An important base triple anchors the substrate helix recognition surface within the Tetrahymena ribozyme active site

Abstract

Key to understanding the structural biology of catalytic RNA is determining the underlying networks of interactions that stabilize RNA folding, substrate binding, and catalysis. Here we demonstrate the existence and functional importance of a Hoogsteen base triple (U300⅐A97-U277), which anchors the substrate helix recognition surface within the Tetrahymena group I ribozyme active site. Nucleotide analog interference suppression analysis of the interacting functional groups shows that the U300⅐A97-U277 triple forms part of a network of hydrogen bonds that connect the P3 helix, the J8͞7 strand, and the P1 substrate helix. Product binding and substrate cleavage kinetics experiments performed on mutant ribozymes that lack this base triple (C A-U, U G-C) or replace it with the isomorphous C ؉ ⅐G-C triple show that the A97 Hoogsteen triple contributes to the stabilization of both substrate helix docking and the conformation of the ribozyme's active site. To accomplish substrate helix docking and catalysis (1, 2), the Tetrahymena ribozyme ( Fig. 1) must fold into a complex tertiary structure with a close-packed catalytic core (3) in which several structural elements come together to jointly recognize and interact with the P1 substrate helix. These elements include the J4͞5 internal loop (4, 5), the J8͞7 strand (6, 7), and the attacking guanosine nucleophile bound within the P7 helix (8). This complex interaction of multiple closepacked helical and single-stranded RNA elements (9) requires several layers of interactions to create the active site of the molecule. The molecular details of these interactions and the underlying principles of RNA tertiary structure stabilization and substrate recognition within the Tetrahymena ribozyme can be readily investigated from a functional perspective by using the techniques of nucleotide analog interference mapping (NAIM) and nucleotide analog interference suppression (NAIS) (7, 10).Recently, the results of NAIM and NAIS experiments to identify the array of hydrogen bonding interactions that stabilize the interaction between the P1 substrate helix and the highly conserved J8͞7 strand within the intron core were reported (7, 11). These data, along with other biochemical structural information (4-6, 12, 13), were used as restraints in building a model of a large section of the ribozyme active site.In conjunction with a G⅐U wobble receptor in the J4͞5 region (4, 5), the J8͞7 strand creates a complementary surface for recognition of the P1 substrate helix (6, 7). The data indicate that the J8͞7 strand forms an extended minor groove triple helical interaction with four 2Ј-OH groups within the 5Ј-exon (P1) helix of the intron. This docking interface includes a tertiary interaction between the 2Ј-oxygen of U300 at the 5Ј-end of J8͞7 and the 2Ј-OH of G26 within the P1 helix. However, the details of the interactions between J8͞7 and the rest of the intron's core remain largely unknown.The single-stranded J8͞7 segment is a particularly critical element of the group I active site. Its ...

Crystal structure of a group I intron splicing intermediate

“…This is not likely to be due to differences between the two introns, because biochemical data suggest that the tertiary contacts to the 5Ј-exon are equivalent (Soukup et al 2002). Significant changes in the J8/7 conformation from that reported in the apoenzyme structure are also consistent with biochemical studies (Szewczak et al 1999). …”

“…This subtle, but important, feature is what distinguishes the IC1 (Tetrahymena) and IC3 (Azoarcus) intron subclasses (Michel and Westhof 1990). Biochemical data suggest that the additional Tetrahymena J8/7 residue (TetU300) makes the same tertiary contact to the substrate helix at TetG26 as is observed between Azoarcus J2/3 residue A39 and C14 (Szewczak et al 1999). Both of these residues are located 6 bp below the 5Ј-splice site in the substrate helix.…”

Comparison of crystal structure interactions and thermodynamics for stabilizing mutations in the Tetrahymena ribozyme

“…1B; Guo et al 2004). In a previous study, U300 was shown to form a base triple interaction with the A97-U277 base pair in the wild-type Tetrahymena ribozyme (Szewczak et al 1999). The interaction between U300 and A97 found in our crystal structure was identical to the one proposed based on biochemical data.…”

Exploration of the Transition State for Tertiary Structure Formation between an RNA Helix and a Large Structured RNA

“…68 Prominent long-range tertiary contacts are indicated by broken lines. 51,52,119 (b) A model for P1 docking shown in the context of the tertiary structure model of the ribozyme. 52 Colors are as for (a).…”