A single adenosine with a neutral pKa in the ribosomal peptidyl transferase center.

Biochemical and crystallographic evidence suggests that 23S ribosomal RNA (rRNA) is the catalyst of peptide bond formation. To explore the mechanism of this reaction, we screened for nucleotides in Escherichia coli 23S rRNA that may have a perturbed pKa (where Ka is the acid constant) based on the pH dependence of dimethylsulfate modification. A single universally conserved A (number 2451) within the central loop of domain V has a near neutral pKa of 7.6 +/- 0.2, which is about the same as that reported for the peptidyl transferase reaction. In vivo mutational analysis of this nucleotide indicates that it has an essential role in ribosomal function. These results are consistent with a mechanism wherein the nucleotide base of A2451 serves as a general acid base during peptide bond formation.

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

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 (U300.A97-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 U300.A97-U277 triple forms part of a network of hydrogen bonds that connect the P3 helix, the J8/7 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(+).G-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. The U300. A97-U277 base triple is not formed in the recently reported crystallographic model of a portion of the group I intron, despite the presence of J8/7 and P3 in the RNA construct [Golden, B. L., Gooding, A. R., Podell, E. R. & Cech, T. R. (1998) Science 282, 259-264]. This, along with other biochemical evidence, suggests that the active site in the crystallized form of the ribozyme is not fully preorganized and that substantial rearrangement may be required for substrate helix docking and catalysis.

A hydrogen-bonding triad stabilizes the chemical transition state of a group I ribozyme.

BACKGROUND: The group I intron is an RNA enzyme capable of efficiently catalyzing phosphoryl-transfer reactions. Functional groups that stabilize the chemical transition state of the cleavage reaction have been identified, but they are all located within either the 5'-exon (P1) helix or the guanosine cofactor, which are the substrates of the reaction. Functional groups within the ribozyme active site are also expected to assist in transition-state stabilization, and their role must be explored to understand the chemical basis of group I intron catalysis. RESULTS: Using nucleotide analog interference mapping and site-specific functional group substitution experiments, we demonstrate that the 2'-OH at A207, a highly conserved nucleotide in the ribozyme active site, specifically stabilizes the chemical transition state by approximately 2 kcal mol-1. The A207 2'-OH only makes its contribution when the U(-1) 2'-OH immediately adjacent to the scissile phosphate is present, suggesting that the 2'-OHs of A207 and U(-1) interact during the chemical step. CONCLUSIONS: These data support a model in which the 3'-oxyanion leaving group of the transesterification reaction is stabilized by a hydrogen-bonding triad consisting of the 2'-OH groups of U(-1) and A207 and the exocyclic amine of G22. Because all three nucleotides occur within highly conserved non-canonical base pairings, this stabilization mechanism is likely to occur throughout group I introns. Although this mechanism utilizes functional groups distinctive of RNA enzymes, it is analogous to the transition states of some protein enzymes that perform similar phosphoryl-transfer reactions.

A minor groove RNA triple helix within the catalytic core of a group I intron.

Close packing of several double helical and single stranded RNA elements is required for the Tetrahymena group I ribozyme to achieve catalysis. The chemical basis of these packing interactions is largely unknown. Using nucleotide analog interference suppression (NAIS), we demonstrate that the P1 substrate helix and J8/7 single stranded segment form an extended minor groove triple helix within the catalytic core of the ribozyme. Because each triple in the complex is mediated by at least one 2'-OH group, this substrate recognition triplex is unique to RNA and is fundamentally different from major groove homopurine-homopyrimidine triplexes. We have incorporated these biochemical data into a structural model of the ribozyme core that explains how the J8/7 strand organizes several helices within this complex RNA tertiary structure.

Identifying RNA minor groove tertiary contacts by nucleotide analogue interference mapping with N2-methylguanosine.

Nucleotide analogue interference mapping (NAIM) is a general biochemical method that rapidly identifies the chemical groups important for RNA function. In principle, NAIM can be extended to any nucleotide that can be incorporated into an in vitro transcript by an RNA polymerase. Here we report the synthesis of 5'-O-(1-thio)-N2-methylguanosine triphosphate (m2GalphaS) and its incorporation into two reverse splicing forms of the Tetrahymena group I intron using a mutant form of T7 RNA polymerase. This analogue replaces one proton of the N2 exocyclic amine with a methyl group, but is as stable as guanosine (G) for secondary structure formation. We have identified three sites of m2GalphaS interference within the Tetrahymena intron: G22, G212, and G303. All three of these guanosine residues are known to utilize their exocyclic amino groups to participate in tertiary hydrogen bonds within the ribozyme structure. Unlike the interference pattern with the phosphorothioate of inosine (IalphaS, an analogue that deletes the N2 amine of G), m2GalphaS substitution did not cause interference at positions attributable to secondary structural stability effects. Given that the RNA minor groove is likely to be widely used for helix packing, m2GalphaS provides an especially valuable reagent to identify RNA minor groove tertiary contacts in less well-characterized RNAs.

The chemical basis of adenosine conservation throughout the Tetrahymena ribozyme.

Adenosines are present at a disproportionately high frequency within several RNA structural motifs. To explore the importance of individual adenosine functional groups for group I intron activity, we performed Nucleotide Analog Interference Mapping (NAIM) with a collection of adenosine analogues. This paper reports the synthesis, transcriptional incorporation, and the observed interference pattern throughout the Tetrahymena group I intron for eight adenosine derivatives tagged with an alpha-phosphorothioate linkage for use in NAIM. All of the analogues were accurately incorporated into the transcript as an A. The sites that interfere with the 3'-exon ligation reaction of the Tetrahymena intron are coincident with the sites of phylogenetic conservation, yet the interference patterns for each analogue are different. These interference data provide several biochemical constraints that improve our understanding of the Tetrahymena ribozyme structure. For example, the data support an essential A-platform within the J6/6a region, major groove packing of the P3 and P7 helices, minor groove packing of the P3 and J4/5 helices, and an axial model for binding of the guanosine cofactor. The data also identify several essential functional groups within a highly conserved single-stranded region in the core of the intron (J8/7). At four sites in the intron, interference was observed with 2'-fluoro A, but not with 2'-deoxy A. Based upon comparison with the P4-P6 crystal structure, this may provide a biochemical signature for nucleotide positions where the ribose sugar adopts an essential C2'-endo conformation. In other cases where there is interference with 2'-deoxy A, the presence or absence of 2'-fluoro A interference helps to establish whether the 2'-OH acts as a hydrogen bond donor or acceptor. Mapping of the Tetrahymena intron establishes a basis set of information that will allow these reagents to be used with confidence in systems that are less well understood.

Complementary sets of noncanonical base pairs mediate RNA helix packing in the group I intron active site.

Helix packing is critical for RNA tertiary structure formation, although the rules for helix-helix association within structured RNAs are largely unknown. Docking of the substrate helix into the active site of the Tetrahymena group I ribozyme provides a model system to study this question. Using a novel chemogenetic method to analyze RNA structure in atomic detail, we report that complementary sets of noncanonical base pairs (a G.U wobble pair and two consecutively stacked sheared A.A pairs) create an RNA helix packing motif that is essential for 5'-splice site selection in the group I intron. This is likely to be a general motif for helix-helix interaction within the tertiary structures of many large RNAs.