Group I Ribozymes: Substrate Recognition, Catalytic Strategies, and Comparative Mechanistic Analysis

Cech, Thomas R.; Herschlag, Daniel

doi:10.1007/978-3-642-61202-2_1

Cited by 59 publications

(35 citation statements)

References 87 publications

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“…Remarkably, deletion of P5abc has only a small effect on the overall folding rate to the native state, demonstrating that this element is not required for the slower folding steps to proceed ef®ciently. We also found that under conditions used in this and several previous folding studies, most of the ribozyme population becomes trapped in an inactive (Cech et al, 1994). Paired structural elements are labeled with the abbreviations P1-P9.…”

Section: Introductionsupporting

confidence: 55%

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New pathways in folding of the Tetrahymena group I RNA enzyme

Russell

Herschlag

1999

Journal of Molecular Biology

103

146

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Section: Introductionsupporting

confidence: 55%

“…The ribozyme uses an exogenous guanosine (G) as a nucleophile, transferring the 3 H portion of an oligonucleotide substrate that mimics the 5 H splice site (S) to give a shorter oligonucleotide product (P) and GA 5 (equation (1) and Figure 2(a); reviewed by Cech & Herschlag, 1997;Narlikar & Herschlag, 1997):…”

Section: Resultsmentioning

confidence: 99%

New pathways in folding of the Tetrahymena group I RNA enzyme

Russell

Herschlag

1999

Journal of Molecular Biology

103

146

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“…Oligonucleotides were made by solid phase synthesis and were supplied by the Protein and Nucleic Acid Facility at Stanford University or were gifts from Dr. L. Beigelman (Ribozyme Pharmaceuticals Inc.). Oligonucleotide substrates were 5′-end-labeled using [γ- 32 P]ATP and T4 polynucleotide kinase and purified by electrophoresis on 24% nondenaturing polyacrylamide gels, as described previously (37). 2′-Aminoguanosine was a gift from Dr. F. Eckstein.…”

Section: Methodsmentioning

confidence: 99%

“…1 This ribozyme catalyzes the transesterification reaction shown in eq 1, in which an exogenous guanosine nucleophile (G) cleaves a specific phosphodiester bond of the oligonucleotide substrate (S) to generate a shorter oligonucleotide product (P; [30][31][32].…”

mentioning

confidence: 99%

Protonated 2‘-Aminoguanosine as a Probe of the Electrostatic Environment of the Active Site of the Tetrahymena Group I Ribozyme

1999

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We have probed the electrostatic environment of the active site of the Tetrahymena group I ribozyme (E) using protonated 2′-aminoguanosine (G NH 3 +), in which the 2′-OH of the guanosine nucleophile (G) is replaced by an -NH 3 + group. At low concentrations of divalent metal ion (2 mM Mg 2+ ), G NH 3 + binds at least 200-fold stronger than G or G NH 2 , with a dissociation constant of e1 µM from the ribozyme‚oligonucleotide substrate‚G NH 3 + complex (E‚S‚G NH 3 +). This strong binding suggests that the -NH 3 + group interacts with negatively charged phosphoryl groups within the active site. Increasing the concentration of divalent metal ion weakens the binding of G NH 3 + to E‚S more than 10 2 -fold. The Mn 2+ concentration dependence suggests that M C , the metal ion that interacts with the 2′-moiety of G in the normal reaction, is responsible for this effect. M C and G NH 3 + compete for binding to the active site; this competition could arise from electrostatic repulsion between the positively charged -NH 3 + and M C and, possibly, from their competition for interaction with active site phosphoryl groups. The reactive phosphoryl group of S increases the competition between M C and G NH 3 +, consistent with a network of interactions involving M C that help position the reactive phosphoryl group and the guanosine nucleophile with respect to one another. The chemical step with bound G NH 3 + is at least 10 4 -fold slower than with G or G NH 2 . These results provide additional support for an integral role of M C in catalysis by the Tetrahymena ribozyme, and demonstrate the utility of the -NH 3 + moiety as an electrostatic probe within a structured RNA.An RNA enzyme can utilize a variety of functional groups that differ in charge and polarity for building its active site and interacting with its substrates. The backbone of RNA is composed of negatively charged phosphoryl groups; this results in repulsive interactions, but also creates numerous potential binding sites for metal ions and other positively charged groups (e.g., 2-15 and references cited therein). The 2′-hydroxyl groups and functional groups on the bases of RNA also provide hydrogen bond donors and acceptors that can interact with each other, with metal ions, and with polar groups on bound ligands (e.g., 3, 4, 6, 8, 9, 11-14, 16-21, and references cited therein). The ring moieties of the bases are nonpolar, creating the potential for hydrophobic interactions with each other and with bound ligands (e.g., 19, 21, and references cited therein).The energetic effects of electrostatic interactions between functional groups within an RNA also depend on the ability of the RNA to position these groups and to limit active site rearrangements. The Tetrahymena group I ribozyme makes extensive interactions with its substrates, which has been suggested to allow precise positioning of substrates and catalytic groups within this RNA active site (22-25 and references cited therein). The interactions within the ribozyme core presumably form an interconnecte...

