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M A Shea

Publications and source records attributed to M A Shea.

At least 55 records · Page 3Linked to original sources
PubMed1986

Energetics of cooperative protein-DNA interactions: comparison between quantitative deoxyribonuclease footprint titration and filter binding.

Using the binding of cI repressor protein to the lambda right and left operators as a model system, we have analyzed the two common experimental techniques for studying the interactions of genome regulatory proteins with multiple, specific sites on DNA. These are the quantitative DNase footprint titration technique [Brenowitz, M., Senear, D. F., Shea, M. A., & Ackers, G. K. (1986) Methods Enzymol. 130, 132-181] and the nitrocellulose filter binding assay [Riggs, A., Suzuki, H., & Bourgeois, S. (1970) J. Mol. Biol. 48, 67-83]. The footprint titration technique provides binding curves that separately represent the fractional saturation for each site. In principle, such data contain the information necessary to determine the thermodynamic constants for local site binding and cooperativity. We show that in practice, this is not possible for all values of the constants in multisite systems, such as the lambda operators. We show how these constants can nevertheless be uniquely determined by using additional binding data from a small number of mutant operators in which the number of binding sites has been reduced. The filter binding technique does not distinguish binding to the individual sites and yields only macroscopic binding parameters which are composite averages of the various local site and cooperativity constants. Moreover, the resolution of even macroscopic constants from filter binding data for multisite systems requires ad hoc assumptions as to a relationship between the number of ligands bound and the filter retention of the complex. Our results indicate that no such relationship exists. Hence, the technique does not permit determination of thermodynamically valid interaction constants (even macroscopic) in multisite systems.

Bacteriophage lambda↗
PubMed1986

"Footprint" titrations yield valid thermodynamic isotherms.

A central issue in gene regulation is the mechanism, and biological function, of the cooperative binding of regulatory protein ligands to specific sites on DNA. To elucidate the physical-chemical basis of these interactions we have developed a thermodynamically rigorous method for conducting DNase I "footprint" (protection) titration experiments. The intrinsic binding constants and also those for cooperative interactions between various sites can be resolved from the individual-site binding curves determined by this technique. Experimental studies of cI-repressor-operator binding have demonstrated that the method provides an accurate representation of the fractional saturation of a binding site. We present individual-site binding curves for a lambda operator with two competent sites that demonstrate the presence of cooperative interactions between the sites. These curves set a lower limit to the magnitude of the cooperative free energy without comparison to single-site mutant operators.

Binding Sites↗
PubMed1985

The OR control system of bacteriophage lambda. A physical-chemical model for gene regulation.

A quantitative model has been developed for processes in the bacteriophage lambda that control the switchover from lysogenic to lytic modes of growth. These processes include the interactions of cI repressor and cro proteins at the three DNA sites of the right operator, OR, the binding of RNA polymerase at promoters PR and PRM, the synthesis of cI repressor and cro proteins, and the degradative action of recA during induction of lysis. The model is comprised of two major physical-chemical components: a statistical thermodynamic theory for relative probabilities of the various molecular configurations of the control system; and a kinetic model for the coupling of these probabilities to functional events, including synthesis of regulatory proteins cI and cro. Using independently evaluated interaction constants and rate parameters, the model was found capable of predicting essential physiological characteristics of the system over an extended time. Sufficiency of the model to predict known physiological properties lends credence to the physical-chemical assumptions used in its construction. Several major physiological characteristics were found to arise as "system properties" through the non-linear, time-dependent, feedback-modulated combinations of molecular interactions prescribed by the model. These include: maintenance of the lysogenic state in the absence of recA-mediated cI repressor degradation; induction of lysis and the phenomenon of subinduction; and autogenous negative control of cro. We have used the model to determine the roles, within the composite system, of several key molecular processes previously characterized by studies in vitro. These include: co-operativity in cI repressor binding to DNA; interactions between repressors and RNA polymerase (positive control); and the monomer-dimer association of cI repressor molecules. A major role of cI repressor co-operativity is found to be that of guaranteeing stability of the lysogenic state against minor changes in cI repressor levels within the cell. The role of positive control seems to be that of providing for a peaked, rather than monotonic, dependence of PRM activity on cI repressor level, while permitting PR activity to be a step function. The model correlates an immense body of studies in vivo and in vitro, and it makes testable predictions about molecular phenomena as well as physiological characteristics of bacteriophage lambda. The approach developed in this study can be extended to include more features of the lambda system and to treat other systems of gene regulation.

