Biomedical subjects

T D Yager

Publications and source records attributed to T D Yager.

11 recordsLinked to original sources

PubMed1992

The elongation-termination decision in transcription.

At any template position, the decision to extend the transcript by one residue or to release the nascent RNA represents a kinetic competition between elongation and termination pathways. This competition is discussed in terms of alternative Eyring transition state barriers; changes in termination efficiency correspond to small changes in the relative heights of these barriers. Elongation complexes are stable at nonterminator positions; a model is presented to explain the destabilization of these complexes at intrinsic termination sites. Functionally analogous effects can operate at rho-dependent terminators. Mechanisms for modulation of termination efficiency by regulatory proteins are described.

Activation Analysis↗

PubMed1992

Physical properties of the Escherichia coli transcription termination factor rho. 1. Association states and geometry of the rho hexamer.

To function as a DNA-RNA helicase in rho-dependent transcript termination, six genetically identical subunits of the Escherichia coli transcription termination protein rho must first assemble into a hexameric complex. To help determine the quaternary structure of this complex, we have studied the association equilibria of the rho protomers. Sedimentation equilibrium, sedimentation velocity, diffusion, X-ray scattering, and neutron-scattering data have been combined to create a "phase diagram" of the association states of this protein as a function of protein concentration and ionic environment. The results show that rho exists predominantly as a hexamer under approximately physiological conditions and that this hexamer is in equilibrium with both lower and higher states of association that may also have physiological relevance. Small-angle X-ray scattering measurements and theoretical calculations indicate that the rho hexamer has a radius of gyration of 50 +/- 3 A. The radius of gyration measured by small-angle neutron scattering in 2H2O is 47 +/- 3 A. These scattering studies also support earlier models of rho as a planar hexagon which have been developed on the basis of electron microscopy. In the following paper in this issue [Geiselmann, J., Seifried, S. E., Yager, T. D., Liang, C., & von Hippel, P. H. (1992)], these results are combined with information on symmetry, subunit interactions, and packing geometry to obtain a model of the quaternary structure of the functional rho hexamer.

DNA↗

PubMed1992

Physical properties of the Escherichia coli transcription termination factor rho. 2. Quaternary structure of the rho hexamer.

Under approximately physiological conditions, the transcription termination factor rho from Escherichia coli is a hexamer of planar hexagonal geometry [Geiselmann, J., Yager, T. D., Gill, S. C., Calmettes, P., & von Hippel, P. H. (1992) Biochemistry (preceding paper in this issue)]. Here we describe studies that further define the quaternary structure of this hexamer. We use a combination of chemical cross-linking and treatment with mild denaturants to show that the fundamental unit within the rho hexamer is a dimer stabilized by an isologous (or pseudoisologous) bonding interface. Three identical dimers of rho interact via a second type of isologous bonding interface to yield a hexamer with C3 or D3 symmetry. Cross-linking and denaturation experiments definitely rule out C6 and C2 symmetry for the rho hexamer. Data from fluorescence quenching, lifetime, and energy transfer experiments also argue against C2 symmetry. The simplest symmetry assignment that is not contradicted by any experimental data is D3; thus we conclude that the rho hexamer has D3 symmetry. We also consider the positioning of the binding sites for RNA and ATP relative to the coordinate reference frame of the D3 hexamer. Fluorescence energy transfer data are presented and integrated with data from the literature to arrive at a self-consistent model for the quaternary structure of the rho hexamer.

Adenosine Triphosphate↗

PubMed1992

Functional interactions of ligand cofactors with Escherichia coli transcription termination factor rho. II. Binding of RNA.

