Metal Ion Requirements for Structure and Catalysis of an RNA Ligase Ribozyme

Abstract

The class I ligase, a ribozyme previously isolated from random sequence, catalyzes a reaction similar to RNA polymerization, positioning its 5′-nucleotide via a Watson-Crick base pair, forming a 3′,5′-phosphodiester bond between its 5′-nucleotide and the substrate, and releasing pyrophosphate. Like most ribozymes, it requires metal ions for structure and catalysis. Here, we report the ionic requirements of this self-ligating ribozyme. 2+ and Co(NH 3 ) 6 3+ inhibit by binding at least two sites, but they appear to productively fill a subset of the required sites. Inhibition is not the result of a significant structural change, since the ribozyme assumes a nativelike structure when folded in the presence of Ca 2+ or Co(NH 3 ) 6 3+ , as observed by hydroxyl-radical mapping. As further support for a nativelike fold in Ca 2+ , ribozyme that has been prefolded in Ca 2+ can carry out the self-ligation very quickly upon the addition of Mg 2+ . Ligation rates of the prefolded ribozyme were directly measured and proceed at 800 min -1 at pH 9.0.Metal ions are essential for the activity of most ribozymes, functioning in electrostatic shielding, structure, and directly in catalysis (1). Natural ribozymes vary widely in their metal ion specificity, from the large ribozymes (group I intron, group II intron, and RNase P 1 ), which require a divalent metal, preferably Mg 2+ , for activity, to the promiscuous selfcleaving ribozymes (hammerhead, hairpin, VS, and HDV), which are active in many different cations (2-13). Hammerhead, hairpin, and VS ribozymes are even active in nonmetal ions such as NH 4 + (12, 13). Ribozymes selected in vitro also exhibit a wide range of metal ion requirements. Some, like the ribonucleolytic leadzyme, are active only in the cations in which they were selected (14-16). Others exhibit activity in the presence of metal ions which were not present during their selection. For example, an acyl transferase ribozyme, which was selected in the presence of Mg 2+ and K + only, is active in a wide variety of cations, including Co(NH 3 ) 6 3+ (17, 18).The class I ligase, which was also selected in the presence of only Mg 2+ and K + , performs a reaction similar to RNA polymerization, positioning its 5′-nucleotide via a WatsonCrick base pair, forming a 3′,5′-phosphodiester bond between its 5′-nucleotide and the substrate, and releasing pyrophosphate (Figure 1) (19). In fact, a version of this ribozyme can act as a general RNA polymerase, accurately extending a primer up to 14 nucleotides (20). Not only does the ligase perform the same reaction as naturally occurring RNA and DNA polymerases, but the stereospecificity of its preference for sulfur substitutions at the nonbridging oxygens on the R-phosphate matches that of these enzymes, raising the possibility that a catalytic mechanism common to all polymerases extends to ribozyme-mediated polymerization as well (21,22). The ligase is also one of the fastest ribozymes, among both natural ribozymes and those selected in vitro (23). These characteristics r...

A class I ligase ribozyme with reduced Mg2+ dependence: Selection, sequence analysis, and identification of functional tertiary interactions

“…In principle, a metal rescue experiment could lend support to the role of some of these oxygens as inner-sphere ligands (Piccirilli et al 1993). However, the ligase is strongly inhibited by the transition metals that are the best candidates for metal rescue (Glasner et al 2002); pilot experiments confirmed that this line of inquiry was unlikely to be productive (data not shown). Instead, the improved ligase crystal structure (Shechner et al 2009) provided a framework for interpreting the interference maps.…”

“…Buffers were the same as described for selection. KCl (200 mM) was included in initial experiments, but omitted in experiments measuring the Mg 2+ -dependence of ligation, and in all reactions in which the concentration of Mg 2+ was <10 mM, because at low Mg 2+ concentration it inhibits the parent ligase (Glasner et al 2002). Omitting KCl did not affect rates of either parent or clones at $10 mM Mg 2+ .…”

“…At 60 mM Mg 2+ and pH 8.0, the cis-acting construct b1-207 reacts with k c = 300 min À1 (Glasner et al 2002), while the trans-acting construct b1-207t reaches k c = 375 min À1 for the multiple turnover reaction (Bergman et al 2000). For the latter construct, k cat /K M is 7 3 10 7 M À1 min À1 , just over an order of magnitude shy of the diffusion-controlled limit.…”

“…For the latter construct, k cat /K M is 7 3 10 7 M À1 min À1 , just over an order of magnitude shy of the diffusion-controlled limit. As fast as class I ligase catalysis is, however, the chemical step remains slower than the folding reaction at pH #7 (Glasner et al 2002). Thus, we decided to target the chemical step for improvement by using a rapid-quench flow apparatus to isolate variants that react rapidly at moderate-to-low pH.…”

Zeptomole detection of a viral nucleic acid using a target-activated ribozyme

“…Although in principle this approach is general to large ribozymes that use extensive Watson-Crick base pairs, we developed it using the extensively characterized Class I ligase motif of Bartel and colleagues Bergman et al 2000;Glasner et al 2000Glasner et al , 2002.…”