Poliovirus RNA-dependent RNA polymerase (3D(pol)). Assembly of stable, elongation-competent complexes by using a symmetrical primer-template substrate (sym/sub)

Detailed studies of the kinetics and mechanism of nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus, 3D(pol), have been limited by the inability to assemble elongation complexes that permit activity to be monitored by extension of end-labeled primers. We have solved this problem by employing a short, symmetrical, heteropolymeric RNA primer-template that we refer to as 'sym/sub'. Formation of 3D(pol)- sym/sub complexes is slow owing to a slow rate of association (0.1 μM-1 s-1) of 3D(pol) and sym/sub and a slow isomerization (0.076 s-1) of the 3D(pol)-sym/sub complex that is a prerequisite for catalytic competence of this complex. Complex assembly is stoichiometric under conditions in which competing reactions, such as enzyme inactivation, are eliminated. Inactivation of 3D(pol) occurs at a maximal rate of 0.051 s-1 at 22 °C in reaction buffer lacking nucleotide. At this temperature, ATP protects 3D(pol) against inactivation with a K0.5 of 37 μM. Once formed, 3D(pol)-sym/sub elongation complexes are stable (t( 1/2 ) = 2 h at 22 °C) and appear to contain only a single polymerase monomer. In the presence of Mg2+, AMP, 2'-dAMP, and 3'-dAMP are incorporated into sym/sub by 3D(pol) at rates of 72, 0.6, and 1 s-1, respectively. After incorporation of AMP, 3D(pol)-sym/sub product complexes have a half-life of 8 h at 22 °C. The stability of 3D(pol)-sym/sub complexes is temperature-dependent. At 30 °C, there is a 2-8-fold decrease in complex stability. Complex dissociation is the rate-limiting step for primer utilization. 3D(pol) dissociates from the end of template at a rate 10-fold faster than from internal positions. The sym/sub system will facilitate mechanistic analysis of 3D(pol) and permit a direct kinetic and thermodynamic comparison of the RNA-dependent RNA polymerase to the other classes of nucleic acid polymerases

Poliovirus RNA-dependent RNA polymerase (3D(pol)) is sufficient for template switching in vitro

We have performed a systematic, quantitative analysis of the kinetics of nucleotide incorporation catalyzed by poliovirus RNA-dependent RNA polymerase, 3D(pol). Homopolymeric primer/templates of defined length were used to evaluate the contribution of primer and template length and sequence to the efficiency of nucleotide incorporation without the complication of RNA structure. Interestingly, thermodynamic stability of the duplex region of these primer/templates was more important for efficient nucleotide incorporation than either primer or template length. Surprisingly, products greater than unit length formed in all reactions regardless of length or sequence. Neither a distributive nor a processive slippage mechanism could be used to explain completely the formation of long products. Rather, the data were consistent with a template-switching mechanism. All of the nucleotide could be polymerized during the course of the reaction. However, very few primers could be extended, suggesting an inverse correlation between the efficiency of primer utilization and that of nucleotide incorporation. Therefore, the greatest fraction of incorporated nucleotide derives from a small fraction of enzyme when radioactive nucleotide and homopolymeric primer/template substrates are employed. The impact of these results on mechanistic studies of 3D(pol)-catalyzed nucleotide incorporation and RNA recombination are discussed

Job Resources Boost Work Engagement, Particularly When Job Demands Are High

This study of 805 Finnish teachers working in elementary, secondary, and vocational schools tested 2 interaction hypotheses. On the basis of the job demands–resources model, the authors predicted that job resources act as buffers and diminish the negative relationship between pupil misbehavior and work engagement. In addition, using conservation of resources theory, the authors hypothesized that job resources particularly influence work engagement when teach

High energy limits of Laplace-type and Dirac-type eigenfunctions and frame flows

We relate high-energy limits of Laplace-type and Dirac-type operators to frame flows on the corresponding manifolds, and show that the ergodicity of frame flows implies quantum ergodicity in an appropriate sense for those operators. Observables for the corresponding quantum systems are matrix-valued pseudodifferential operators and therefore the system remains non-commutative in the high-energy limit. We discuss to what extent the space of stationary high-energy states behaves classically.Comment: 26 pages, latex2

Poliovirus RNA-dependent RNA polymerase (3D(pol)). Divalent cation modulation of primer, template, and nucleotide selection

