Characterization of the Azoarcus ribozyme: tight binding to guanosine and substrate by an unusually small group I ribozyme

Kuo, Louis Y.; Davidson, Leslie A.; Pico, Stacy

doi:10.1016/s0167-4781(99)00200-6

Cited by 39 publications

(44 citation statements)

References 36 publications

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“…We designed and constructed a ribozyme from the minimal active form of the Neurospora mtLSU intron (38) by eliminating the 5Ј and 3Ј exons (39)(40)(41) (Fig. 15A, which is published as supporting information on the PNAS web site).…”

Section: Cyt-19-mediated Unwinding Of the P1 Duplex Is Inhibited By Tmentioning

confidence: 99%

Nonspecific binding to structured RNA and preferential unwinding of an exposed helix by the CYT-19 protein, a DEAD-box RNA chaperone

Tijerina

Bhaskaran

Russell

2006

Proc. Natl. Acad. Sci. U.S.A.

163

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We explore the interactions of CYT-19, a DExD͞H-box protein that functions in folding of group I RNAs, with a well characterized misfolded species of the Tetrahymena ribozyme. Consistent with its function, CYT-19 accelerates refolding of the misfolded RNA to its native state. Unexpectedly, CYT-19 performs another reaction much more efficiently; it unwinds the 6-bp P1 duplex formed between the ribozyme and its oligonucleotide substrate. Furthermore, CYT-19 performs this reaction 50-fold more efficiently than it unwinds the same duplex free in solution, suggesting that it forms additional interactions with the ribozyme, most likely using a distinct RNA binding site from the one responsible for unwinding. This site can apparently bind double-stranded RNA, as attachment of a simple duplex adjacent to P1 recapitulates much of the activation provided by the ribozyme. Unwinding the native P1 duplex does not accelerate refolding of the misfolded ribozyme, implying that CYT-19 can disrupt multiple contacts on the RNA, consistent with its function in folding of multiple RNAs. Further experiments showed that the P1 duplex unwinding activity is virtually the same whether the ribozyme is misfolded or native but is abrogated by formation of tertiary contacts between the P1 duplex and the body of the ribozyme. Together these results suggest a mechanism for CYT-19 and other general DExD͞H-box RNA chaperones in which the proteins bind to structured RNAs and efficiently unwind loosely associated duplexes, which biases the proteins to disrupt nonnative base pairs and gives the liberated strands an opportunity to refold.group I RNA ͉ RNA folding ͉ RNA unwinding ͉ Tetrahymena ribozyme E ssentially all cellular processes that are mediated by structured RNAs also require one or more DExD͞H-box proteins (1). These proteins use the energy from ATP binding and hydrolysis to accelerate RNA structural transitions, which can represent folding steps toward the native state or conformational switches between functional forms. The requirement for proteins presumably arises because RNA base pairs and other local structure can be highly stable even in the absence of enforcing structure, such that folding steps or rearrangements that require significant unfolding require assistance to proceed efficiently (2-4).Despite their ubiquitous presence, key questions about the functions of DExD͞H-box proteins remain largely unanswered. First, what interactions direct different DExD͞H-box proteins to their physiological substrates? All of these proteins share a core ''helicase'' domain containing a set of conserved motifs, and most have additional domains, a few of which have been shown to recognize substrate RNAs or RNA-protein complexes (reviewed in ref. 5). On this basis, targeting has been proposed as a general role for these domains, and the specific interactions that target one DExD͞H-box protein have been delineated (6-9). Nevertheless, in general, the interactions that direct DExD͞H box proteins to their substrates remain to be identified.Second, h...

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Section: Cyt-19-mediated Unwinding Of the P1 Duplex Is Inhibited By Tmentioning

confidence: 99%

Nonspecific binding to structured RNA and preferential unwinding of an exposed helix by the CYT-19 protein, a DEAD-box RNA chaperone

Tijerina

Bhaskaran

Russell

2006

Proc. Natl. Acad. Sci. U.S.A.

163

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“…The Azoarcus intron is of the same group IC subclass as the more extensively investigated Tetrahymena intron, and both introns appear to follow an equivalent reaction mechanism (Reinhold-Hurek and Shub 1992;Tanner and Cech 1996;Kuo et al 1999;Kuo and Piccirilli 2001). The bacterial intron retains the same core secondary structural elements, i.e., helices P1 through P10 and the joiner segments (J) that connect the helices, yet it is the smallest known group I ribozyme due primarily to a lack of peripheral domains and the absence of an open reading frame.…”

