1. Evaluation Summary:

    The plasticity of RNA folds and their ability to response to changes in selective pressure is a key aspect of understanding the evolution of life on this planet. The Class I ligase is a remarkably fast RNA ligase ribozyme that has been harnessed by a number of laboratories to power RNA polymerization. Thought by many to be the immutable catalytic core required for polymerization, Portillo et al. demonstrate evolutionary trajectories that result in a new and catalytically enhanced ligase core. An accumulation of mutations results in a the formation of a new pseudoknot structure immediately outside the active site of the ligase core. This new structure appears to more optimally position the P7-P6-P3 coaxially stacked stems of the ligase core with respect to the primer template substrate. Tracking the emergence of this new fold, which is correlated with an enhancement in RNA polymerization activity, is novel and interesting.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

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  2. Reviewer #1 (Public Review):

    Tracking the evolutionary optimization a catalytic RNA, via the emergence of a new structural fold is an exciting development. This work captures the emergence of a new pseudoknot in the catalytic core of a two domain RNA polymerase ribozyme. The work is convincing, timely and will be of significant interest to evolutionary biologists. Biologically speaking, the emergence of new folds is frozen in time. So we can admire the fact that RNaseP has evolutionary flexibility in its substrate recognition domain and not in its catalytic core. We can appreciate that the ribosome through its detailed set of A-minor interactions encodes its evolutionary progression from a simple and ancient gene duplication to the very complex enzyme we see today. Despite having these powerful arcs of evolutionary history we rarely, if ever, get to see the emergence of new RNA structures, that demonstrably improve RNA functionality. Tracking in 'real-time' such evolutionary progressions therefore provides powerful insights into how RNA folds are optimized after their initial emergence. Thus in one sense the initial emergence of the RNA polymerase ribozyme described here happened all at once from random sequence. A new domain called the accessory domain conferred to the ligase core the ability of polymerization (Johnston et al, 2001). How such catalytic systems evolve after this initial 'birth' is key to understanding the evolution of catalytic RNAs early in the evolution life. This work provides a glimpse as to how such optimizations take place and was a pleasure to read.

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  3. Reviewer #2 (Public Review):

    Portillo et al describe continuing progress in improving a polymerase ribozyme and furthermore, analyse the structural changes that underlie these improvements over the course of in vitro evolution. They describe the most active polymerase ribozyme yet, which is now able to synthesize the highly structured RNA ligase catalytic core with good efficiency.

    Mapping of the structural changes using in line probing they find a distinct structural remodelling of part of the catalytic core of the polymerase ribozyme that correlates with enhanced catalytic activity. Such progress is encouraging as it suggests that - contrary to previous suggestions - the polymerase ribozyme catalytic core does not occupy an isolated fitness peak in the adaptive landscape and is impervious to further evolutionary optimization. Furthermore, these results suggest that there is, in principle, no obstacle to reaching ever more active polymerase ribozymes ultimately capable of self-replication. Once again the ceiling is raised for RNA replicase capabilities boding well for the potential of an RNA-based genetic system.

    The key claims of the manuscript are well supported by the data, but several important questions are raised that require further discussion and some experimental investigation:

    1. The authors show that the rearrangement correlates with improved activity, but there is little data on how the structural rearrangement alters and improves polymerase activity. Previous generations of polymerase ribozymes were particularly limited in substrate / template binding and processivity and generally had modest fidelity, and the rearrangement could be influencing any of these parameters (see points 2 and 5 below). This requires some investigation, perhaps through product sequencing or assay of activity in the absence of template tether. What are the reasons for the improved activity and remaining limitations that prevent ribozyme self-synthesis?

    2. The parental class I ligase ribozyme synthesized by 52-2 is reported to have a catalytic rate of 0.3 h-1 or as stated in the manuscript" ...accelerating the rate ... of ...ligation by 1,500-fold...". I think this statement is potentially misleading for non-experts in the field, giving the impression of an impressively active product. The original class I ligase is one of the most powerful RNA catalysts ever described, reaching catalytic rates of up to 375 min-1 (Biochemistry 39 : 3115) under optimal conditions and therefore four to five orders of magnitude faster. It may well be that the precise construct and conditions used here conspire to give a somewhat slower catalytic rate, but it seems rather unlikely that it would be compromised to such an extent. Activity should be compared to an equivalent construct transcribed using T7 RNA polymerase and assayed under equivalent conditions. To better understand any discrepancy in activity between protein-prepared, 52-2-prepared and 24-3 prepared RNA, the authors should sequence the 52-2 synthesis products. This would also provide information on ribozyme fidelity (see 1).

    3. The authors state "Tertiary structural remodeling of RNA is known to occur in nature, ..., but .... structural innovation had not previously been observed in an experimental setting." This statement should be tempered in the light of previous studies such as morphing one ribozyme activity / structure into another (Science 289: 44), evolution of a new GTP binding fold from the canonical ATP aptamer (Rna 9: 1456) or indeed experiments from the senior author's own laboratory on the evolution of ribozymes with a reduced nucleotide content (e.g. Nature, 402: 323).

