Christof K. Biebricher

In vitro recombination and terminal elongation of RNA by Q beta replicase.

SV‐11 is a short‐chain [115 nucleotides (nt)] RNA species that is replicated by Q beta replicase. It is reproducibly selected when MNV‐11, another 87 nt RNA species, is extensively amplified by Q beta replicase at high ionic strength and long incubation times. Comparing the sequences of the two species reveals that SV‐11 contains an inverse duplication of the high‐melting domain of MNV‐11. SV‐11 is thus a recombinant between the plus and minus strands of MNV‐11 resulting in a nearly palindromic sequence. During chain elongation in replication, the chain folds consecutively to a metastable secondary structure of the RNA, which can rearrange spontaneously to a more stable hairpin‐form RNA. While the metastable form is an excellent template for Q beta replicase, the stable RNA is unable to serve as template. When initiation of a new chain is suppressed by replacing GTP in the replication mixture by ITP, Q beta replicase adds nucleotides to the 3′ terminus of RNA. The replicase uses parts of the RNA sequence, preferentially the 3′ terminal part for copying, thereby creating an interior duplication. This reaction is about five orders of magnitude slower than normal template‐instructed synthesis. The reaction also adds nucleotides to the 3′ terminus of some RNA molecules that are unable to serve as templates for Q beta replicase.

Ice and the origin of life

Sea ice occurs abundantly at the polar caps of the Earth and, probably, of many other planets. Its static and dynamic properties that may be important for prebiotic and early biotic reactions are described. It concentrates substrates and has many features that are important for catalytical actions. We propose that it provided optimal conditions for the early replication of nucleic acids and the RNA world. We repeated a famous prebiotic experiment, the poly-uridylic acid-instructed synthesis of polyadenylic acid from adenylic acid imidazolides in artificial sea ice, simulating the dynamic variability of real sea ice by cyclic temperature variation. Poly(A) was obtained in high yield and reached nucleotide chain lengths up to 400 containing predominantly 3′→ 5′ linkages.

Kinetic analysis of template-instructed and de novo RNA synthesis by Qβ replicase

The kinetics of template-free and template-instructed RNA synthesis by Qβ replicase were investigated. Template-instructed RNA synthesis has different growth rates in the exponential (excess enzyme) and the linear (excess template) phase of growth. In the absence of exogenous template, Qβ replicase synthesizes self-replicating RNA after an initial lag phase (“template-free” synthesis). The lag time can be determined by extrapolating the growth curve to the time of appearance of the first self-replicating strand. Growth rates in the exponential and linear phase, and especially the times of the lag phase for nucleotide incorporations under identical template-free conditions, show considerable scattering in contrast to the deterministic behavior of template-instructed synthesis. Evaluation of the kinetic data reveals that the time lag of template-free synthesis is strongly dependent on the concentration of the nucleoside triphosphate and the enzyme. The lag time is approximately inversely proportional to the powers 2.75 of the nucleotide and 2.5 of the enzyme concentration, respectively, both being lower limit values. The rate of template-instructed RNA synthesis is linearly proportional to the enzyme concentration and less than linearly proportional to the triphosphate concentration, in accordance with a substrate dependence of a Michaelis-Menten type of mechanism. The kinetic data cannot be reconciled with the proposition that template-free synthesis is due to low concentrations of templates present as impurities in the incorporation mixture and giving rise to autocatalytic RNA synthesis by a template-instructed mechanism. The data strongly favor the de novo mechanism proposed by Sumper & Luce (1975).

Product analysis of RNA generated de novo by Qβ replicase

Qβ replicase synthesizes self-replicating RNA in the absence of exogenous template after a certain lag time (“template-free synthesis”). The products of the template-free RNA synthesis have been investigated by gel electrophoresis and fingerprinting techniques. It has been found that a multitude of self-replicating RNA species appears in the early phases of reaction with variable lengths and sequences. Template-free synthesis in different samples under completely identical conditions yields RNA products with very different and unrelated fingerprints. The early products rapidly undergo an evolution process that alters the phenotypic properties of the self-replicating RNA, and leads to a concomitant increase of replication efficiency. Fingerprints and electrophoretic mobilities of the self-replicating RNA species are altered discontinuously during the evolution process. The evolution process ends with the selection of optimized self-replicating RNA species, whose phenotypes are conserved even after many serial transfers. Some optimized RNA species and midivariant RNA apparently have related sequences, since they contain many identical spots in their fingerprints. The properties of the RNA species produced by template-free synthesis match those of 6 S RNA found in Qβ-infected Escherichia coli cells. The results are in full agreement with the finding of Sumper & Luce (1975), who have presented evidence that Qβ replicase synthesized RNA de novo in the absence of exogenous template.

