Processivity of Ribozyme-Catalyzed RNA Polymerization
Abstract
The "RNA world" hypothesis proposes that early in the evolution of life, before the appearance of DNA or protein, RNA was responsible both for encoding genetic information and for catalyzing biochemical reactions. Ribo-organisms living in the RNA world would have replicated their RNA genomes by using an RNA polymerase ribozyme. Efforts to provide experimental support for the RNA world hypothesis have focused on producing such a polymerase, and in vitro evolution methods have led to the isolation of a polymerase ribozyme that catalyzes primer extension which is accurate and general, but slow. To understand the reaction of this ribozyme, we developed a method of measuring polymerase processivity that is particularly useful in the case of an inefficient polymerase. This method allowed us to demonstrate that the polymerase ribozyme, despite its inefficiency, is partially processive. It is currently limited by a low affinity for the primer-template duplex, but once it successfully binds the primer-template duplex in the productive alignment, it catalyzes an extension reaction that is so rapid that it can occur multiple times during the short span of a single binding event. This finding contributes to the understanding of one of the more sophisticated activities yet to be generated de novo in the laboratory and sheds light on the parameters to be targeted for further optimization.
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RPRs generally depend upon tethering to template due to their poor affinity for primer/template duplex.
“Due to their poor affinity for primer/template duplex (Lawrence and Bartel, 2003), RPRs generally depend upon such tethering to template (Attwater et al., 2010; Wochner et al., 2011; Horning and Joyce, 2016), which enhances local ribozyme concentration and promotes formation of the RPR-primer/template holoenzyme (Attwater et al., 2010; Attwater et al., 2013a).”
From Ribozyme-catalysed RNA synthesis using triplet building blocks (2018) · cites this paper as Lawrence and Bartel, 2003
The ribozyme has a catalytic rate for NTP addition of at least 1.5 min-1, but its Km is so high that it requires about 2 h to complete each NTP addition under certain conditions.
“The ribozyme has a catalytic rate for NTP addition of at least 1.5 min -1 , but its K m is so high that, even in the presence of micromolar concentrations of oligonucleotides and millimolar concentrations of NTPs, it requires about 2 h to complete each NTP addition”
From Protocells and RNA Self-Replication (2018) · cites this paper as Lawrence and Bartel 2003
R18 is not capable of self-replication because its synthetic capacity is limited, partly by weak primer/template duplex binding, and because it is unstable in the high Mg2+ concentration needed for polymerase activity.
“However, the activity of R18 falls short of that required for self-replication in both its limited synthetic capacity (exacerbated by a low affinity for substrate primer/template duplex14) as well as its poor stability in the presence of the high concentration of Mg2+ ions (200 mM) required for polymerase activity9,15,16.”
From In-ice evolution of RNA polymerase ribozyme activity (2013) · cites this paper as [14]
Cites
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“9, placing it among lowprocessivity proteinaceous polymerases such as yeast polymeraseη (12).”
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“The G93A and C97A mutations that provide the main cold-specific activity boost are located in the single-stranded linker region of the ribozyme between the catalytic core (1-90) and the processivity domain (105-187).”
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- Authors
- Lawrence, M. S., & Bartel, D. P.
- Affiliations
- Whitehead Institute for Biomedical Research (US)
- Corresponding author
- Bartel, David P.
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2003-07-01
- Volume
- 42
- Issue
- 29
- Pages
- 8748-8755
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- OpenAlex
- W2020781311
- Cited by
- 62
- References
- 3
- Citations by year
- 2026: 1 · 2025: 3 · 2024: 1 · 2023: 1 · 2022: 2 · 2021: 5 · 2020: 1 · 2019: 1 · 2018: 3 · 2017: 1 · 2016: 1 · 2015: 2 · 2014: 2 · 2013: 1
- FWCI
- 2.11
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institute of General Medical Sciences (GM61835)
- Topics
- Physical Sciences › Physics and Astronomy › Astronomy and Astrophysics › Origins and Evolution of Life
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Evolution and Genetic Dynamics
- Keywords
- RNA world, polymerase processivity, primer extension, in vitro evolution, RNA replication, catalytic efficiency, RNA catalysis
- MeSH terms
- Catalysis, Kinetics, Nucleic Acid Conformation, Protein Binding, RNA, DNA-Directed RNA Polymerases, Time Factors, RNA, Catalytic
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