Witnessing the structural evolution of an RNA enzyme

Abstract

An RNA polymerase ribozyme that has been the subject of extensive directed evolution efforts has attained the ability to synthesize complex functional RNAs, including a full-length copy of its own evolutionary ancestor. During the course of evolution, the catalytic core of the ribozyme has undergone a major structural rearrangement, resulting in a novel tertiary structural element that lies in close proximity to the active site. Through a combination of site-directed mutagenesis, structural probing, and deep sequencing analysis, the trajectory of evolution was seen to involve the progressive stabilization of the new structure, which provides the basis for improved catalytic activity of the ribozyme. Multiple paths to the new structure were explored by the evolving population, converging upon a common solution. Tertiary structural remodeling of RNA is known to occur in nature, as evidenced by the phylogenetic analysis of extant organisms, but this type of structural innovation had not previously been observed in an experimental setting. Despite prior speculation that the catalytic core of the ribozyme had become trapped in a narrow local fitness optimum, the evolving population has broken through to a new fitness locale, suggesting that further improvement of polymerase activity may be achievable.

Cross-Chiral, RNA-Catalyzed Exponential Amplification of RNA

“…This approach has proven to be powerful in optimizing the activity of a homochiral RNA polymerase ribozyme. 5,24,25 By comparison, the cross-chiral polymerase is a relatively young enzyme that has not had yet the benefit of extensive evolutionary optimization.…”

Experimental exploration of a ribozyme neutral network using evolutionary algorithm and deep learning

“…effects on their fitness. Contrasting evidence can also be found in other experimental studies that demonstrated that artificial evolution can be used to engineer a ribozyme that adopts a new structure while retaining its function 21 , or that acquires a new function 22,23 .…”

Cryo-EM structure and functional landscape of an RNA polymerase ribozyme

“…Thus the t1 domain and its KL interactions may together serve to hold J1/3 in this out-stretched conformation, as a longer or untethered single-stranded templatebinding strand would likely be more dynamic and adopt a variety of conformations, increasing the entropic cost of template interaction. Analysis of the evolution of the related 52-2 polymerase ribozyme (which used NTPs as substrates) 7 suggest the emergence of a pseudoknot structure involving P7 and the J1/3 equivalent, which . CC-BY-NC-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.…”

“…RNA catalysts (ribozymes) occupy central structural and catalytic roles in the function of modern cells including tRNA processing (RNaseP), mRNA splicing (spliceosome, group I / II self-splicing introns) and translation (ribosome peptidyl transferase center) 15 . In addition, a much wider variety of ribozyme activities not found in nature has been discovered by in vitro evolution, including polymerase ribozymes (PR) that are capable of synthesizing a complementary strand on an RNA template 3-7,11 . The capacity for RNA-catalyzed RNA-templated synthesis and replication is widely believed to have been a central pillar of the emergence of life’s first genetic system and even life itself.…”

“…The emergence of an RNA molecule capable of replicating itself and other RNA sequences is a central pillar of hypotheses regarding the origin of life 1,2 . In vitro evolution has yielded polymerase ribozymes (PR) that can copy a range of RNA templates using nucleotide [3][4][5][6][7][8][9][10] or trinucleotide triphosphates (triplets) 11 as substrates and may give rise to a replicase activity. However, our understanding of PR function is encumbered by a lack of structural information beyond the progenitor class I ligase (cIL) ribozyme [12][13][14] .…”