979 research outputs found
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
Accumulation of a nod Gene Inducer, the Flavonoid Naringenin, in the Cytoplasmic Membrane of Rhizobium leguminosarum biovar viciae Is Caused by the pH-Dependent Hydrophobicity of Naringenin
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
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
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