Section: Introductionmentioning

confidence: 99%

Crystal structure of a group I intron splicing intermediate

Adams

Stahley²,

Gill³

et al. 2004

RNA

116

133

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A recently reported crystal structure of an intact bacterial group I self-splicing intron in complex with both its exons provided the first molecular view into the mechanism of RNA splicing. This intron structure, which was trapped in the state prior to the exon ligation reaction, also reveals the architecture of a complex RNA fold. The majority of the intron is contained within three internally stacked, but sequence discontinuous, helical domains. Here the tertiary hydrogen bonding and stacking interactions between the domains, and the single-stranded joiner segments that bridge between them, are fully described. Features of the structure include: (1) A pseudoknot belt that circumscribes the molecule at its longitudinal midpoint; (2) two tetraloop-tetraloop receptor motifs at the peripheral edges of the structure; (3) an extensive minor groove triplex between the paired and joiner segments, P6-J6/6a and P3-J3/4, which provides the major interaction interface between the intron's two primary domains (P4-P6 and P3-P9.0); (4) a six-nucleotide J8/7 single stranded element that adopts a µ-shaped structure and twists through the active site, making critical contacts to all three helical domains; and (5) an extensive base stacking architecture that realizes 90% of all possible stacking interactions. The intron structure was validated by hydroxyl radical footprinting, where strong correlation was observed between experimental and predicted solvent accessibility. Models of the pre-first and pre-second steps of intron splicing are proposed with full-sized tRNA exons. They suggest that the tRNA undergoes substantial angular motion relative to the intron between the two steps of splicing.

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“…The group I self-splicing reaction has been studied in the greatest depth with shortened ribozyme constructs derived from the self-splicing intron ( (1,5,8,20,21,30,(35)(36)(37), see also: (38,39)). The most commonly studied of these constructs is the L-21 ScaI ribozyme ( Figure 1B).…”

mentioning

confidence: 99%

Probing the Role of a Secondary Structure Element at the 5‘- and 3‘-Splice Sites in Group I Intron Self-Splicing: The Tetrahymena L-16 ScaI Ribozyme Reveals a New Role of the G·U Pair in Self-Splicing

2007

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Several ribozyme constructs have been used to dissect aspects of the group I self-splicing reaction. The Tetrahymena L-21 ScaI ribozyme, the best studied of these intron analogs, catalyzes a reaction analogous to the first step of self-splicing, in which a 5′-splice site analog (S) and guanosine (G) are converted into a 5′-exon analog (P) and GA. This ribozyme preserves the active site but lacks a short 5′-terminal segment (called the IGS extension herein) that forms dynamic helices, called the P1 extension and P10 helix. The P1 extension forms at the 5′-splice site in the first step of self-splicing and P10 forms at the 3′-splice site in the second step of self-splicing. To dissect the contributions from the IGS extension and the helices it forms we have investigated the effects of each of these elements at each reaction step. These experiments were performed with the L-16 ScaI ribozyme, which retains the IGS extension, and with 5′-and 3′-splice site analogs that differ in their ability to form the helices. The presence of the IGS extension strengthens binding of P by 40-fold, even when no new base pairs are formed. This large effect was especially surprising, as binding of S is essentially unaffected for S analogs that do not form additional base pairs with the IGS extension. Analysis of a U•U pair immediately 3′ to the cleavage site suggests that a previously identified deleterious effect from a dangling U residue on the L-21 ScaI ribozyme arises from a fortuitous active site interaction and has implications for RNA tertiary structure specificity. Comparisons of the affinities of 5′-splice site analogs that form only a subset of base pairs reveal that inclusion of the conserved G•U base pair at the cleavage site of group I introns destabilizes the P1 extension >100-fold relative to the stability of a helix with all Watson-Crick base pairs. Previous structural data with model duplexes and the recent intron structures suggest that this effect can be attributed to partial unstacking of the P1 extension at the G•U step. These results suggest a previously unrecognized role of the G•U wobble pair in self-splicing: breaking cooperativity in base pair formation between P1 and the P1 extensions. This effect may facilitate replacement of the P1 extension with P10 after the first chemical step of self-splicing and release of the ligated exons after the second step of self-splicing.The group I intron from Tetrahymena thermophila, the first catalytic RNA to be discovered, folds into a specific structure and performs a self-splicing reaction that includes chemical steps as well as RNA conformational rearrangements. This reaction is simple, relative to pre-mRNA † This work was supported by NIH grant GM49243. K.K. was supported in part by a predoctoral fellowship from the Boehringer Ingelheim NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript splicing and other RNA-mediated processes, and may serve as a tractable system to learn more about RNA catalysis and its functional conformational change...

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Characterization of the Azoarcus ribozyme: tight binding to guanosine and substrate by an unusually small group I ribozyme

Cited by 39 publications

References 36 publications

Nonspecific binding to structured RNA and preferential unwinding of an exposed helix by the CYT-19 protein, a DEAD-box RNA chaperone

Nonspecific binding to structured RNA and preferential unwinding of an exposed helix by the CYT-19 protein, a DEAD-box RNA chaperone

Crystal structure of a group I intron splicing intermediate

Probing the Role of a Secondary Structure Element at the 5‘- and 3‘-Splice Sites in Group I Intron Self-Splicing: The Tetrahymena L-16 ScaI Ribozyme Reveals a New Role of the G·U Pair in Self-Splicing

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