Frequent use of the same tertiary motif by self-folding RNAs.

Costa, María; Michel, François

doi:10.1002/j.1460-2075.1995.tb07111.x

Cited by 337 publications

(376 citation statements)

References 44 publications

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“…This lag was not observed with the parent ligase (13). A similar phenomenon was encountered with the Group II intron and was attributed to a Mg 2+ -induced conformation change (14). Rates were determined using the part of the curve that best fit eq 1.…”

Section: Methodsmentioning

confidence: 57%

Recognition of Nucleoside Triphosphates during RNA-Catalyzed Primer Extension

et al. 2000

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In support of the idea that certain RNA molecules might be able to catalyze RNA replication, a ribozyme was previously generated that synthesizes short segments of RNA in a reaction modeled after that of proteinaceous RNA polymerases. Here, we describe substrate recognition by this polymerase ribozyme. Altering base or sugar moieties of the nucleoside triphosphate only moderately affects its utilization, provided that the alterations do not disrupt Watson-Crick pairing to the template. Correctly paired nucleotides have both a lower K m and a higher k cat , suggesting that differential binding and orientation each play roles in discriminating matched from mismatched nucleotides. Binding of the pyrophosphate leaving group appears weak, as evidenced by a very inefficient pyrophosphate-exchange reaction, the reverse of the primer-extension reaction. Indeed, substitutions at the γ-phosphate can be tolerated, although poorly. Thio substitutions of oxygen atoms at the reactive phosphate exert effects similar to those seen with cellular polymerases, leaving open the possibility of an active site analogous to those of protein enzymes. The polymerase ribozyme, derived from an efficient RNA ligase ribozyme, can achieve the very fast k cat of the parent ribozyme when the substrate of the polymerase (GTP) is replaced by an extended substrate (pppGGA), in which the GA dinucleotide extension corresponds to the second and third nucleotides of the ligase. This suggests that the GA dinucleotide, which had been deleted when converting the ligase into a polymerase, plays an important role in orienting the 5′-terminal nucleoside. Polymerase constructs that restore this missing orientation function should achieve much more efficient and perhaps more accurate RNA polymerization.The RNA world hypothesis proposes that early life relied on catalytic RNAs rather than protein enzymes for catalysis (1). Much of the appeal of this theory comes from the notion that ribozymes, which could have served as their own genes, would have been much simpler to duplicate than proteins (2-5). Hence, a vital component of the RNA world scenario is an RNA polymerase ribozyme responsible for replicating the essential ribozymes of early life (including itself). Whether there might be RNA sequences that can promote RNA polymerization with sufficient accuracy and efficiency for RNA self-replication is one of the fundamental unanswered questions related to the RNA world hypothesis (1, 6). Finding such a sequence would support the idea of the RNA world and provide a key component for the synthesis of simple living cells (7).The class I ligase ( Figure 1A) is a promising starting point for efforts to develop a self-replicating ribozyme. This ribozyme ligates two RNAs, generating a 3′,5′-phosphodiester linkage and releasing pyrophosphate, a reaction similar to that of RNA and DNA polymerases (8). Engineered forms of the ligase can polymerize short segments of RNA ( Figure 1C). In the presence of appropriate RNA templates and nucleoside triphosphates (NTP...

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Section: Methodsmentioning

confidence: 57%

Recognition of Nucleoside Triphosphates during RNA-Catalyzed Primer Extension

et al. 2000

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“…Additionally, a GAAA tetraloop in P5abc docks into its tetraloop receptor by both cross-helical base stacking and extensive hydrogen bonding. Similar GAAA tetraloop/tetraloop receptor interactions are thought to occur in other large RNAs including group I and group II introns and ribosomal RNA (5). Thus, the P4-P6 domain crystal structure provides a basis for examining the formation and stability of tertiary interactions that characterize helix packing in RNA.…”