Allosteric Regulation↗
PubMed1984

Cosmic ray exposure factors for Shuttle altitudes derived from calculated cut-off rigidities.

The allowed cosmic radiation flux accessible to an earth-orbiting spacecraft is a complex function of the satellite position and the geomagnetic cutoff characteristics at each zenith and azimuth angle at each position. We have determined cosmic ray exposure factors for the galactic cosmic ray spectrum for typical shuttle altitudes and inclinations up to 50 degrees. We have utilized d world grid of trajectory-derived cutoff rigidity calculations at 400 km altitude to determine geomagnetic transmission functions that permit a simple and direct calculation of the allowed cosmic ray spectrum to a 400 km satellite orbit. If the interplanetary cosmic ray spectrum is multiplied by the orbit-averaged geomagnetic transmission function the result is the allowed cosmic ray spectrum at the spacecraft.

Altitude↗
PubMed1983

Free energy coupling within macromolecules. The chemical work of ligand binding at the individual sites in co-operative systems.

Individual-site binding curves such as those obtainable from techniques of DNase footprinting or nuclear magnetic resonance spectroscopy can be used to monitor structurally localized events within biopolymers. This paper discusses thermodynamic aspects of individual-site ligand binding for co-operative systems where the binding of ligand at a local site is coupled to binding of the same ligand species at other sites within the macromolecule. Individual-site binding isotherms have the following properties. (1) They provide a direct indication of the role played by the particular site in the overall binding reaction. (2) They can be used to determine the energetic contribution of loading the site regardless of the complexity of the system. (3) They can be used to resolve microscopic equilibrium constants and co-operativity constants in cases where the classical isotherm is incapable of such resolution. The microscopic constants bear a complex relation to the chemical work of loading each individual site. For a system with two interacting sites we derive analytical relationships between the individual-site loading energies and the microscopic constants. These relationships prescribe, for any values of the microscopic constants, how the co-operative energy is partitioned between events at the two sites. At fixed ligand activity the binding free energy can be estimated directly from an individual-site isotherm. This quantity, which is also a composite of the microscopic constants, provides a useful measure of site--site interaction. Several examples and applications are discussed for these properties of individual-site binding reactions.

Binding Sites↗
PubMed1982

Quantitative model for gene regulation by lambda phage repressor.

A statistical thermodynamic model has been developed to account for the cooperative interactions of the bacteriophage lambda repressor with the lambda right operator. The model incorporates a general theory for quantitatively interpreting cooperative site-specific equilibrium binding data. Values for all interaction parameters of the model have been evaluated at 37 degrees C, 0.2 M KCl, from results of DNase protection experiments in vitro [A. D. Johnson, B. J. Meyer, & M. Ptashne, Proc. Natl. Acad. Sci. USA (1979) 76, 5061-5065]. With these values, the model predicts repression curves at the divergent promoters PR and PRM that control transcription of genes coding for the regulatory proteins cro and repressor, respectively. At physiological repressor concentrations, repression at PR is predicted to be nearly complete whereas PRM is predicted to remain highly active. The results demonstrate the importance of cooperative interactions between repressor dimers bound to the adjacent operator sites OR1 and OR2 in maintaining a stable lysogenic state and in allowing efficient switchover to the lytic state during induction.

Bacteriophage lambda↗
PubMed1971

Transport as the rate-limiting step in the incorporation of uridine into mengovirus ribonucleic acid in Novikoff rat hepatoma cells.

