A map of the binding site for catalytic domain 5 in the core of a group II intron ribozyme

Konforti, Boyana; Liu, Qiaolian; Pyle, Anna Marie

doi:10.1093/emboj/17.23.7105

Cited by 26 publications

(31 citation statements)

References 44 publications

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“…RT-AMV (Roche) was used to map the location of cross-links, following standard RT mapping procedures (Stern et al 1988;Konforti et al 1998b;Juzumiene et al 2001). Several DNA primers complementary to different regions of the RNA were used to scan the entire intron.…”

Section: Rt Mapping Of Cross-linksmentioning

confidence: 99%

Three essential and conserved regions of the group II intron are proximal to the 5′-splice site

Lencastre¹,

Pyle²

2007

RNA

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Despite the central role of group II introns in eukaryotic gene expression and their importance as biophysical and evolutionary model systems, group II intron tertiary structure is not well understood. In order to characterize the architectural organization of intron ai5g, we incorporated the photoreactive nucleotides s 4 U and s 6 dG at specific locations within the intron core and monitored the formation of cross-links in folded complexes. The resulting data reveal the locations for many of the most conserved, catalytically important regions of the intron (i.e., the J2/3 linker region, the IC1(i-ii) bulge in domain 1, the bulge of D5, and the 59-splice site), showing that all of these elements are closely colocalized. In addition, we show by nucleotide analog interference mapping (NAIM) that a specific functional group in J2/3 plays a role in first-step catalysis, which is consistent with its apparent proximity to other first-step components. These results extend our understanding of active-site architecture during the first step of group II intron self-splicing and they provide a structural basis for spliceosomal comparison.

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Section: Rt Mapping Of Cross-linksmentioning

confidence: 99%

Three essential and conserved regions of the group II intron are proximal to the 5′-splice site

Lencastre¹,

Pyle²

2007

RNA

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“…To identify the essential residues within the QR domain, we performed S1 nuclease mapping, Pb 2ϩ cleavage (Krzyzosiak et al+, 1988;Behlen et al+, 1990;Pan et al+, 1991), and chemical modifications using 1-cyclohexyl-3-(2-morpholinomethyl)carbodiimide metho-ptoluenesulfonate (CMCT; Moazed et al+, 1986;Green et al+, 1997), DMS (Murphy & Cech, 1994;Konforti et al+, 1998), and kethoxal (Moazed et al+, 1986) on the AD02 ribozyme+ The S1 and Pb 2ϩ cleavage probe the accessibility of 29-OH and 39-bridging phosphate on ribose ring, whereas the chemical modifications explore the accessibility of N1 of purine and N3 of pyrim-idine+ The structural effects of 59-glutaminylation were also investigated to provide insight into the amino acid binding site+ S1 nuclease cleavage, Pb 2ϩ -induced cleavage, and the chemical modifications were observed in the singlestranded regions of our proposed secondary structure (Fig+ 2A,B)+ These results together with compensatory mutations in the L6b-IGS (Lee et al+, 2000) and the P5 stem (Y+ Bessho & H+ Suga, unpubl+ results) have affirmed the secondary structure+ When AD02 was aminoacylated, some bases in the P6b-L6b region showed distinct chemical modification profiles from those observed before the aminoacylation (Fig+ 2A,C)+ U125 is particularly noteworthy: The Pb 2ϩ -induced cleavage of cleavage by 59-glutaminylation, respectively+ B: Summary of structural probing by chemical modifications using CMCT (square), DMS (circle), and kethoxal (arrowhead)+ Filled squares, circles, and arrowheads denote strong modification, and opened symbols denote mild modification+ The filled circle on A116 and the opened circles on G107, G112, and A114 indicate strong or mild protection of chemical modification upon 59-glutaminylation, respectively+ The filled square on U125 indicates the CMCT modification only upon 59-glutaminylation+ C: Chemical modifications of P6b-L6b region+ Bases discussed in the text are highlighted by arrowheads+ Two time points were taken for each reagent; 1 and 3 h for Pb 2ϩ ; 5 and 30 min for CMCT, DMS, and kethoxal+ that a dynamic change in the pairing configuration of A111:U125 occurs upon 59-glutaminylation, but only U125 bulges out from the strand (more insight into the roles of this base pair is discussed in the section below)+ Because the interaction of L6b and IGS brings the P6b region into close proximity to the 59-OH nucleophile (Fig+ 1A), these findings provide a strong argument for the glutamine binding site being located near the A111:U125 pair+…”