The rho protein of Escherichia coli interacts with the nascent RNA transcript while RNA polymerase is paused at specific rho-dependent termination sites on the DNA template, and (in a series of steps that are still largely undefined) brings about transcript termination at these sites. In this paper we characterize the interactions of rho with RNA and relate these interactions to the quaternary structure of the functional form of rho. We use CD spectroscopy and analytical ultracentrifugation to determine the binding interactions of rho with RNA ligands of defined length ([rC]n where n > or = 6). Rho binds to long RNA chains as a hexamer characterized by D3 symmetry. Each hexamer binds approximately 70 residues of RNA. We show by ultracentrifugation and dynamic laser light scattering that, in the presence of RNA ligands less than 22 nucleotide residues in length, rho changes its quaternary structure and becomes a homogeneous dodecamer. The dodecamer contains six strong binding sites for short RNA ligands: i.e., one site for every two rho protomers. The measured association constant of these short RNAs to rho increases with increasing (rC)n length, up to n = 9, suggesting that the binding site of each rho protomer interacts with 9 RNA nucleotide residues. Oligo (rC) ligands bound to the strong RNA binding sites on the rho dodecamer do not significantly stimulate the RNA-dependent ATPase activity of rho. Based on these features of the rho-RNA interaction and other experimental data we propose a molecular model of the interaction of rho with its cofactors.

Binding Sites↗

PubMed1992

The single-nucleotide addition cycle in transcription: a biophysical and biochemical perspective.

This review has summarized the known features of the single-nucleotide addition reaction cycle in transcription. The reader will have noted that the information available is very incomplete, and that, in some cases, related experiments seem to lead to contradictory conclusions. We have tried to point out these discrepancies as they occur and to indicate areas where more experimentation is needed. We look forward to the day when all the microscopic steps of the single-nucleotide addition cycle can be identified and defined in thermodynamic, kinetic, and structural terms. At that point, we can begin to understand the principles that relate these parameters to template position and to the pathway of formation of a specific complex. It should be possible to provide specific molecular interpretations for observed effects on activation barrier heights to elongation and termination (154, 155) and to begin to understand the molecular bases of the regulation in these phases of transcription. Much work remains before this happy situation can be totally realized, but we feel that now the problem can at least be approached at this level. We hope that this review helps to illuminate the difficulties that remain.

Binding Sites↗

PubMed1991

Escherichia coli sigma 70 and NusA proteins. II. Physical properties and self-association states.

In this paper we examine the physical properties and potential for self-association of the Escherichia coli transcription factors, sigma 70 and NusA. We show, by a combination of chemical crosslinking, equilibrium and velocity sedimentation, quasi-elastic light scattering, and small-angle X-ray scattering that NusA exists as a monomer at KCl concentrations between 0.01 and 1.5 M, and that sigma 70 exists as a monomer at KCl concentrations between 0.1 and 1.5 M. The shape and hydration characteristics of each of these monomeric proteins are also examined. The results serve as background for the companion paper in which a thermodynamic analysis is made of the interactions of these transcription factor with E. coli core RNA polymerase in solution and as a component of the functional transcription complex.

Bacterial Proteins↗

PubMed1991

Transcript elongation and termination are competitive kinetic processes.

In this paper, we develop a kinetic approach to predict the efficiency of termination at intrinsic (factor independent) terminators of Escherichia coli and related organisms. In general, our predictions agree well with experimental results. Our analysis also suggests that termination efficiency can readily be modulated by protein factors and environmental variables that shift the kinetic competition toward either elongation or termination. A quantitative framework for the consideration of such regulatory effects is developed and the strengths and limitations of the approach are discussed.

DNA, Bacterial↗

PubMed1991

A thermodynamic analysis of RNA transcript elongation and termination in Escherichia coli.