mentioning

confidence: 75%

Assembly of an Exceptionally Stable RNA Tertiary Interface in a Group I Ribozyme

1999

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Group I intron RNAs contain a core of highly conserved helices flanked by peripheral domains that stabilize the core structure. In the Tetrahymena group I ribozyme, the P4, P5, and P6 helices of the core pack tightly against a three-helix subdomain called P5abc. Chemical footprinting and the crystal structure of the Tetrahymena intron P4-P6 domain revealed that tertiary interactions between these two parts of the domain create an extensive solvent-inaccessible interface. We have examined the formation and stability of this tertiary interface by providing the P5abc segment in trans to a Tetrahymena ribozyme construct that lacks P5abc (E ∆P5abc ). Equilibrium gel shift experiments show that the affinity of the P5abc and E ∆P5abc RNAs is exceptionally strong, with a K d of ∼100 pM at 10 mM MgCl 2 (at 37°C). Chemical and enzymatic footprinting shows that the RNAs are substantially folded prior to assembly of the complex. Solvent accessibility mapping reveals that, in the absence of P5abc, the intron RNA maintains a nativelike fold but its active-site helices are not tightly packed. Upon binding of P5abc, the catalytic core becomes more tightly packed through indirect effects of the tertiary interface formation. This two-component system facilitates quantitative examination of individual tertiary contacts that stabilize the folded intron.Large RNA molecules, like proteins, readily form specific molecular shapes adapted for ligand binding and catalysis. Catalytic RNAs including group I and group II introns and RNase P have compact interiors, involving close association of several segments of secondary structure. Close packing of RNA helices requires extensive screening of the phosphodiester backbone by cations in solution (1, 2). Furthermore, noncanonical base pairs and hairpin loops provide distinct arrays of hydrogen bond donors and acceptors and irregular surface features that serve as recognition sites for RNA helices in the formation of higher order structures (3)(4)(5)(6)(7)(8). How tertiary contacts, charge screening, and other factors stabilize RNA helix packing is not well understood.The X-ray crystal structure of the P4-P6 domain from the Tetrahymena thermophila group I intron provided the first detailed view of an extended tertiary interface in RNA (9). In the 160 nucleotide domain, a flexible internal loop, J5/5a, allows the backbone to make a ∼180°bend, enabling parallel association of two coaxially stacked helical segments (Figure 1). One-half of the P4-P6 domain, a three-helix subdomain called P5abc, binds at two distinct locations to the other half of the domain, constituted by the coaxially stacked P5, P4, and P6 helices (Figure 1). The A-rich bulge of P5abc contacts the minor groove of helix P4 by backbone and base-mediated hydrogen bonds. Additionally, a GAAA tetraloop in P5abc docks into its tetraloop receptor by both cross-helical base stacking and extensive hydrogen bonding. Similar GAAA tetraloop/tetraloop receptor interactions are thought to occur in other large RNAs including group I...

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“…The tetraloop/tetraloop receptor motif is perhaps the most famous RNA motif. It was first identified by phylogeny in group I and II introns by Michel, Westhof, and colleagues and observed for the first time in the seminal crystal structure of the P4-P6 domain of the Tetrahymena group I intron, in which this motif reinforces the side-by-side packing architecture of RNA helices [84][85][86]. This motif and others are ubiquitous in the ribosome and other structured RNAs [87][88][89][90][91].…”

Section: Simple Forces Underlying the Complex Behavior Of Rnamentioning

confidence: 99%

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

Chu

Herschlag

2008

Current Opinion in Structural Biology

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SummaryThe rapid development of our understanding of the diverse biological roles fulfilled by non-coding RNA has motivated interest in the basic macromolecular behavior, structure, and function of RNA. We focus on two areas in the behavior of complex RNAs. First, we present advances in the understanding of how RNA folding is accomplished in vivo by presenting a mechanism for the action of DEAD-box proteins. Members of this family are intimately associated with almost all cellular processes involving RNA, mediating RNA structural rearrangements and chaperoning their folding. Next, we focus on advances in understanding and characterizing the basic biophysical forces that govern the folding of complex RNAs. Ultimately we expect that a confluence and synergy between these approaches will lead to profound understanding of RNA and its biology.The explosion in our knowledge about noncoding RNA (ncRNA) -RNA that is not translated into protein -has expanded interest in RNA beyond the traditional RNA community. Diverse ncRNAs include the recently-discovered riboswitches, whose novel gene-regulation function is induced by the binding of small metabolites [1]; most of the so-called "Human Accelerated Regions" (HAR) of the human genome, whose recent evolution may be linked to the emergence of the human brain [2]; and many ncRNAs of unknown function [3]. These discoveries underscore the need to understand the fundamental macromolecular behavior of RNA, its structure and dynamics, and how these RNAs are handled and exploited in biology.Study of catalytic RNAs, which allow detection of correct folding via activity measurements, has established basic thermodynamic and kinetic properties of RNA folding. Large catalytic RNAs such as the group I intron from Tetrahymena thermophila and the RNase P RNA from Bacillus subtilis fold at vastly different rates under different conditions upon addition of Mg 2+ and have been shown to fold via multiple parallel pathways, in which different molecules follow different routes with different rates and intermediates, to a common final folded structure [4,5]. These observations support the common view that RNAs traverse rugged energy landscapes as they fold [6,7]. However, little is known about the true shape of these

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Frequent use of the same tertiary motif by self-folding RNAs.

Cited by 337 publications

References 44 publications

Recognition of Nucleoside Triphosphates during RNA-Catalyzed Primer Extension

Recognition of Nucleoside Triphosphates during RNA-Catalyzed Primer Extension

Assembly of an Exceptionally Stable RNA Tertiary Interface in a Group I Ribozyme

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

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