Workshop 2: Origins of Life 16:30 - 18:00 Wednesday, 27th May, 2020 Talk Session Chair Peter Unrau
200 An RNA polymerase ribozyme that synthesizes its own ancestor
David P Horning1, Katrina Tjhung1, Maxim Shokhirev1, Yu-Ting Huang2, Saikat Bala3, John Chaput3, Gerald Joyce1 1The Salk Institute, La Jolla, CA, USA. 2University of California, San Diego, La Jolla, CA, USA. 3University of California, Irvine, Irvine, CA, USA
Abstract
In the RNA world, which is thought to have preceded the invention of genetically-encoded proteins, the propagation of heritable information across successive generations would have been controlled by an RNA polymerase ribozyme that copies RNA molecules. Building RNA-based life in the laboratory will require a polymerase ribozyme with sufficient activity to copy RNAs as complex as itself and with sufficient accuracy to maintain heritable information against deleterious mutations. The class I polymerase ribozyme has now undergone more than fifty generations of directed in vitro evolution for the ability to synthesize other functional RNA molecules. This has yielded highly active polymerases that are able to synthesize other functional ribozymes, including the polymerase's own ancestor, the class I ligase. Directed evolution has changed the polymerase sequence by twenty six mutations and remodeled its secondary structure, stabilizing a region of the ribozyme that binds to the template-primer complex. Catalytic promiscuity of the polymerase has also expanded, enabling the RNA-templated synthesis of DNA, threose nucleic acid (TNA), or arabinose nucleic acid (ANA), as well as the synthesis of RNA from DNA and TNA templates. These results suggest that polymerase ribozymes could copy the complex, functional RNA structures that would be needed to sustain an RNA organism, and could catalyze information transfer between nucleic-acid-like molecules that would enable evolutionary transitions between early genetic systems. However, greater efficiency is needed for the polymerase to copy itself, and the current modest fidelity of the polymerase severely limits the amount of functional information it can copy. The former challenge can be addressed by dividing the polymerase into multiple, shorter fragments that are more readily copied and self- assemble into a functional ribozyme. The improved efficiency of the polymerase can, in turn, be used to impose greater pressure on fidelity by requiring the polymerase to synthesize more complex RNAs that contain a larger number of nucleotides that are critical for function. This bootstrapping process is analogous to what is thought to have driven the evolution of more complex genomes in the RNA world.
Presenting author email [email protected]
Topic category
Origins of Life and evolution 359 Selection of an RNA Polymerase Ribozyme with Promoter-like Selectivity and Improved Processivity
Razvan Cojocaru, Peter Unrau Simon Fraser University, Burnaby, BC, Canada
Abstract
The “RNA World” appears likely to have required some form of conditional gene expression analogous to the sigma dependent gene expression found in modern day bacteria. We wondered if the emergence of such promoter-like activity might in fact be naturally linked to polymerase processivity. To explore this hypothesis, we used a 'sigma-factor' like RNA primer to activate the 5' ends of a diverse pool of RNA polymerase ribozymes based on the B6.61 polymerase. By design, this primer can be conditionally stripped from the polymerase upon encountering a template containing a complementary 'promoter' sequence, localizing the polymerase to a specific template sequence. Upon recognizing the promoter-binding site, this strategy can allow successful pool molecules to undergo a structural rearrangement that can lead to increased polymerase processivity. After 27 rounds of selection a polymerase ribozyme was isolated that can extend 90+ nt of a randomly selected template in as little as 24 hours. This polymerase favorably distinguishes circular from linear templates, indicating that processivity is dependent, at least partially, on a sliding type motion along the template. Consistent with these findings the polymerase cannot effectively extend a short promoter-template, but extension is rescued by lengthening the short template with oligo(A) at either terminus. Of equal interest, hybridizing the RNA primer initially to the promoter-template rather than the ribozyme causes a dramatic decrease in extension efficiency indicating that the polymerase localizes to and then extends template sequence in a mechanistically coupled fashion. Finally, and of high utility to an “RNA World”, we found that this polymerase can also synthesize at least 7-nt of the sigma-like primer using its own sequence as a template. This self-priming primer synthesis, encodes the ribozyme’s sequence into its own sigma-like primer, providing a simple way for a replicase ribozyme to specifically copy its own genome over that of potentially competing replicases. Such self-priming, promoter-linked replication activity could also give rise to conditional expression of genes essential to the reproductive success of a specific replicase. Together, these mechanisms might have played an integral part in the evolution of an “RNA World” prior to membrane based cellular compartmentalization.
