Multiple Unfolding Events during Native Folding of the Tetrahymena Group I Ribozyme

Despite the ubiquitous nature of misfolded intermediates in RNA folding, little is known about their physical properties or the folding transitions that allow them to continue folding productively. Folding of the Tetrahymena group I ribozyme includes sequential accumulation of two intermediates, termed I_trap and misfolded (M). Here, we probe the structure and folding transition of I_trap and compare them to those of M. Hydroxyl radical and dimethyl sulfate footprinting show that both I_trap and M are extensively structured and crudely resemble the native RNA. However, regions of the core P3-P8 domain are more exposed to solvent in I_trap than in M. I_trap rearranges to continue folding nearly 1000-fold faster than M, and urea accelerates folding of I_trap much less than M. Thus, the rate-limiting transition from I_trap requires a smaller increase in exposed surface. Mutations that disrupt peripheral tertiary contacts give large and nearly uniform increases in re-folding of M, whereas the same mutations give at most modest increases in folding from I_trap. Intriguingly, mutations within the peripheral element P5abc give 5- to 10-fold accelerations in escape from I_trap, whereas ablation of P13, which lies on the opposite surface in the native structure, near the P3-P8 domain, has no effect. Thus, the unfolding required from I_trap appears to be local, whereas the unfolding of M appears to be global. Further, the modest effects from several mutations suggest that there are multiple pathways for escape from I_trap and that escape is aided by loosening nearby native structural constraints, presumably to facilitate local movements of nucleotides or segments that have not formed native contacts. Overall, these and prior results suggest a model in which the global architecture and peripheral interactions of the RNA are achieved relatively early in folding. Multiple folding and re-folding events occur on the predominant pathway to the native state, with increasing native core interactions and cooperativity as folding progresses.     

Structural Rearrangements Linked to Global Folding Pathways of the Azoarcus Group I Ribozyme

Stable RNAs must fold into specific three-dimensional structures to be biologically active, yet many RNAs form metastable structures that compete with the native state. Our previous time-resolved footprinting experiments showed that Azoarcus group I ribozyme forms its tertiary structure rapidly (τ < 30 ms) without becoming significantly trapped in kinetic intermediates. Here, we use stopped-flow fluorescence spectroscopy to probe the global folding kinetics of a ribozyme containing 2-aminopurine in the loop of P9. The modified ribozyme was catalytically active and exhibited two equilibrium folding transitions centered at 0.3 and 1.6 mM Mg²⁺, consistent with previous results. Stopped-flow fluorescence revealed four kinetic folding transitions with observed rate constants of 100, 34, 1, and 0.1 s^− 1 at 37 °C. From comparison with time-resolved Fe(II)-ethylenediaminetetraacetic acid footprinting of the modified ribozyme under the same conditions, these folding transitions were assigned to formation of the I_C intermediate, tertiary folding and docking of the nicked P9 tetraloop, reorganization of the P3 pseudoknot, and refolding of nonnative conformers, respectively. The footprinting results show that 50-60% of the modified ribozyme folds in less than 30 ms, while the rest of the RNA population undergoes slow structural rearrangements that control the global folding rate. The results show how small perturbations to the structure of the RNA, such as a nick in P9, populate kinetic folding intermediates that are not observed in the natural ribozyme.     

Directional Selection and Variation in Finite Populations

Predictions are made of the equilibrium genetic variances and responses in a metric trait under the joint effects of directional selection, mutation and linkage in a finite population. The "infinitesimal model" is analyzed as the limiting case of many mutants of very small effect, otherwise Monte Carlo simulation is used. If the effects of mutant genes on the trait are symmetrically distributed and they are unlinked, the variance of mutant effects is not an important parameter. If the distribution is skewed, unless effects or the population size is small, the proportion of mutants that have increasing effect is the critical parameter. With linkage the distribution of genotypic values in the population becomes skewed downward and the equilibrium genetic variance and response are smaller as disequilibrium becomes important. Linkage effects are greater when the mutational variance is contributed by many genes of small effect than few of large effect, and are greater when the majority of mutants increase rather than decrease the trait because genes that are of large effect or are deleterious do not segregate for long. The most likely conditions for "Muller's ratchet" are investigated.     

