It is generally expected that the kinetics of reactions inside livingDNA ͉ in vivo ͉ molecular crowding ͉ temperature oscillation ͉ optical lock-in microscopy
A poorly understood step in the transition from a chemical to a biological world is the emergence of self-replicating molecular systems. We study how a precursor for such a replicator might arise in a hydrothermal RNA reactor, which accumulates longer sequences from unbiased monomer influx and random ligation. In the reactor, intra- and intermolecular base pairing locally protects from random cleavage. By analyzing stochastic simulations, we find temporal sequence correlations that constitute a signature of information transmission, weaker but of the same form as in a true replicator.
The NS3 helicase (NS3h) of hepatitis C virus (HCV) is a 3 0 to 5 0 SF2 RNA and DNA helicase that is essential for the replication of HCV. We have examined the kinetic mechanism of translocation of NS3h along single stranded nucleic acid with bases rU, dU and dT and found that the rate of translocation is dependent upon both base and sugar moieties. We find that the approximate rates of translocation are 3 nt/s (oligo-dT), 35 nt/s (oligo-dU), and 42 nt/s (oligo-rU). These macroscopic translocation rates correspond well to differences in the binding affinity of the translocating NS3h protein to the respective substrates. The values of K M for NS3h translocating at a saturating ATP concentration are: 3.3 (5 0.4) mM nucleotide (poly-dT), 27 (5 2) mM nucleotide (poly-dU), and 36 (5 2) mM nucleotide (poly-rU). Despite the differences in translocation rates and binding affinities, the ATP coupling stoichiometry for NS3h translocation is identical for all three substrates, with a value of ~2 nt per ATP consumed. The identical periodicity of ATP consumption implies a similar mechanism for NS3h translocation along each substrate. This data, together with our independently determined values of K D for NS3h binding to poly-dT (220 5 20 nM nucleotide) and poly-dU (430 5 30 nM nucleotide), suggest that the differences in the macroscopic translocation rates may be explained by differences in the entropic contribution to the binding free energy of NS3h to the different nucleic acid substrates. This conclusion is consistent with observations from a previously published crystal structure of NS3h in complex with a short oligonucleotide (Kim, et al (1998) Structure 6:89-100).
Evolving systems rely on the storage and replication of genetic information. Here we present an autonomous, purely thermally driven replication mechanism. A pool of hairpin molecules, derived from transfer RNA replicates the succession of a two-letter code. Energy is first stored thermally in metastable hairpins. Thereafter, energy is released by a highly specific and exponential replication with a duplication time of 30 s, which is much faster than the tendency to produce false positives in the absence of template. Our experiments propose a physical rather than a chemical scenario for the autonomous replication of protein encoding information in a disequilibrium setting.
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