Processive RNA polymerization and promoter recognition in an RNA World

Science ◽  
2021 ◽  
Vol 371 (6535) ◽  
pp. 1225-1232
Author(s):  
Razvan Cojocaru ◽  
Peter J. Unrau
Keyword(s):  
Rna World ◽  
Promoter Sequence ◽  
Second Step ◽  
Promoter Recognition ◽  
Rna Polymerization ◽  
Evolution Of Life ◽  
Strand Invasion ◽  
Critical Requirement ◽  
Self Replication ◽  
Rna Promoter

Early life is thought to have required the self-replication of RNA by RNA replicases. However, how such replicases evolved and subsequently enabled gene expression remains largely unexplored. We engineered and selected a holopolymerase ribozyme that uses a sigma factor–like specificity primer to first recognize an RNA promoter sequence and then, in a second step, rearrange to a processive elongation form. Using its own sequence, the polymerase can also program itself to polymerize from certain RNA promoters and not others. This selective promoter–based polymerization could allow an RNA replicase ribozyme to define “self” from “nonself,” an important development for the avoidance of replicative parasites. Moreover, the clamp-like mechanism of this polymerase could eventually enable strand invasion, a critical requirement for replication in the early evolution of life.

scholarly journals Enzymatic RNA Production from NTPs Synthesized from Nucleosides and Trimetaphosphate

2021 ◽  
Author(s):  
Fabio Chizzolini ◽  
Alexandra Kent ◽  
Luiz F. M. Passalacqua ◽  
Andrej Lupták
Keyword(s):  
Rna Synthesis ◽  
Rna World ◽  
Rna Replication ◽  
High Energy ◽  
Rna Degradation ◽  
T7 Rna Polymerase ◽  
Nucleotide Synthesis ◽  
Enthalpy Of Activation ◽  
Rna Polymerization ◽  
Synthetic Approaches

<p>A mechanism of nucleoside triphosphorylation would have been critical in an evolving “RNA world” to provide high-energy substrates for reactions such as RNA polymerization. However, synthetic approaches to produce ribonucleoside triphosphoates (rNTPs) have suffered from conditions such as high temperatures or high pH that lead to increased RNA degradation, as well as substrate production that cannot sustain replication. We demonstrate that cyclic trimetaphosphate (cTmp) can react with nucleosides to form rNTPs under mild, prebiotically-relevant conditions, with second-order rate constants ranging from 1.7 x 10<sup>–6</sup> to 6.5 x 10<sup>–6</sup> M<sup>–1</sup> s<sup>–1</sup>. The ATP reaction shows a linear dependence on pH and Mg<sup>2+</sup>, and an enthalpy of activation of 88 ± 4 kJ/mol. At millimolar nucleoside and cTmp concentrations, the rNTP production rate is sufficient to facilitate RNA synthesis by both T7 RNA polymerase and a polymerase ribozyme. We suggest that the optimized reaction of cTmp with nucleosides may provide a viable connection between prebiotic nucleotide synthesis and RNA replication.</p>

scholarly journals RNA Back and Forth: Looking through Ribozyme and Viroid Motifs

Viruses ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 283 ◽  
Author(s):  
Marie-Christine Maurel ◽  
Fabrice Leclerc ◽  
Jacques Vergne ◽  
Giuseppe Zaccai
Keyword(s):  
Rna World ◽  
Hammerhead Ribozyme ◽  
Intramolecular Interactions ◽  
Modern World ◽  
Widespread Occurrence ◽  
Living Fossils ◽  
Evolution Of Life ◽  
Catalytically Active ◽  
World Hypothesis ◽  
Rna World Hypothesis

Current cellular facts allow us to follow the link from chemical to biochemical metabolites, from the ancient to the modern world. In this context, the “RNA world” hypothesis proposes that early in the evolution of life, the ribozyme was responsible for the storage and transfer of genetic information and for the catalysis of biochemical reactions. Accordingly, the hammerhead ribozyme (HHR) and the hairpin ribozyme belong to a family of endonucleolytic RNAs performing self-cleavage that might occur during replication. Furthermore, regarding the widespread occurrence of HHRs in several genomes of modern organisms (from mammals to small parasites and elsewhere), these small ribozymes have been regarded as living fossils of a primitive RNA world. They fold into 3D structures that generally require long-range intramolecular interactions to adopt the catalytically active conformation under specific physicochemical conditions. By studying viroids as plausible remains of ancient RNA, we recently demonstrated that they replicate in non-specific hosts, emphasizing their adaptability to different environments, which enhanced their survival probability over the ages. All these results exemplify ubiquitous features of life. Those are the structural and functional versatility of small RNAs, ribozymes, and viroids, as well as their diversity and adaptability to various extreme conditions. All these traits must have originated in early life to generate novel RNA populations.

scholarly journals Mutational Analysis of the Promoter Recognized by Chlamydia and Escherichia coli σ28 RNA Polymerase

