New ligase-derived RNA polymerase ribozymes

Lawrence, Michael S.; Bartel, David P.

doi:10.1261/rna.2110905

Cited by 58 publications

(47 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…While a number of strategies to create such a polymerase have been explored, none has been as successful as those making use of the Class I ligase ribozyme as a core catalytic component (Bartel et al 1991;Bartel and Szostak 1993;Johnston et al 2001;McGinness et al 2002;Lawrence and Bartel 2005). This ribozyme, originally isolated from a high diversity random sequence pool of RNA, is one of the fastest known ligase ribozymes (Bergman et al 2000).…”

Section: Introductionmentioning

confidence: 99%

Characterization of the B6.61 polymerase ribozyme accessory domain

Wang¹,

Cheng²,

Unrau³

2011

RNA

View full text Add to dashboard Cite

The ''RNA world'' hypothesis rests on the assumption that RNA polymerase ribozymes can replicate RNA without the use of protein. In the laboratory, in vitro selection has been used to create primitive versions of such polymerases. The best variant to date is a ribozyme called B6.61 that can extend a RNA primer template by 20 nucleotides (nt). This polymerase has two domains: the recently crystallized Class I ligase core, responsible for phosphodiester bond formation, and the poorly characterized accessory domain that makes polymerization possible. Here we find that the accessory domain is specified by a 37-nt bulged stem-loop structure. The accessory domain is positioned by a tertiary interaction between the terminal AL4 loop of the accessory and the J3/4 triloop found within the ligase core. This docking interaction is associated with an unwinding of the A3 and A4 helixes that appear to facilitate the correct positioning of an essential 8-nt purine bulge found between the two helices. This, together with other constraints inferred from tethering the accessory domain to a range of sites on the ligase core, indicates that the accessory domain is draped over the vertex of the ligase core tripod structure. This geometry suggests how the purine bulge in the polymerase replaces the P2 helix in the Class I ligase with a new structure that may facilitate the stabilization of incoming nucleotide triphosphates.

show abstract

Section: Introductionmentioning

confidence: 99%

Characterization of the B6.61 polymerase ribozyme accessory domain

Wang¹,

Cheng²,

Unrau³

2011

RNA

View full text Add to dashboard Cite

show abstract

“…However, polymerization efficiency of current ribozymes is too low for self-replication: No primer extension longer than 20 nucleotides (nt) has been observed for a polymerase ribozyme (Zaher and Unrau 2007), whereas the lengths of all known polymerase ribozymes are in the range of 190 nt (Johnston et al 2001;Lawrence and Bartel 2005;Zaher and Unrau 2007). The limiting factor for polymerization efficiency is a low substrate binding affinity, with a K M in the millimolar range (Lawrence and Bartel 2003).…”

Section: Introductionmentioning

confidence: 99%

“…In addition, the effective rates of nucleotide addition are sequence dependent, differing by factors of more than 100-fold, which causes the accumulation of specific polymerization intermediates (Lawrence and Bartel 2003;Müller and Bartel 2003). All 1 of the 10 existing polymerase ribozymes (Johnston et al 2001;Lawrence and Bartel 2005;Zaher and Unrau 2007) appear to suffer from the same problem.…”

Section: Introductionmentioning

confidence: 99%

Improved polymerase ribozyme efficiency on hydrophobic assemblies

Müller

Bartel²

2008

RNA

Self Cite

View full text Add to dashboard Cite

During an early step in the evolution of life, RNA served both as genome and as catalyst, according to the RNA world hypothesis. For self-replication, the RNA organisms must have contained an RNA that catalyzes RNA polymerization. As a first step toward recapitulating an RNA world in the laboratory, a polymerase ribozyme was generated previously by in vitro evolution and design. However, the efficiency of this ribozyme is about 100-fold too low for self-replication because of a low affinity of the ribozyme to its primer/template substrate. To improve the substrate interactions by colocalizing ribozyme and substrate on micelles, we attached hydrophobic anchors to both RNAs. We show here that the hydrophobic anchors led to aggregates with the expected size of the corresponding micelles. The micelle formation increased the polymerization yield of full-length products by 3-to 20-fold, depending on substrates and reaction conditions. With the best-characterized substrate, the improvement in polymerization efficiency was primarily due to reduced sequence-specific stalling on partially extended substrates. We discuss how, during the origin of life, micellar ribozyme aggregates could have acted as precursors to membraneencapsulated life forms.

show abstract

“…Significant progress has be been made in recent years towards the construction of artificial replicase ribozymes (Ekland & Bartel, 1996;Johnston et al, 2001;McGinness & Joyce, 2003;Lawrence & Bartel, 2005). While to date, no ribozyme is known that could faithfully replicate another copy of itself, this goal seems to be within experimental reach.…”

Section: Molecular Replicatorsmentioning

confidence: 99%

“…The model of Szostak et al (Szostak et al, 2001) consists of a vesicle containing an RNA genome that contains an RNA-replicase ribozyme (e.g. an advanced version of the molecule described in (Johnston et al, 2001;Paul & Joyce, 2003;Lawrence & Bartel, 2005)) and a functionality that influences the fitness of the vesicle. The construct of Pohorille & Deamer (Pohorille & Deamer, 2002), which is even closer to a modern cell, includes transcription and translation functionalities.…”

Section: Introductionmentioning

confidence: 99%