Structural analysis of self-replicating RNA synthesized by Qβ replicase

Self-replication of the RNA variant MNV-11 (86 base-pairs) by Qβ replicase leads, in the presence of salt, to stepwise selection of the more salt-resistant variants SV-11 (113 base-pairs) and MDV-1 (220 base-pairs). The structures of these and two other unrelated salt-resistant variants are compared. All variants can exist as double-stranded or as single-stranded RNA. The co-operative thermal denaturation of double-stranded RNAs requires high temperatures, around or even above 100°C. Double-stranded RNA molecules have high negative electric dichroisms and their polarizabilities and rotational diffusion constants show the same length dependences as double-stranded DNAs if RNA is assumed to be in the A-form and DNA in the B-form. The single-stranded RNAs also have sharp melting points at high temperatures and rather high hyperchromicities, revealing strong secondary structuring of the single-stranded RNA. The more salt-resistant variants have still higher melting points and hyperchromicities. Single-stranded RNAs melt a few degrees lower than double-strands of the same species, except for single-stranded MDV-1, which melts at the same temperature as its double-stranded form. Concomitant with increasing secondary structure of the single-stranded RNA is a decrease in their rates of annealing to form double strands. Single-stranded variants are both the immediate products of replication (replicas) and the preferred templates. SV-11 has two single-stranded RNA forms, only one of which is able to serve as an active template for Qβ replicase. Its inactive single-stranded form is more stable and apparently has a hairpin structure. The single-stranded RNAs active in replication have vanishing electric dichroisms, indicating defined tertiary structures that do not allow an independent orientation of stems and loops in the electrical field.

Darwinian Selection of Self-Replicating RNA Molecules

Molecular biology has brought a wealth of information in support of Darwin’s theory of natural selection. Investigations of the cellular machinery have supplied striking evidence for the unity of life on earth, resulting from its common origin, revealed the molecular basis of the genotype and its propagation, and shown the awesome complexity of its expression into the phenotype.

Molecular evolution of RNA in vitro

Experimental studies of RNA evolution in vitro are reviewed in the context of Eigens 1971 theory and its subsequent extensions. Current research activity and future prospects for using automated molecular biology techniques for in vitro evolution experiments are surveyed.

Template-free generation of RNA species that replicate with bacteriophage T7 RNA polymerase.

A large variety of different RNA species that are replicated by DNA‐dependent RNA polymerase from bacteriophage T7 have been generated by incubating high concentrations of this enzyme with substrate for extended time periods. The products differed from sample to sample in molecular weight and sequence, their chain lengths ranging from 60 to 120. The mechanism of autocatalytic amplification of RNA by T7 RNA polymerase proved to be analogous to that observed with viral RNA‐dependent RNA polymerases (replicases): only single‐stranded templates are accepted and complementary replica strands are synthesized. With enzyme in excess, exponential growth was observed; linear growth resulted when the enzyme was saturated by RNA template. The plus strands, present at 90% of the replicating RNA species, were found to have GG residues at both termini. Consensus sequences were not found among the sequences of the replicating RNA species. The secondary structures of all species sequenced turned out to be hairpins. The RNA species were specifically replicated by T7 RNA polymerase; they were not accepted as templates by the RNA polymerases from Escherichia coli or bacteriophage SP6 or by Qbeta replicase; T3 RNA polymerase was partially active. Template‐free production of RNA was completely suppressed by addition of DNA to the incubation mixture. When both DNA and RNA templates were present, transcription and replication competed, but T7 RNA polymerase preferred DNA as a template. No replicating RNA species were detected in vivo in cells expressing T7 RNA polymerase.

Lethal mutants and truncated selection together solve a paradox of the origin of life.

Background Many attempts have been made to describe the origin of life, one of which is Eigens cycle of autocatalytic reactions [Eigen M (1971) Naturwissenschaften 58, 465–523], in which primordial life molecules are replicated with limited accuracy through autocatalytic reactions. For successful evolution, the information carrier (either RNA or DNA or their precursor) must be transmitted to the next generation with a minimal number of misprints. In Eigens theory, the maximum chain length that could be maintained is restricted to nucleotides, while for the most primitive genome the length is around . This is the famous error catastrophe paradox. How to solve this puzzle is an interesting and important problem in the theory of the origin of life. Methodology/Principal Findings We use methods of statistical physics to solve this paradox by carefully analyzing the implications of neutral and lethal mutants, and truncated selection (i.e., when fitness is zero after a certain Hamming distance from the master sequence) for the critical chain length. While neutral mutants play an important role in evolution, they do not provide a solution to the paradox. We have found that lethal mutants and truncated selection together can solve the error catastrophe paradox. There is a principal difference between prebiotic molecule self-replication and proto-cell self-replication stages in the origin of life. Conclusions/Significance We have applied methods of statistical physics to make an important breakthrough in the molecular theory of the origin of life. Our results will inspire further studies on the molecular theory of the origin of life and biological evolution.

Mutation, Competition, and Selection as Measured with Small RNA Molecules

Publisher Summary Evolutionary success depends on two components of the phenotype: those that determine survival in the prevailing environment and those that establish the rate of producing viable offspring. The combination of these two determines the population trend called fitness. Species interact by competition for resources (predation or symbiosis); individuals of the same species influence one another socially, and the individuals themselves may be composed of large numbers of specialized cells, all containing the same genetic information, that have to cooperate with one another for the organism they compose to survive and reproduce. Evolution is a dynamic self-organization process in which causal correlations between the performance of the process as a whole and its component subprocesses are not identifiable. In Darwinian evolution, selection is complemented by mutation. Mutation can be studied quantitatively if selection is excluded by restricting amplification to a single replication round. Mutation rates can be measured and is defined as the probability of incorporating a noncognate base per incorporation event. Many quantitative insights into the nature of evolution have been gained from studying the model system provided by Qβ replicase. For quantitative studies of natural selection under controlled conditions, amplification of RNA by Qβ replicase is still unsurpassed.