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Structure-guided engineering of the regioselectivity of RNA ligase ribozymes.

ABSTRACT: Ribozyme-catalyzed RNA synthesis is central to the RNA world hypothesis. No natural RNA polymerase ribozymes have been discovered. However, ribozymes that catalyze the requisite chemistry, generating a new phosphodiester through attack of a terminal hydroxyl of an RNA on the alpha-phosphate of a triphosphate-activated oligonucleotide, have been isolated by in vitro selection. These experiments often yield ribozymes that generate 2'-5' phosphodiesters rather than conventional 3'-5' linkages. We have determined crystal structures of the duplex formed by the template segment of a representative 2'-5' RNA ligase ribozyme, the class II ligase, and its ligation product. The structures reveal a product-template duplex with a G x A pair at the ligation junction. This sheared pair is flanked on one side by a minor groove-broadening wedge comprised of two unpaired nucleotides. The reported structure of an independently isolated 3'-5' ligase ribozyme, the L1 ligase, shows a product-template duplex that shares the G x A pair with the class II ligase. However, this G x A pair is flanked by G x U wobbles, rather than an unpaired wedge. We demonstrate that these structural differences of the substrate-template duplexes are largely responsible for the divergent regioselectivity of the two ribozymes, independent of their catalytic moieties, by constructing chimeras. The L1 ligase with a class II substrate-template duplex shows a 30-fold increase in 2'-5' bond synthesis, while the class II ligase with an L1 substrate-template duplex produces 3'-5' bonds exclusively. These results demonstrate how local geometry inherent to the substrate-template duplexes controls the regioselectivity of ribozyme-catalyzed RNA ligation reactions.

SUBMITTER: Pitt JN

PROVIDER: S-EPMC2678027 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Structure-guided engineering of the regioselectivity of RNA ligase ribozymes.

Pitt Jason N JN Ferré-D'Amaré Adrian R AR

Journal of the American Chemical Society 20090301 10

Ribozyme-catalyzed RNA synthesis is central to the RNA world hypothesis. No natural RNA polymerase ribozymes have been discovered. However, ribozymes that catalyze the requisite chemistry, generating a new phosphodiester through attack of a terminal hydroxyl of an RNA on the alpha-phosphate of a triphosphate-activated oligonucleotide, have been isolated by in vitro selection. These experiments often yield ribozymes that generate 2'-5' phosphodiesters rather than conventional 3'-5' linkages. We h ...[more]

PMID: 19220054

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Project description:BACKGROUND: RNA ligases are essential reagents for many methods in molecular biology including NextGen RNA sequencing. To prevent ligation of RNA to itself, ATP independent mutant ligases, defective in self-adenylation, are often used in combination with activated pre-adenylated linkers. It is important that these ligases not have de-adenylation activity, which can result in activation of RNA and formation of background ligation products. An additional useful feature is for the ligase to be active at elevated temperatures. This has the advantage or reducing preferences caused by structures of single-stranded substrates and linkers. RESULTS: To create an RNA ligase with these desirable properties we performed mutational analysis of the archaeal thermophilic RNA ligase from Methanobacterium thermoautotrophicum. We identified amino acids essential for ATP binding and reactivity but dispensable for phosphodiester bond formation with 5' pre-adenylated donor substrate. The motif V lysine mutant (K246A) showed reduced activity in the first two steps of ligation reaction. The mutant has full ligation activity with pre-adenylated substrates but retained the undesirable activity of deadenylation, which is the reverse of step 2 adenylation. A second mutant, an alanine substitution for the catalytic lysine in motif I (K97A) abolished activity in the first two steps of the ligation reaction, but preserved wild type ligation activity in step 3. The activity of the K97A mutant is similar with either pre-adenylated RNA or single-stranded DNA (ssDNA) as donor substrates but we observed two-fold preference for RNA as an acceptor substrate compared to ssDNA with an identical sequence. In contrast, truncated T4 RNA ligase 2, the commercial enzyme used in these applications, is significantly more active using pre-adenylated RNA as a donor compared to pre-adenylated ssDNA. However, the T4 RNA ligases are ineffective in ligating ssDNA acceptors. CONCLUSIONS: Mutational analysis of the heat stable RNA ligase from Methanobacterium thermoautotrophicum resulted in the creation of an ATP independent ligase. The K97A mutant is defective in the first two steps of ligation but retains full activity in ligation of either RNA or ssDNA to a pre-adenylated linker. The ability of the ligase to function at 65°C should reduce the constraints of RNA secondary structure in RNA ligation experiments.

Dataset Information

Structure-guided engineering of the regioselectivity of RNA ligase ribozymes.

Publications

Structure-guided engineering of the regioselectivity of RNA ligase ribozymes.

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