Presenting author email [email protected]
Topic category
Origins of Life and evolution 169 Experimental fitness landscapes surrounding a dual-function RNA intersection sequence: further implications for the evolution of new RNA folds and functions
Eric Hayden1, Devin Bendixsen2 1Boise State University, Boise, ID, USA. 2Stockholm University, Stockholm, Sweden
Abstract
The diversification of life has been possible because of numerous molecular innovations – defined as molecules with new structures and functions. The numerous RNA molecules that are critical to all life suggest that RNA innovations have remained important since life’s origins. Experimental RNA fitness landscapes are a recent approach to explain evolutionary outcomes by measuring the effects of numerous mutational neighbors and predicting the evolutionary consequences of mutational pathways to higher fitness. While these fitness landscapes can help explain evolutionary constraints and dynamics, they do not explain the emergence of innovations. I will describe our recent efforts using high-throughput sequencing to construct two experimental landscapes for two different RNA functions that exist in the same neighboring sequence space. We use this data as a proxy for a fitness landscape at an evolutionary innovation. We also demonstrate the use of computational simulations of sequences competing for survival to explore the challenges to this evolutionary innovation under different evolutionary scenarios. The experimental landscapes are very rugged, and our results emphasize the importance of local fitness peaks in constraining rates of adaptation. Nevertheless, the numerous points where the two landscapes intersect suggests that historic innovations in natural RNA molecules may have also been facilitated by the existence of multi-functional RNA sequences.
Presenting author email [email protected]
Topic category
Origins of Life and evolution 374 Ribozyme mediated repair of damaged and mismatched RNA sequences
Alexandra Kent, Lucy Yang, Ivee Lee, Andrej Luptak University of California at Irvine, Irvine, CA, USA
Abstract
The RNA world hypothesis is predicated upon the ability of RNA to self-replicate. Because of the centrality of phosphodiester bond formation to genome replication, a polymerase enzyme was likely one of the first enzymes encoded by the earliest cells. To date, however, even the most evolved RNA polymerases have fallen short of this ideal due to high mutational burden and aborted sequences. One of the first phenotypes that could lend an evolutionary advantage to an early protocell would be the ability to replicate with higher fidelity. Here we describe an example of ribozyme-mediated repair of damaged and mismatched RNA sequences. Under conditions of high concentrations of pyrophosphate, a polymerase ribozyme is capable of “chewing back” along a 2´-3´-cyclic-phosphate–terminated RNA sequence or mismatch–terminated sequence to subsequently allow for further extension along a template. We hypothesize that addition of pyrophosphate to prebiotic replication could have increased overall genome fidelity, as well as rescue cyclic phosphate terminated sequences arising from RNA degradation.
Presenting author email [email protected]
Topic category
Origins of Life and evolution 198 Making Prebiotic Chemistry Simple: RNA First
Steven Benner, Elisa Biondi, Jan Spacek Foundation for Applied Molecular Evolution, Alachua, FL, USA
Abstract
This talk describes experiments showing that oligomeric RNA was a likely, and possibly an inevitable, molecule to emerge on a Hadean Earth ~4.35 billion years ago. These experiments are grounded in strongly constrained models for the Hadean mantle, whose fayalite–magnetite fugacity delivered volcanic CO2, water, SO2, and other gasses into a redox neutral atmosphere. Well-accepted photochemistry in that upper atmosphere must have created large amounts (~ 1 mg/year/m2) of HCHO (formaldehyde) and catalytic (ppm) amounts of glycolaldehyde. Those simple carbohydrates could not have avoided reaction with SO2 to form metastable sulfonate adducts which, if rained into a constrained aquifer on a semi-arid subaerial surface could not not have matured to give 5-carbon carbohydrates, including ribose, as metastable end-products, if that surface contained borate minerals.
Laboratory experiments show that phosphate anhydrides almost certainly available on any semi-arid surface containing phosphorus transform these matured carbohydrates to phosphorylated carbohydrates that react directly with nucleobases to give nucleosides. Laboratory experiments also show that analogous phosphorylation mineralogy generates nucleoside 5'-mono-, di-, and tri-phosphates, with borate controlling regiochemistry. Laboratory experiments also show that silicate mineral assemblies catalyze the continuous formation of oligomeric RNA that accumulates over months.
The model is further supported by the impact history of early Earth. Impactors transiently generated atmospheres productive for the synthesis of nucleobase precursors. Formation of nucleobases from these is well-documented. The length of the productive period depends on impactor size and frequency, both now well constrained. Haze in productive atmospheres protected nascent RNA on the surface from photodestruction.
Mars exploration suggests that required mineralogy (especially borate) was available even before large-scale plate tectonics. The recent discovery of meteoritic ribose likewise is consistent with the model.
While they demystify the prebiotic formation of RNA, these data leave open two prominent challenges: (a) Chirality. To support Darwinism, a biopolymer must have a polyelectrolyte backbone and interchangeable building blocks that fit a Schrödingerian "aperiodic crystal." The process outlined here offers the first structural feature, but not the second. (b) Poor catalytic versatility of standard RNA. Greater versatility may be possible with more RNA building blocks, now know to support Darwinism.
Presenting author email [email protected]
Topic category
Origins of Life and evolution