2006 ◽  
Vol 188 (15) ◽  
pp. 5524-5531 ◽  
Author(s):  
Hilda Hiu Yin Yu ◽  
Elizabeth G. Di Russo ◽  
Megan A. Rounds ◽  
Ming Tan
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Mutational Analysis ◽  
Promoter Sequence ◽  
Promoter Element ◽  
Developmental Gene ◽  
Spacer Length ◽  
E Coli ◽  
Promoter Recognition ◽  
Developmental Gene Regulation

ABSTRACT σ28 RNA polymerase is an alternative RNA polymerase that has been postulated to have a role in developmental gene regulation in Chlamydia. Although a consensus bacterial σ28 promoter sequence has been proposed, it is based on a relatively small number of defined promoters, and the promoter structure has not been systematically analyzed. To evaluate the sequence of the σ28-dependent promoter, we performed a comprehensive mutational analysis of the Chlamydia trachomatis hctB promoter, testing the effect of point substitutions on promoter activity. We defined a −35 element recognized by chlamydial σ28 RNA polymerase that resembles the consensus −35 sequence. Within the −10 element, however, chlamydial σ28 RNA polymerase showed a striking preference for a CGA sequence at positions −12 to −10 rather than the longer consensus −10 sequence. We also observed a strong preference for this CGA sequence by Escherichia coli σ28 RNA polymerase, suggesting that this previously unrecognized motif is the critical component of the −10 promoter element recognized by σ28 RNA polymerase. Although the consensus spacer length is 11 nucleotides (nt), we found that σ28 RNA polymerase from both Chlamydia and E. coli transcribed a promoter with either an 11- or 12-nt spacer equally well. Altogether, we found very similar results for σ28 RNA polymerase from C. trachomatis and E. coli, suggesting that promoter recognition by this alternative RNA polymerase is well conserved among bacteria. The preferred σ28 promoter that we defined in the context of the hctB promoter is TAAAGwwy-n11/12-ryCGAwrn, where w is A or T, r is a purine, y is a pyrimidine, n is any nucleotide, and n11/12 is a spacer of 11 or 12 nt.

Nucleic acids: function and potential for abiogenesis

2017 ◽  
Vol 50 ◽  
Author(s):  
Falk Wachowius ◽  
James Attwater ◽  
Philipp Holliger
Keyword(s):  
Nucleic Acids ◽  
Prebiotic Chemistry ◽  
Molecular Level ◽  
Rna World ◽  
Information Storage ◽  
Molecular Systems ◽  
Rna Molecules ◽  
Functional Potential ◽  
Self Replication ◽  
Uniform Composition

AbstractThe emergence of functional cooperation between the three main classes of biomolecules – nucleic acids, peptides and lipids – defines life at the molecular level. However, how such mutually interdependent molecular systems emerged from prebiotic chemistry remains a mystery. A key hypothesis, formulated by Crick, Orgel and Woese over 40 year ago, posits that early life must have been simpler. Specifically, it proposed that an early primordial biology lacked proteins and DNA but instead relied on RNA as the key biopolymer responsible not just for genetic information storage and propagation, but also for catalysis, i.e. metabolism. Indeed, there is compelling evidence for such an ‘RNA world’, notably in the structure of the ribosome as a likely molecular fossil from that time. Nevertheless, one might justifiably ask whether RNA alone would be up to the task. From a purely chemical perspective, RNA is a molecule of rather uniform composition with all four bases comprising organic heterocycles of similar size and comparable polarity and pKa values. Thus, RNA molecules cover a much narrower range of steric, electronic and physicochemical properties than, e.g. the 20 amino acid side-chains of proteins. Herein we will examine the functional potential of RNA (and other nucleic acids) with respect to self-replication, catalysis and assembly into simple protocellular entities.

scholarly journals Deep sequencing of nonenzymatic RNA primer extension

2020 ◽  
Author(s):  
Daniel Duzdevich ◽  
Christopher E. Carr ◽  
Jack W. Szostak
Keyword(s):  
Sample Preparation ◽  
Deep Sequencing ◽  
Rna World ◽  
Primer Extension ◽  
Proof Of Concept ◽  
Sequencing Data ◽  
Experimental Conditions ◽  
Custom Software ◽  
Key Features ◽  
Self Replication

ABSTRACTLife emerging in an RNA world is expected to propagate RNA as hereditary information, requiring some form of primitive replication without enzymes. Nonenzymatic template-directed RNA primer extension is a model of the polymerisation step in this posited form of replication. The sequence space accessed by primer extension dictates potential pathways to self-replication and, eventually, ribozymes. Which sequences can be accessed? What is the fidelity of the reaction? Does the recently-illuminated mechanism of primer extension affect the distribution of sequences that can be copied? How do sequence features respond to experimental conditions and prebiotically relevant contexts? To help answer these and related questions, we here introduce a deep-sequencing methodology for studying RNA primer extension. We have designed and vetted special RNA constructs for this purpose, honed a protocol for sample preparation and developed custom software that sorts and analyses raw sequencing data. We apply this new methodology to proof-of-concept controls, and demonstrate that it works as expected and reports on key features of the sequences accessed by primer extension.

