Structure and Function Converge To Identify a Hydrogen Bond in a Group I Ribozyme Active Site

Forconi, Marcello; Sengupta, Raghuvir N.; Liu, Mao-Chin; Sartorelli, Alan C.; Piccirilli, Joseph A.; Herschlag, Daniel

doi:10.1002/ange.200903006

Cited by 8 publications

(25 citation statements)

References 23 publications

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“…1D; Stahley and Strobel 2005;Lipchock and Strobel 2008). Models for this discrepancy have been described (Discussion; see Hougland et al 2006) and we note that the remaining interactions inferred from the functional and structural data are in excellent agreement (Hougland et al 2006;Forconi et al 2008Forconi et al , 2009 …”

Section: Use Ofsupporting

confidence: 68%

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

Sengupta

Schie

Giambaşu

et al. 2015

RNA

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Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such "off-pathway" species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2 ′ -and 3 ′ -deoxy (-H) and −amino (-NH 2 ) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3 ′ -OH making a nonproductive interaction with an active site metal ion termed M A and with the adjacent 2 ′ -OH making no interaction. Upon S binding, a rearrangement occurs that allows both -OH groups to contact a different active site metal ion, termed M C , to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA's difficulty in specifying a unique conformation and highlighting Nature's potential to use local transitions of RNA in complex function.

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Section: Use Ofsupporting

confidence: 68%

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

Sengupta

Schie

Giambaşu

et al. 2015

RNA

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“…Their results implicated a subset of atoms important for guanosine binding and reactivity that was largely confirmed by subsequent structural (Adams et al 2004;Golden et al 2005;Guo et al 2005;Stahley and Strobel 2005) and functional data (Michel et al 1989;Sjogren et al 1997;Forconi et al 2009Forconi et al , 2011. However, one uncertainty has remained-regarding the role of the N7 atom of G.…”

Section: Introductionmentioning

confidence: 90%

“…The structure of the exogenous guanosine binding site in group I introns reveals apparent stacking interactions that sandwich the bound guanosine in addition to the hydro- (Michel et al 1989;Forconi et al 2009) and structural (Adams et al 2004;Golden et al 2005;Guo et al 2005;Stahley and Strobel 2005) data. (B) The same interactions are present in the Azoarcus group I ribozyme structural model 3BO3.…”

Section: Duplex Stabilities Of the Analogs Correlate With The Observementioning

confidence: 99%

“…This ability, coupled with an initial dearth of structural information, led to extensive early structure-function studies of RNAs (see Waring 1989;Christian and Yarus 1992;Sontheimer et al 1997;Strobel and Shetty 1997;Blount and Uhlenbeck 2005;Das and Piccirilli 2005;Hougland et al 2005;Soukup and Soukup 2009;Chirkova et al 2010, and references therein). The power of these approaches to identify atomic-level interactions has been confirmed by subsequent X-ray structural models, and the functional studies have at times resolved uncertainties from structural models and, more generally, have greatly extended our understanding of the energetic and functional properties of these RNAs (e.g., Blount and Uhlenbeck 2005;Hougland et al 2006;Forconi et al 2009).…”

Section: Introductionmentioning

confidence: 98%

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Exploring purine N7 interactions via atomic mutagenesis: The group I ribozyme as a case study

Forconi¹,

Benz-Moy²,

Gleitsman³

et al. 2012

RNA

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Atomic mutagenesis has emerged as a powerful tool to unravel specific interactions in complex RNA molecules. An early extensive study of analogs of the exogenous guanosine nucleophile in group I intron self-splicing by Bass and Cech demonstrated structure-function relationships analogous to those seen for protein ligands and provided strong evidence for a wellformed substrate binding site made of RNA. Subsequent functional and structural studies have confirmed these interacting sites and extended our understanding of them, with one notable exception. Whereas 7-methyl guanosine did not affect reactivity in the original study, a subsequent study revealed a deleterious effect of the seemingly more conservative 7-deaza substitution. Here we investigate this paradox, studying these and other analogs with the more thoroughly characterized ribozyme derived from the Tetrahymena group I intron. We found that the 7-deaza substitution lowers binding by~20-fold, relative to the cognate exogenous guanosine nucleophile, whereas binding and reaction with 7-methyl and 8-aza-7-deaza substitutions have no effect. These and additional results suggest that there is no functionally important contact between the N7 atom of the exogenous guanosine and the ribozyme. Rather, they are consistent with indirect effects introduced by the N7 substitution on stacking interactions and/or solvation that are important for binding. The set of analogs used herein should be valuable in deciphering nucleic acid interactions and how they change through reaction cycles for other RNAs and RNA/protein complexes.

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“…[41c, 127,129] Group II introns and the spliceosome: An atomic mutation cycle similar to the one described above for group I introns has also been applied to the spliced exons reopening reaction of group II introns.…”

Section: Group I Intronsmentioning

confidence: 99%

Understanding Catalysis of Phosphate‐Transfer Reactions by the Large Ribozymes

Lönnberg

2011

Chemistry A European J

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Large ribozymes are unique among catalytic RNA molecules in that their reactions involve intermolecular nucleophilic attack on an RNA phosphodiester linkage. Crystal structures of near-atomic resolution are now available for the group I and group II self-splicing introns and the RNA subunit of RNase P. The structural data agrees well with the earlier models proposed on the basis of biochemical studies and the evidence at hand suggests that all of the large ribozymes utilize a mechanism in which coordination of Mg(II) ions reduces the negative charge on the scissile phosphodiester linkage, as well as assists both the nucleophilic attack and the departure of the leaving group.

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Structure and Function Converge To Identify a Hydrogen Bond in a Group I Ribozyme Active Site

Cited by 8 publications

References 23 publications

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

Exploring purine N7 interactions via atomic mutagenesis: The group I ribozyme as a case study

Understanding Catalysis of Phosphate‐Transfer Reactions by the Large Ribozymes

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