Interaction between Natural Selection for Heterozygotes and Directional Selection

Significant correlations between allelic frequencies and environmental variables in a number of insect species have been demonstrated by multivariate techniques. Since many environmental variables show a strong relationship to geographic location and since gene flow between populations can also produce patterns of gene frequencies which are related to the geographic location, both selection and gene-flow hypotheses are consistent with the observed correlations. The genetic variables can be corrected for geographic location and so for linear gene-flow patterns. If, after correction, the genetic variables still show significant correlations with similarly corrected environmental variables, then these correlations are consistent with hypotheses of selection but not of gene flow. The data of Johnson and Schaffer (1973) have been reanalyzed using the method of canonical correlation after correction for geographical location by means of multiple regression. Five of the nine loci studied exhibit significant canonical correlations. These results, under the assumption of linear gene flow, support hypotheses of selective action of environmental variables in the genotype-environment relationships observed.     

Low Selection Pressure Aids the Evolution of Cooperative Ribozyme Mutations in Cells

Understanding the evolution of functional RNA molecules is important for our molecular understanding of biology. Here we tested experimentally how two evolutionary parameters, selection pressure and recombination, influenced the evolution of an evolving RNA population. This was done using four parallel evolution experiments that employed low or gradually increasing selection pressure, and recombination events either at the end or dispersed throughout the evolution. As model system, a trans-splicing group I intron ribozyme was evolved in Escherichia coli cells over 12 rounds of selection and amplification, including mutagenesis and recombination. The low selection pressure resulted in higher efficiency of the evolved ribozyme populations, whereas differences in recombination did not have a strong effect. Five mutations were responsible for the highest efficiency. The first mutation swept quickly through all four evolving populations, whereas the remaining four mutations accumulated later and more efficiently under low selection pressure. To determine why low selection pressure aided this evolution, all evolutionary intermediates between the wild type and the 5-mutation variant were constructed, and their activities at three different selection pressures were determined. The resulting fitness profiles showed a high cooperativity among the four late mutations, which can explain why high selection pressure led to inefficient evolution. These results show experimentally how low selection pressure can benefit the evolution of cooperative mutations in functional RNAs.     

Spatial Autocorrelation of Genotypes under Directional Selection

The spatial distributions of genetic variation under selection-mutation equilibrium within populations that have limited dispersal are investigated. The results show that directional selection with moderate strength rapidly reduces the amount of genetic structure and spatial autocorrelations far below that predicted for selectively neutral loci. For the latter, homozygotes are spatially clustered into separate areas or patches, each consisting of several hundred homozygotes. When selection is added the patches of the deleterious homozygotes are much smaller, in the range of 25 to 50 individuals. Selection also reduces temporal correlations. Also investigated are the effects of random replacement processes, such as mutation, immigration, and long-distance migration, on spatial and temporal correlations. The detection of natural selection through spatial pattern analysis is discussed, and applied to data from populations of the morning glory, Ipomoea purpurea.     

Multiplicative Genotype-Environment Interaction as a Cause of Reversed Response to Directional Selection

In experiments with directional selection on a quantitative character a "reversed response" to selection is occasionally observed, when selection of individuals for a higher (lower) value of the character results in a lower (higher) value of the character among their offspring. A sudden change in environments or random drift is often assumed to be responsible for this. It is demonstrated in this paper that these two causes cannot account for the reversed response at least in some of the experiments. Multiplicative genotype-environment interaction is discussed as a possible cause of a reversed response to directional selection. Such interaction entails either disruptive or stabilizing genotypic selection, even when the phenotypic selection is directional.     

Genetic Variation in a Heterogeneous Environment. II. Temporal Heterogeneity and Directional Selection

The maintenance of genetic variation is investigated in a finite population where selection at an autosomal locus with two alleles varies temporally between two environments and the heterozygote has an intermediate fitness value. When there is additive gene action and equal selection in both environments, the autocorrelation between subsequent environments must be negative for more maintenance of genetic variation than for neutrality. The maximum maintenance occurs when there is equal selection in the two environments and the autocorrelation approaches ^-1.0 (for a stochastic model), or when there is short repeating cycle such as one related to seasons. Also comparison of the effects of stochastic variation in selection in finite and infinite populations is made by using Monte Carlo simulation. One situation was found where temporal environmental variation maintains genetic variation very effectively even in a small population and that is when there is evolution of dominance, i.e., the heterozygote is closer in fitness to the favored homozygote than the other homozygote. An important conclusion is that in a finite population genetic tracing of environmental change, particularly when there is a positive autocorrelation between environments or a long environmental cycle, leads to an increased loss of genetic variation making such a response undesirable in the long term, a result different from that in infinite populations.     