We have analyzed the divalent cation specificity of poliovirus RNA- dependent RNA polymerase, 3D(pol). The following preference was observed: Mn2+ > Co2+ > Ni2+ > Fe2+ > Mg2+ > Ca2+ > Cu2+, and Zn2+ was incapable of supporting 3D(pol)-catalyzed nucleotide incorporation. In the presence of Mn2+, 3D(pol) activity was increased by greater than 10-fold relative to that in the presence of Mg2+. Steady-state kinetic analysis revealed that the increased activity observed in the presence of Mn2+ was due, primarily, to a reduction in the K(M) value for 3D(pol) binding to primer/template, without any significant effect on the K(M) value for nucleotide. The ability of 3D(pol) to catalyze RNA synthesis de novo was also stimulated approximately 10-fold by using Mn2+, and the enzyme was now capable of also utilizing a DNA template for primer-independent RNA synthesis. Interestingly, the use of Mn2+ as divalent cation permitted 3D(pol) activity to be monitored by following extension of 5'-32P-end- labeled, heteropolymeric RNA primer/templates. The kinetics of primer extension were biphasic because of the enzyme binding to primer/template in both possible orientations. When bound in the incorrect orientation, 3D(pol) was capable of efficient addition of nucleotides to the blunt-ended duplex; this activity was also apparent in the presence of Mg2+. In the presence of Mn2+, 3D(pol) efficiently utilized dNTPs, ddNTPs, and incorrect NTPs. On average, three incorrect nucleotides could be incorporated by 3D(pol). The ability of 3D(pol) to incorporate the correct dNTP, but not the correct ddNTP, was also observed in the presence of Mg2+. Taken together, these results provide the first glimpse into the nucleotide specificity and fidelity of the poliovirus polymerase and suggest novel alternatives for the design of primer/templates to study the mechanism of 3D(pol)-catalyzed nucleotide incorporation

Single-nucleotide resolution of RNA strands in the presence of their RNA complements

Double-stranded (ds)RNA is important for a variety of biological systems. The discovery of the dsRNA-binding motif (dsRBM), coupled with the occurrence of this motif in a wide variety of functionally diverse proteins, has led to increased interest and study of - dsRNA (6,14). For example, the dsRNA- activated protein kinase (PKR), an enzyme involved in the cellular antiviral response, contains two tandem copies of the dsRBM. In addition, the dsRNA adenine deaminases (dsRADs) contain three tandem copies of this motif (7). Likewise, the study of the RNA-dependent RNA polymerase (RdRP) activity associated with RNA virus transcriptases and replicases also requires the use of dsRNA. In each of these systems, the length of the typical RNA used is in the 10–80 bp range (1,9)

Heavy flavor diffusion in weakly coupled N=4 Super Yang-Mills theory

We use perturbation theory to compute the diffusion coefficient of a heavy quark or scalar moving in N=4 SU(N_c) Super Yang-Mills plasma to leading order in the coupling and the ratio T/M<<1. The result is compared both to recent strong coupling calculations in the same theory and to the corresponding weak coupling result in QCD. Finally, we present a compact and simple formulation of the Lagrangian of our theory, N=4 SYM coupled to a massive fundamental N=2 hypermultiplet, which is well-suited for weak coupling expansions.Comment: 22 pages, 4 figures; v3: error corrected in calculations, figures and discussion modified accordingl

Single-channel transmission in gold one-atom contacts and chains

We induce superconductivity by proximity effect in thin layers of gold and study the number of conduction channels which contribute to the current in one-atom contacts and atomic wires. The atomic contacts and wires are fabricated with a Scanning Tunneling Microscope. The set of transmission probabilities of the conduction channels is obtained from the analysis of the $I(V)$ characteristic curve which is highly non-linear due to multiple Andreev reflections. In agreement with theoretical calculations we find that there is only one channel which is almost completely open.Comment: 4 pages, 2 figures. To be published in Phys. Rev. B, Rapid Communications (2003

Perturbations of anti-de Sitter black holes

I review perturbations of black holes in asymptotically anti-de Sitter space. I show how the quasi-normal modes governing these perturbations can be calculated analytically and discuss the implications on the hydrodynamics of gauge theory fluids per the AdS/CFT correspondence. I also discuss phase transitions of hairy black holes with hyperbolic horizons and the dual superconductors emphasizing the analytical calculation of their properties.Comment: 25 pages, 4 figures, prepared for the proceedings of the 5th Aegean Summer School "From Gravity to Thermal Gauge Theories: the AdS/CFT Correspondence," Milos, Greece, September 2009