In the first part of this paper we present a thermodynamic analysis of the elongation phase of transcription in Escherichia coli. The stability of the elongation complex is described by a "free energy of formation" function (delta G zero f) that is a sum of terms for forming (i) a locally denatured 17-base-pair DNA "bubble"; (ii) a constant-length hybrid between the 3'-terminal 12-nucleotide residues of the RNA transcript and the corresponding region of the DNA template strand; and (iii) a set of binding interactions between the polymerase and certain DNA and RNA residues within and near the "transcription bubble". The transcriptional elongation complex is very stable at most positions along a natural DNA template and moves in a highly processive fashion. At these positions, the delta G zero f function provides a quantitative measure of the stability of the elongation complex. Besides allowing for the polymerization of the RNA transcript, the elongation complex also serves to define the context within which transcript termination occurs. In the second part of the paper the thermodynamic analysis is extended to discriminate between template positions at which the elongation complex is stable and positions at which it is rendered relatively unstable by the presence of a string of rU residues at the 3'-terminus of the RNA together with the formation of a specific RNA hairpin just upstream of this point. Most factor-independent (intrinsic) termination events are thermodynamically disallowed at the former positions and are thermodynamically allowed at the latter positions. The extended form of the analysis closely predicts the exact sites of termination at a number of intrinsic terminators (and attenuators) in the E. coli genome. It also correctly predicts bidirectional function for a number of bidirectional terminators. In some cases it may identify terminators that are similar to the intrinsic type but that require additional protein factors, unusual polymerase-nucleic acid interactions, or rate-limiting conformational changes in order to function. Finally, it successfully locates intrinsic terminators within a number of E. coli operons and discriminates between these terminators and the surrounding DNA sequence.

Algorithms↗

PubMed1991

The Human Genome Project: creating an infrastructure for biology and medicine.

The Human Genome Project (HGP) is an international effort to map and sequence the human genome. It combines skills from diverse fields of biological and technological research, thus establishing deeper interactions between scientific disciplines. The combination of these skills should stimulate many advances in both pure and applied fields of research and give rise to new, interdisciplinary training programs. Some critics say that the HGP will damage biomedical research; however, we argue that it will bring new funds to the field and create a large ripple effect by providing new research opportunities through its discoveries.

Chromosome Mapping↗

PubMed1990

Thermodynamic analysis of the transcription cycle in E. coli.

The E. coli RNA transcription cycle can be divided into three major phases, which are generally called initiation, elongation, and termination. In this paper, we review recent biophysical studies of the interactions of the transcriptional regulatory proteins, sigma 70 and NusA, with themselves and with core RNA polymerase in solution, as well as with core polymerase within the transcription complex. The different affinities of sigma 70 and NusA for core RNA polymerase at various stages in the transcription cycle, together with other quantitative data, are then used to construct a partial free energy diagram for the overall transcription process. This thermodynamic framework, which is interrupted by at least two irreversible steps, can be used to rationalize physiological aspects of the transcription cycle and its regulation, as well as to identify crucial points at which our knowledge is still incomplete.

Bacterial Proteins↗

PubMed1989

Salt-induced release of DNA from nucleosome core particles.

At elevated salt concentrations, the structure of chromatin is destabilized. This paper is concerned with the processes by which DNA is released from nucleosome core particles in free, uncomplexed form. Our experiments indicate that the DNA release reaction has distinctly different characteristics below and above approximately 0.75 M NaCl. Below this concentration of salt, release of the histones from the DNA is highly cooperative, so that no dissociation intermediates are even seen. Above this salt concentration, histone release is not so cooperative; H2A and H2B are released from the DNA more readily than are H3 and H4. This results in an apparently heterogeneous population of (H2A, H2B)-depleted intermediate species sedimenting at rates between that of free DNA and that of intact core particles. Dissociation of core particles at NaCl concentrations below 0.75 M is readily reversible. Reassociation of DNA and histones from higher salt concentrations is nearly quantitative if carried out by gradual decrease of salt concentration, but rapid dilution to low salt results in the formation of a fraction of metastable nucleosome multimers. To help organize our description of the DNA release process, we introduce a stability diagram for the core particle, defined with respect to the independent variables of salt concentration and particle concentration. We draw upon our own experimental work and also upon the work of several other laboratories. We distinguish five major regions in this diagram.

Chromatin↗