Section: Nuclease and Chemical Modification Mappingmentioning

confidence: 99%

A minihelix-loop RNA acts as a trans-aminoacylation catalyst

Lee

Suga

2001

RNA

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We previously reported a bifunctional ribozyme that catalyzes self-aminoacylation and subsequent acyl-transfer to a tRNA. The ribozyme selectively recognizes a biotinyl-glutamine substrate, and charges the tRNA molecule in trans. Structurally, there are two catalytic domains, referred to as glutamine-recognition (QR) and acyl-transferase (ATRib). We report here the essential catalytic core of the QR domain as determined by extensive biochemical probing, mutation, and structural minimization. The minimal core of the QR domain is a 29-nt helix-loop RNA, which is also able to glutaminylate ATRib in trans. Its amino acid binding site is embedded in an 11-nt cluster that is adjacent to the loop that interacts with the ATRib domain. Our study shows that a minihelix-loop RNA can act as a trans-aminoacylation catalyst, which lends support for the critical role of minihelix-loops in the early evolution of the aminoacylation system.

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“…The group I, group II, and HDV ribozymes as well as tRNA (Ϸ400, 600, 90, and 76 nt, respectively) fold cooperatively, form structures with solvent protected cores, and appear to have active site clefts akin to those observed in protein enzymes (19)(20)(21)(22)(23)(24)(25)(26). Thus, it was suggested that larger RNAs, like proteins, could preform ligand-binding sites.…”

mentioning

confidence: 99%

Extraordinarily slow binding of guanosine to the Tetrahymena group I ribozyme: Implications for RNA preorganization and function

Karbstein

Herschlag

2003

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

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The Tetrahymena ribozyme derived from the self-splicing group I intron binds a 5 -splice site analog (S) and guanosine (G), catalyzing their conversion to a 5 -exon analog (P) and GA. Herein, we show that binding of guanosine is exceptionally slow, limiting the reaction at near neutral pH. Our results implicate a conformational rearrangement on guanosine binding, likely because the binding site is not prearranged in the absence of ligand. The fast accommodation of guanosine (10 2 to 10 3 ⅐s ؊1 ) and prior structural data suggest local rather than global rearrangements, raising the possibility that folding of this and perhaps other large RNAs is not fully cooperative. Guanosine binding is accelerated by addition of residues that form helices, referred to as P9.0 and P10, immediately 5 and 3 to the guanosine. These rate enhancements provide evidence for binding intermediates that have the adjacent helices formed before accommodation of guanosine into its binding site. Because the ability to form the P9.0 and P10 helices distinguishes the guanosine at the correct 3 -splice site from other guanosine residues, the faster binding of the correct guanosine can enhance specificity of 3 -splice site selection. Thus, paradoxically, the absence of a preformed binding site and the resulting slow guanosine binding can contribute to splicing specificity by providing an opportunity for the adjacent helices to increase the rate of binding of the guanosine specifying the 3 -splice site.I n 1894, Fischer proposed that biological recognition could be likened to the fit between a key and lock (1). More than a century later, this view has been refined to incorporate dynamic aspects of protein structure and function. Indeed, nearly all enzymes appear to undergo some motion in the course of catalysis, often in the form of a loop or domain closure that enhances binding interactions (2). More extensive conformational reordering can occur in allosteric or signaling proteins, and recently extreme examples of proteins that may have unfolded resting states have been identified (3,4). Whereas the modern view of protein structure encompasses these dynamics, a large database of protein structures with and without bound ligand suggests that binding sites are largely preformed in most cases and that closure, if it occurs, provides additional binding interactions (e.g., refs. 5-11).In contrast, structural studies with many RNAs reveal extensive local reordering of the structure on ligand binding. These conformational transitions include changes in hydrogen bonding as well as base stacking to accommodate the bound ligand; e.g., binding of the arginine ligand to Tar RNA induces formation of a base triple and unstacking of residues in an internal loop to allow interaction with the arginine (12-14). The frequent observation of such structural changes on ligand binding to the characterized RNAs (Յ40 nt) has led to the notion that small RNAs have great difficulty in preforming ligand-binding sites (refs. 15-18 and refs. therein).Several observations sug...

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A map of the binding site for catalytic domain 5 in the core of a group II intron ribozyme

Cited by 26 publications

References 44 publications

Three essential and conserved regions of the group II intron are proximal to the 5′-splice site

Three essential and conserved regions of the group II intron are proximal to the 5′-splice site

A minihelix-loop RNA acts as a trans-aminoacylation catalyst

Extraordinarily slow binding of guanosine to the Tetrahymena group I ribozyme: Implications for RNA preorganization and function

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