scholarly journals RNA Back and Forth: Looking Through Ribozyme and Viroid Motifs

2019 ◽  
Author(s):  
Marie-Christine Maurel ◽  
Fabrice Leclerc ◽  
Jacques Vergne ◽  
Giuseppe Zaccai
Keyword(s):  
Rna World ◽  
Hammerhead Ribozyme ◽  
Intramolecular Interactions ◽  
Modern World ◽  
Hairpin Ribozyme ◽  
Living Fossils ◽  
Evolution Of Life ◽  
Catalytically Active ◽  
World Hypothesis ◽  
Rna World Hypothesis

Current cellular facts allow us to follow the link from chemical to biochemical metabolites, from the ancient to the modern world. In this context, the "RNA world" hypothesis proposes that early in the evolution of life, the ribozyme was responsible for the storage and transfer of genetic information and for the catalysis of biochemical reactions. Accordingly, the hammerhead ribozyme (HHR) and the hairpin ribozyme, belong to a family of endonucleolytic RNAs performing self-cleavage that might occur during replication. Furthermore, regarding the ultraconserved occurrence of HHR in several genomes of modern organisms (from mammals to small parasites and elsewhere), these small ribozymes have been regarded as living fossils of a primitive RNA world. They fold into 3D structures that generally require long-range intramolecular interactions to adopt the catalytically active conformation under specific physicochemical conditions. By studying viroids as plausible remains of ancient RNA, we recently demonstrated that they replicate in non-specific hosts, emphasizing their adaptability to different environments, which enhanced their survival probability over the ages. All these results exemplify ubiquitously features of life. Those are the versatility and efficiency of small RNAs, viroids and ribozymes, as well as their diversity and adaptability to various extreme conditions. All these traits must have originated in early life to generate novel RNA populations.

scholarly journals Enzymatic RNA Production from NTPs Synthesized from Nucleosides and Trimetaphosphate

2021 ◽  
Author(s):  
Fabio Chizzolini ◽  
Alexandra Kent ◽  
Luiz F. M. Passalacqua ◽  
Andrej Lupták
Keyword(s):  
Rna Synthesis ◽  
Rna World ◽  
Rna Replication ◽  
High Energy ◽  
Rna Degradation ◽  
T7 Rna Polymerase ◽  
Nucleotide Synthesis ◽  
Enthalpy Of Activation ◽  
Rna Polymerization ◽  
Synthetic Approaches

<p>A mechanism of nucleoside triphosphorylation would have been critical in an evolving “RNA world” to provide high-energy substrates for reactions such as RNA polymerization. However, synthetic approaches to produce ribonucleoside triphosphoates (rNTPs) have suffered from conditions such as high temperatures or high pH that lead to increased RNA degradation, as well as substrate production that cannot sustain replication. We demonstrate that cyclic trimetaphosphate (cTmp) can react with nucleosides to form rNTPs under mild, prebiotically-relevant conditions, with second-order rate constants ranging from 1.7 x 10<sup>–6</sup> to 6.5 x 10<sup>–6</sup> M<sup>–1</sup> s<sup>–1</sup>. The ATP reaction shows a linear dependence on pH and Mg<sup>2+</sup>, and an enthalpy of activation of 88 ± 4 kJ/mol. At millimolar nucleoside and cTmp concentrations, the rNTP production rate is sufficient to facilitate RNA synthesis by both T7 RNA polymerase and a polymerase ribozyme. We suggest that the optimized reaction of cTmp with nucleosides may provide a viable connection between prebiotic nucleotide synthesis and RNA replication.</p>

scholarly journals Deep sequencing of non-enzymatic RNA primer extension

2020 ◽  
Vol 48 (12) ◽  
pp. e70-e70 ◽  
Author(s):  
Daniel Duzdevich ◽  
Christopher E Carr ◽  
Jack W Szostak
Keyword(s):  
Sample Preparation ◽  
Deep Sequencing ◽  
Rna World ◽  
Primer Extension ◽  
Proof Of Concept ◽  
Sequencing Data ◽  
Experimental Conditions ◽  
Custom Software ◽  
Key Features ◽  
Self Replication

Abstract Life emerging in an RNA world is expected to propagate RNA as hereditary information, requiring some form of primitive replication without enzymes. Non-enzymatic template-directed RNA primer extension is a model of the copying step in this posited form of replication. The sequence space accessed by primer extension dictates potential pathways to self-replication and, eventually, ribozymes. Which sequences can be accessed? What is the fidelity of the reaction? Does the recently illuminated mechanism of primer extension affect the distribution of sequences that can be copied? How do sequence features respond to experimental conditions and prebiotically relevant contexts? To help answer these and related questions, we here introduce a deep-sequencing methodology for studying RNA primer extension. We have designed and vetted special RNA constructs for this purpose, honed a protocol for sample preparation and developed custom software that analyzes sequencing data. We apply this new methodology to proof-of-concept controls, and demonstrate that it works as expected and reports on key features of the sequences accessed by primer extension.

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