Template-Directed Ligation of Tethered Mononucleotides by T4 DNA Ligase for Kinase Ribozyme Selection

Background

In vitro selection of kinase ribozymes for small molecule metabolites, such as free nucleosides, will require partition systems that discriminate active from inactive RNA species. While nucleic acid catalysis of phosphoryl transfer is well established for phosphorylation of 5′ or 2′ OH of oligonucleotide substrates, phosphorylation of diffusible small molecules has not been demonstrated.

Methodology/Principal Findings

This study demonstrates the ability of T4 DNA ligase to capture RNA strands in which a tethered monodeoxynucleoside has acquired a 5′ phosphate. The ligation reaction therefore mimics the partition step of a selection for nucleoside kinase (deoxy)ribozymes. Ligation with tethered substrates was considerably slower than with nicked, fully duplex DNA, even though the deoxynucleotides at the ligation junction were Watson-Crick base paired in the tethered substrate. Ligation increased markedly when the bridging template strand contained unpaired spacer nucleotides across from the flexible tether, according to the trends: A₂>A₁>A₃>A₄>A₀>A₆>A₈>A₁₀ and T₂>T₃>T₄>T₆≈T₁>T₈>T₁₀. Bridging T''s generally gave higher yield of ligated product than bridging A''s. ATP concentrations above 33 µM accumulated adenylated intermediate and decreased yields of the gap-sealed product, likely due to re-adenylation of dissociated enzyme. Under optimized conditions, T4 DNA ligase efficiently (>90%) joined a correctly paired, or T∶G wobble-paired, substrate on the 3′ side of the ligation junction while discriminating approximately 100-fold against most mispaired substrates. Tethered dC and dG gave the highest ligation rates and yields, followed by tethered deoxyinosine (dI) and dT, with the slowest reactions for tethered dA. The same kinetic trends were observed in ligase-mediated capture in complex reaction mixtures with multiple substrates. The “universal” analog 5-nitroindole (dNI) did not support ligation when used as the tethered nucleotide.

Conclusions/Significance

Our results reveal a novel activity for T4 DNA ligase (template-directed ligation of a tethered mononucleotide) and establish this partition scheme as being suitable for the selection of ribozymes that phosphorylate mononucleoside substrates.     

Moment Analysis for the Probit Model of Directional Selection

An iteration relation is given for the moments of the surviving individuals after probit directional selection if the individuals before selection are normally distributed. The proportion, mean, variance, skewness, and kurtosis are expressed in closed form.     

Dominance Genetic Variance for Traits Under Directional Selection in Drosophila serrata

In contrast to our growing understanding of patterns of additive genetic variance in single- and multi-trait combinations, the relative contribution of nonadditive genetic variance, particularly dominance variance, to multivariate phenotypes is largely unknown. While mechanisms for the evolution of dominance genetic variance have been, and to some degree remain, subject to debate, the pervasiveness of dominance is widely recognized and may play a key role in several evolutionary processes. Theoretical and empirical evidence suggests that the contribution of dominance variance to phenotypic variance may increase with the correlation between a trait and fitness; however, direct tests of this hypothesis are few. Using a multigenerational breeding design in an unmanipulated population of Drosophila serrata, we estimated additive and dominance genetic covariance matrices for multivariate wing-shape phenotypes, together with a comprehensive measure of fitness, to determine whether there is an association between directional selection and dominance variance. Fitness, a trait unequivocally under directional selection, had no detectable additive genetic variance, but significant dominance genetic variance contributing 32% of the phenotypic variance. For single and multivariate morphological traits, however, no relationship was observed between trait–fitness correlations and dominance variance. A similar proportion of additive and dominance variance was found to contribute to phenotypic variance for single traits, and double the amount of additive compared to dominance variance was found for the multivariate trait combination under directional selection. These data suggest that for many fitness components a positive association between directional selection and dominance genetic variance may not be expected.     

