A novel mechanism for protein-assisted group I intron splicing

Solem, Amanda; Chatterjee, Piyali; Caprara, Mark G.

doi:10.1017/s1355838202029321

Cited by 33 publications

(74 citation statements)

References 48 publications

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“…This dissociation rate is close to the rate of C-terminal fragment facilitated splicing under conditions where protein dissociation is rate limiting (k splice = 0.004 min −1 ; Fig. 4C; Solem et al 2002). Thus, the slowly dissociating species in the k off experiments likely represents the splicing competent complex.…”

Section: Maturase-active Peptidessupporting

confidence: 60%

“…As discussed elsewhere, the slow splicing species may represent a population of RNA that undergoes a slow folding step required for RNA catalysis or protein binding (Solem et al 2002). In the presence of full-length protein, 90% of the A. nidulans COB pre-RNA spliced at a rate of 2.1 min −1 (Solem et al 2002;Fig. 3).…”

Section: Properties Of the C Terminus/a Nidulans Cob Pre-rna Complexmentioning

confidence: 99%

“…4A,B). Equilibrium binding experiments for full-length I-AniI do not report the same K d as that calculated from k off /k on (∼50 pM; Solem et al 2002) for technical and mechanistic reasons, but is a rapid and useful assay to estimate relative affinities between mutant proteins or intron RNAs (see Materials and Meth- Note that the band above the 5Ј exon is generated by cleavage of an alternative 5ЈSS (U + 2 in the intron). The band above the ligated exons is generated by ligation of this alternative 5Ј exon to the 3Ј exon.…”

Section: Properties Of the C Terminus/a Nidulans Cob Pre-rna Complexmentioning

confidence: 99%

“…Finally, comparison of the threedimensional structures between a dual function maturase/ endonuclease from Aspergillus nidulans (I-AniI) with a number of other homing endonucleases shows that both types of proteins share a conserved overall topology (Bolduc et al 2003). While the mechanisms by which homing endonucleases recognize and cleave their target sequences are well established, how such proteins bind with high specificity to their intron RNA substrates and promote RNA splicing is only beginning to be uncovered (Ho and Waring 1999;Solem et al 2002).…”

Section: Introductionmentioning

confidence: 99%

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A C-terminal fragment of an intron-encoded maturase is sufficient for promoting group I intron splicing

Downing

Brady²,

Caprara³

2005

RNA

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Group I introns often encode proteins that catalyze site-specific DNA hydrolysis. Some of these proteins have acquired the ability to promote splicing of their cognate intron, but whether these two activities reside in different regions of the protein remains obscure. A crystal structure of I-AniI, a dual function intron-encoded protein, has shown that the protein has two pseudo-symmetric domains of equal size. Each domain contacts its DNA substrate on either side of two cleavage sites. As a first step to identify the RNA binding surface, the N-and C-terminal domains of I-AniI were separately expressed and tested for promoting the splicing of the mitochondrial (mt) COB pre-RNA. The N-terminal protein showed no splicing activation or RNA binding, suggesting that this domain plays a minimal role in activity or is improperly folded. Remarkably, the 16-kDa C-terminal half facilitates intron splicing with a rate similar to that of the full-length protein. Both the C-terminal fragment and full-length proteins bind tightly to the COB intron. RNase footprinting shows that the C-terminal and full-length proteins bind to the same regions and induce the same conformational changes in the COB intron. Together, these results show that the C-terminal fragment of I-AniI is necessary and sufficient for maturase activity and suggests that I-AniI acquired splicing function by utilizing a relatively small protein surface that likely represents a novel RNA binding motif. This fragment of I-AniI represents the smallest group I intron splicing cofactor described to date.

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Section: Maturase-active Peptidessupporting

confidence: 60%

Section: Properties Of the C Terminus/a Nidulans Cob Pre-rna Complexmentioning

confidence: 99%

Section: Properties Of the C Terminus/a Nidulans Cob Pre-rna Complexmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

A C-terminal fragment of an intron-encoded maturase is sufficient for promoting group I intron splicing

Downing

Brady²,

Caprara³

2005

RNA

Self Cite

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“…Finally, we emphasize that conformational reorganization occurs via Brownian motion, involving multiple bond rotations, so that all binding processes are complex, multistep processes (63)(64)(65)(66)(67)(68). For binding of large protein and RNA ligands, the mechanisms and principles described above for binding of G and G analogs may be combined: there may be multiple intermediates that can use localized and facilitated tertiary structure capture in different steps.…”

Section: Discussionmentioning

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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