Convergent donor and acceptor substrate utilization among kinase ribozymes

Abstract

Accommodation of donor and acceptor substrates is critical to the catalysis of (thio)phosphoryl group transfer, but there has been no systematic study of donor nucleotide recognition by kinase ribozymes, and there is relatively little known about the structural requirements for phosphorylating internal 2′OH. To address these questions, new self-phosphorylating ribozymes were selected that utilize ATP(gammaS) or GTP(gammaS) for 2′OH (thio)phosphorylation. Eight independent sequence families were identified among 57 sequenced isolates. Kinetics, donor nucleotide recognition and secondary structures were analyzed for representatives from each family. Each ribozyme was highly specific for its cognate donor. Competition assays with nucleotide analogs showed a remarkable convergence of donor recognition requirements, with critical contributions to recognition provided by the Watson–Crick face of the nucleobase, lesser contributions from donor nucleotide ribose hydroxyls, and little or no contribution from the Hoogsteen face. Importantly, most ribozymes showed evidence of significant interaction with one or more donor phosphates, suggesting that—unlike most aptamers—these ribozymes use phosphate interactions to orient the gamma phosphate within the active site for in-line displacement. All but one of the mapped (thio)phosphorylation sites are on unpaired guanosines within internal bulges. Comparative structural analysis identified three loosely-defined consensus structural motifs for kinase ribozyme active sites.

Lewis acid catalysis of phosphoryl transfer from a copper(II)-NTP complex in a kinase ribozyme

“…The present work details the metal ion and pH requirements for phosphoryl transfer by ribozyme K28(1-77)C. This 58 nt RNA is a variant of ribozyme K28, which was originally selected as a 126 nt species for thiophosphoryl transfer activity using GTPγS as donor (52). Ribozyme K28(1-77)C folds into a compact pseudoknot and transfers a phosphoryl group from GTP (or a thiophosphoryl group from GTPγS) onto itself at two different sites in the primary sequence (Figure 1A) (53).…”

“…Ribozyme K28(1-77)C folds into a compact pseudoknot and transfers a phosphoryl group from GTP (or a thiophosphoryl group from GTPγS) onto itself at two different sites in the primary sequence (Figure 1A) (53). The original selection and functional analysis of ribozyme K28 and its derivatives were performed in the presence of alkaline earth metal ions (Mg 2+ and Ca 2+ ), transition metal ions (Mn 2+ and Cu 2+ ) and monovalent cations (K + and Na + ) and was buffered to near neutrality with hydroxyethylpiperazine ethane sulphonate (HEPES, pH 7.5) (52). In the present work, we sought to determine the contributions of each of these components to RNA-catalyzed phosphoryl transfer.…”

Potent Inhibition of HIV-1 Reverse Transcriptase and Replication by Nonpseudoknot, “UCAA-motif” RNA Aptamers

“…Secondary structures in solution were assessed by enzymatic digestion as described. 29 For each reaction, 50,000–200,000 cpm of 5′ radiolabeled RNA was digested under native conditions at 37 °C with ribonuclease T1 (0.005 U/µl for 2 min; Ambion; Life Technologies, Grand Island, NY), or S1 nuclease (4.75 U/µl for 10 min; New England Biolabs, Ipswich, MA), or ribonuclease V1 (5 × 10 –5 U/µl for 8 min; Ambion). All reactions were quenched with equal volumes of colorless gel loading buffer (10 mol/l urea, 15 mmol/l EDTA) and quickly cooled in a dry ice/ethanol bath.…”

Assembly and activation of a kinase ribozyme

“…The recent discovery of metabolite-linked and cofactor-linked RNAs in cell extracts Kowtoniuk et al 2009) further raises the possibility that ribozymes may have important roles in metabolic transformations of small molecules in modern biology. Phosphoryl transfer is of special interest because of its ubiquitous role in cellular biology, and phosphoryl transfer ribozymes (and deoxyribozymes) have been isolated and described by us (Rhee and Burke 2004;Saran et al 2005Saran et al , 2006Biondi et al 2010) and by others Szostak 1994, 1995;Li and Breaker 1999;Wang et al 2002;Achenbach et al 2005;Curtis and Bartel 2005;Chiuman and Li 2006;Li 2007, 2008). Nevertheless, there is still relatively little known about the mechanisms by which nucleic acids catalyze phosphoryl transfer or the structures that they use to do so.…”