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“…All group I ribozymes in nature are coded by group I introns, and most of them carry out reactions leading to excision of intron RNA and ligation of flanking exons (Cech & Herschlag, 1996)+ Group I ribozymes are characterized by a common secondary structure of about 10 paired segments (P1-P10), which include the 120-nt catalytic core, and usually one or more optional segments (P11-P17) (Lehnert et al+, 1996)+ Three-dimensional structure models of the core have been proposed based on comparative sequence and mutation analyses (Michel & Westhof, 1990) and have been updated recently to include new structural information (Jaeger et al+, 1996a;Lehnert et al+, 1996)+ The core consists of two juxtaposed principal domains, P4-P6 (P4, P5, and P6) and P3-P8 (P3, P7, and P8), which are connected by single-stranded regions and a base triple (Michel & Westhof, 1990;Cech et al+, 1994;Doudna & Cech, 1995;Tanner & Cech, 1997;Tanner et al+, 1997)+ The P3-P8 domain contains the P7 guanosine binding site, which is an essential component of the catalytic site+ In the Tetrahymena intron, P4-P6 is able to self-assemble (Doudna & Cech, 1995), and its structure recently has been determined and characterized from RNA crystals (Cate et al+, 1996)+ P4-P6 directs the tertiary folding of P3-P8 (Doherty & Doudna, 1997) and subsequently creates a cleft between the principal domains where the more flexible substrate duplex is located+ Group I introns are classified into at least 11 subgroups within four main groups (IA, IB, IC, and ID), based on distinct primary sequence motifs and characteristic structural features in peripheral regions (Michel & Westhof, 1990;Jaeger et al+, 1996a)+ The peripheral regions are divergent both in sequence and structure, and several of them are known to be important for stabilizing the catalytic core structure by tertiary interactions (Jaeger et al+, 1991;Michel & Shub, 1992;Lehnert et al+, 1996)+ Complete 3D structure models of group I splicing-ribozymes have been proposed recently (Lehnert et al+, 1996) and show that most peripheral regions appear to be projected away from the catalytic core+ In about one third of all known group I splicing-ribozymes, one of the peripheral regions harbors an open reading frame (ORF)+ The two nucleolar introns DiSSU1 and NaSSU1 from the distantly related protists Didymium iridis and some Naegleria species, respectively, contain complex peripheral insertions in regular group I splicing-ribozymes+ These insertions consist of a small group I-like ribozyme (GIR1), followed by an ORF …”

Section: Introductionmentioning

confidence: 99%

Group I-like ribozymes with a novel core organization perform obligate sequential hydrolytic cleavages at two processing sites

Einvik

Nielsen

Westhof

et al. 1998

RNA

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A new category of self-splicing group I introns with conserved structural organization and function is found among the eukaryotic microorganisms Didymium and Naegleria. These complex rDNA introns contain two distinct ribozymes with different functions: a regular group I splicing-ribozyme and a small internal group I-like ribozyme (GIR1), probably involved in protein expression. GIR1 was found to cleave at two internal sites in an obligate sequential order. Both sites are located 39 of the catalytic core. GIR1-catalyzed transesterification reactions could not be detected. We have compared all available GIR1 sequences and propose a common RNA secondary structure resembling that of group I splicing-ribozymes, but with some important differences. The GIR1s lack most peripheral sequence components, as well as a P1 segment, and, at approximately 160-190 nt, they are the smallest functional group I ribozymes known from nature. All GIR1s were found to contain a novel 6-bp pseudoknot (P15) within their catalytic core region. Experimental support of the proposed structure was obtained from the Didymium GIR1 by RNA structure probing and site-directed mutagenesis. Three-dimensional modeling indicates a compactly folded ribozyme with the functionally essential P15 exposed in the cleft between the two principal domains P3-P8 and P4-P6.

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Group I Ribozymes: Substrate Recognition, Catalytic Strategies, and Comparative Mechanistic Analysis

Cited by 59 publications

References 87 publications

New pathways in folding of the Tetrahymena group I RNA enzyme

New pathways in folding of the Tetrahymena group I RNA enzyme

Protonated 2‘-Aminoguanosine as a Probe of the Electrostatic Environment of the Active Site of the Tetrahymena Group I Ribozyme

Group I-like ribozymes with a novel core organization perform obligate sequential hydrolytic cleavages at two processing sites

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