The incorporation of uridine into the nucleotide pool of actinomycin-treated, mengovirus-infected Novikoff rat hepatoma cells in culture follows simple Michaelis-Menten kinetics, and the apparent V(max) and K(m) values are similar to those for uridine transport by uninfected cells. Incorporation of uridine into mengovirus-specific ribonucleic acid (RNA) also follows Michaelis-Menten kinetics, and the apparent K(m) (about 10 mum) is approximately the same as for uridine transport. Inhibition of uridine transport by the presence of adenosine, persantin, or phenethyl alcohol inhibits simultaneously and to the same extent the incorporation of uridine into the nucleotide pool and into viral RNA, without affecting viral RNA synthesis per se. Phenethyl alcohol, however, also inhibits virus maturation. The inhibition of uridine incorporation into the nucleotide pool and into viral RNA is of the simple competitive type, indicating that transport into the cells is the rate-limiting step in the incorporation of uridine into mengovirus RNA. The results also indicate that treatment with actinomycin D or mengovirus infection does not affect uridine transport.

Biological Transport↗
PubMed1971

Effects of elevated temperatures on mengovirus ribonucleic acid synthesis and virus production in Novikoff rat hepatoma cells.

The production of mengovirus in Novikoff rat hepatoma cells is progressively reduced with an increase in incubation temperature of the cells from 34 to 40 C, in spite of the fact that about the same amounts of single-stranded and double-stranded viral ribonucleic acid (RNA) are synthesized at 34, 37, and 40 C; the rate of overall protein synthesis is as high at 40 C as at 37 C. At 40 C, progeny viral RNA accumulates in an undegraded form without being incorporated into virus particles. The results suggest that virus maturation is preferentially inhibited at supraoptimal temperatures. At 42 C, on the other hand, no viral RNA is produced and no viral RNA polymerase activity is detectable in cell lysates. Failure of infected cells to form viral RNA polymerase at 42 C is probably due to an impairment of protein synthesis since most of the polyribosomes are rapidly lost during incubation at 42 C and the rate of amino acid incorporation into protein is 70% lower at 42 C than at 37 C. When infected cells are shifted from 37 to 42 C during the period of active viral RNA synthesis, viral RNA polymerase activity is rapidly lost from the cells, and viral RNA synthesis ceases within 45 min. In contrast, the RNA polymerase is as active in vitro at 42 C as at 37 C, and the activity is relatively stable at 42 C.

Amino Acids↗
PubMed1970

Effect of mengovirus replication on choline metabolism and membrane formation in novikoff hepatoma cells.

Infection of Novikoff rat hepatoma cells (subline NlSL-67) with mengovirus resulted in a two- to threefold increase in the rate of choline incorporation into membrane phosphatidylcholine at about 3 hr after infection, without affecting the rate of transport of choline into the cell or its phosphorylation. The time course of virus-stimulated phosphatidylcholine synthesis was compared with the time courses of other virus-induced processes during a single cycle of replication. The formation of viral ribonucleic acid (RNA) polymerase and of viral RNA commenced about 1 hr earlier than the virus-stimulated choline incorporation. Further, isopycnic centrifugation of cytoplasmic extracts indicated that the excess of phosphatidylcholine synthesized by infected cells is not located in the membrane structures associated with the viral RNA replication complex, but with structures of a lower density (1.08 to 1.14 g/cc). These membrane structures probably represent the smooth vesicles which accumulate in the cytoplasm of infected cells during the period of increased phosphatidylcholine synthesis between 3 and 5 hr after infection. They are formed with both newly synthesized phosphatidylcholine and phosphatidylcholine present prior to infection. However, concomitant protein synthesis is not required for the stimulated synthesis of membranes; the effect was not inhibited by treating the cells with inhibitors of protein synthesis at 3 hr after infection, although virus production was inhibited about 90% and virus-induced cell degeneration was markedly reduced and delayed. Production of mature virus began normally at about the same time as the stimulation of phosphatidylcholine synthesis. Treatment of infected cells with puromycin at 2 hr, on the other hand, completely inhibited the stimulation of phosphatidylcholine synthesis.

Animals↗