Evidence for Directional Selection at a Novel Major Histocompatibility Class I Marker in Wild Common Frogs (Rana temporaria) Exposed to a Viral Pathogen (Ranavirus)

Whilst the Major Histocompatibility Complex (MHC) is well characterized in the anuran Xenopus, this region has not previously been studied in another popular model species, the common frog (Rana temporaria). Nor, to date, have there been any studies of MHC in wild amphibian host-pathogen systems. We characterise an MHC class I locus in the common frog, and present primers to amplify both the whole region, and specifically the antigen binding region. As no more than two expressed haplotypes were found in over 400 clones from 66 individuals, it is likely that there is a single class I locus in this species. This finding is consistent with the single class I locus in Xenopus, but contrasts with the multiple loci identified in axolotls, providing evidence that the diversification of MHC class I into multiple loci likely occurred after the Caudata/Anura divergence (approximately 350 million years ago) but before the Ranidae/Pipidae divergence (approximately 230 mya). We use this locus to compare wild populations of common frogs that have been infected with a viral pathogen (Ranavirus) with those that have no history of infection. We demonstrate that certain MHC supertypes are associated with infection status (even after accounting for shared ancestry), and that the diseased populations have more similar supertype frequencies (lower F_ST) than the uninfected. These patterns were not seen in a suite of putatively neutral microsatellite loci. We interpret this pattern at the MHC locus to indicate that the disease has imposed selection for particular haplotypes, and hence that common frogs may be adapting to the presence of Ranavirus, which currently kills tens of thousands of amphibians in the UK each year.     

Increased Ribozyme Activity in Crowded Solutions

Noncoding RNAs must function in the crowded environment of the cell. Previous small-angle x-ray scattering experiments showed that molecular crowders stabilize the structure of the Azoarcus group I ribozyme, allowing the ribozyme to fold at low physiological Mg²⁺ concentrations. Here, we used an RNA cleavage assay to show that the PEG and Ficoll crowder molecules increased the biochemical activity of the ribozyme, whereas sucrose did not. Crowding lowered the Mg²⁺ threshold at which activity was detected and increased total RNA cleavage at high Mg²⁺ concentrations sufficient to fold the RNA in crowded or dilute solution. After correcting for solution viscosity, the observed reaction rate was proportional to the fraction of active ribozyme. We conclude that molecular crowders stabilize the native ribozyme and favor the active structure relative to compact inactive folding intermediates.     

Modular engineering of a Group I intron ribozyme

All Group I intron ribozymes contain a conserved core region consisting of two helical domains, P4–P6 and P3–P7. Recent studies have demonstrated that the elements required for catalysis are concentrated in the P3–P7 domain. We carried out in vitro selection experiments by using three newly constructed libraries on a variant of the T4 td Group I ribozyme containing only a P3–P7 domain in its core. Selected variants with new peripheral elements at L7.1, L8 or L9 after nine cycles efficiently catalyzed the reversal reaction of the first step of self-splicing. The variants from this selection contained a short sequence complementary to the substrate RNA without exception. The most active variant, which was 3-fold more active than the parental wild-type ribozyme, was developed from the second selection by employing a clone from the first selection. The results show that the P3–P7 domain can stand as an independent catalytic module to which a variety of new domains for enhancing the activity of the ribozyme can be added.     

The Quantitative Genetic Consequences of Pleiotropy under Stabilizing and Directional Selection

The independence of two phenotypic characters affected by both pleiotropic and nonpleiotropic mutations is investigated using a generalization of M. Slatkin's stepwise mutation model of 1987. The model is used to determine whether predictions of either the multivariate normal model introduced in 1980 by R. Lande or the house-of-cards model introduced in 1985 by M. Turelli can be regarded as typical of models that are intermediate between them. We found that, under stabilizing selection, the variance of one character at equilibrium may depend on the strength of stabilizing selection on the other character (as in the house-of-cards model) or not (as in the multivariate normal model) depending on the types of mutations that can occur. Similarly, under directional selection, the genetic covariance between two characters may increase substantially (as in the house-of-cards model) or not (as in the multivariate normal model) depending on the kinds of mutations that are assumed to occur. Hence, even for the simple model we consider, neither the house-of-cards nor the multivariate normal model can be used to make predictions, making it unlikely that either could be used to draw general conclusions about more complex and realistic models.