rapidly "scans" (in a locally correlated fashion) adjacent sites until the operator is located or the repressor dissociates from the chain. Dissociation of the RO complex follows the same two-step process in reverse. Quantitative comparisons are made between these results and the theoretical predictions of the two facilitating translocation mechanisms (one-dimensional "sliding" along the DNA double helix and direct transfer between DNA segments) developed in the first paper of this series [Berg, O. G., Winter, R. B., & von Hippel, P. H. (1981) Biochemistry (first paper of three in this issue)]. We conclude that the experimental data for the "faster-than-diffusion-controlled" interaction of repressor and operator can be quantitatively modeled by a two-step process in which sliding is the dominant transfer mechanism. Molecular models of the initial nonspecific binding event (including "hopping") as well as sliding and interchain transfer are discussed, and the possible roles of facilitated translocation mechanisms of the diffusion-driven type in this and other in vitro and in vivo protein--nucleic acid interaction processes are considered.

DNA, Bacterial↗