    4. The authors state "... an RNA polymerase ribozyme was seen to undergo a tertiary structural change, similar to changes inferred to have occurred in nature based on phylogenetic analysis (Bokov & Steinberg 2009, Fox 2010)." This statement should be rephrased and different citations used. The changes seen in the polymerase ribozyme do not obviously resemble the structural changes inferred to have happened during ribosome evolution such as hierarchical domain addition and insertion of expansion segments. Considering the focus of the paper, the authors should more directly compare the rearrangement seen here to inferred examples of transformation from biological RNAs. Does in vitro evolution offer different routes of structural changes to biological evolution? Such a significant structural rearrangement during evolution whilst maintaining activity is somewhat surprising. Certainly, the notion that structure is conserved to support activity during sequence divergence is deeply embedded in our understanding of protein evolution. More extensive discussion of these possibilities is needed to strengthen the paper, together with analysis of the implications for e.g., library design during RNA in vitro evolution.

    5. The new P8 helix element is well supported by the data, although I have some doubts as to whether A16 should be assigned as part of this duplex. As the authors identify, A16 has functions beyond base-pairing, and in the parental ligase makes critical contacts to the primer-template using its Watson-Crick face. How is this compensated for? If the J1/3 a-minor interactions are preserved, might the role of the new stem be to position these residues and thus the primer/template?

    6. It would be of great interest, although potentially technically challenging, to investigate to what extent structural changes affect the apo form / ground state of the polymerase ribozyme compared to the holoenzyme (with primer-template / NTPs bound). This would be of particular relevance in the context of a recently described variant of the same polymerase ribozyme (Science 371: 1225), which has been proposed to undergo a significant structural rearrangement at the level of the holoenzyme towards a highly processive conformation.

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  4. Reviewer #3 (Public Review):

    The authors have monitored an additional phase in the in vitro evolution of polymerase ribozymes derived from the 1994 Bartel & Szostak ligase ribozyme. Prior phases had appended large domains and engineered or selected abilities to synthesize small amounts of large RNAs with simple repeats and/or to synthesize large functional RNAs (aptamer, ribozyme, tRNA). A large amount of work has gone into this system, both because of its robust catalysis and because of the potential for polymerase ribozymes to drive in vitro evolution of molecular systems composed entirely of RNA (for origins-of-life or SynBio applications).

    The present work heavily mutagenized a single clone from the Rnd 38 population (10% per position), then carried out 14 more cycles of selection for synthesis of a functional hammerhead ribozyme with mutagenic PCR in the amplification steps. and slightly lower magnesium concentrations than prior rounds (down from 200 mM to 50 mM). The resulting species was 3x to 23x more active in 50 mM Mg2+ than was the wt ribozyme or the Rnd 38 clone from which it was derived.

    Major Strengths:

    The primary 'new' finding of the paper is the acquisition of a new structure feature in which a portion of a stem (p7) and loop (L7) gains pairing interactions with a previously unpaired region (J2/3?) to form a new pseudoknot that was not present in the 'wt' ribozyme. This feature is supported by in-line probing data, by kinetics of ribozymes carrying disruptive and compensatory mutation, and by RNASeq data from 19 of the 52 rounds of the selection that document the appearance and development of this feature. Thus, the major claims are well supported by the data.

    An intriguing observation that is not significantly developed is that the 52-2 ribozyme exhibits a burst phase for nucleotide incorporation that may be as much as 100x faster than the later processive elongation rates. Many questions immediately come to mind: Why does it slow down after the initial burst? How many nucleotides can be made at 'burst speed'? Does a structural accommodation accompany the change from burst phase to processive elongation phase? If so, when and why does it occur, and what are the structural differences between ribozymes in these two phases? No doubt these questions will be explored in future studies.

    Figure 5 is one of my favorite parts of the paper. Eighteen clusters with >1% representation in any given round (among the 19 rounds analyzed) were identified, and their fractional representations were plotted as a heat map across the 52 Rnds of the selection (5B). The potential of each sequence to form stem P8 is also shown (5A). While I would like to see a network analysis defining evolutionary precursor-product relationships, I cannot trace the evolutionary ancestor of any of the sequences shown (with a few exceptions in the early Rnds). That is not surprising for the last few sequences, since they arose from heavy (10%) mutagenesis of clone 38-6 and are likely independent of each other. However, as noted by the authors and in the figure, stem P8 was established long before Rnd 38 - including several clones from as early as Rnd 11! Very cool. The lack of evident (by eye) evolutionary connections among these species indirectly supports the claim in the abstract that "multiple paths to the new structure were explored by the evolving population."

    Minor Weakness:

    The only significant weakness is that while the paper does demonstrate that the pseudoknot is necessary, it is not clear whether it is sufficient. Specifically, there is no evaluation of whether rate improvements can be obtained simply by introducing any of the derived P8 pseudoknots into earlier forms of the ribozyme, or if instead the pseudoknot must be accompanied by secondary mutations elsewhere in the molecule.

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