A specific monovalent metal ion integral to the AA platform of the RNA tetraloop receptor

Abstract

Metal ions are essential for the folding and activity of large catalytic RNAs. While divalent metal ions have been directly implicated in RNA tertiary structure formation, the role of monovalent ions has been largely unexplored. Here we report the first specific monovalent metal ion binding site within a catalytic RNA. As seen crystallographically, a potassium ion is coordinated immediately below AA platforms of the Tetrahymena ribozyme P4-P6 domain, including that within the tetraloop receptor. Interference and kinetic experiments demonstrate that potassium ion binding within the tetraloop receptor stabilizes the folding of the P4-P6 domain and enhances the activity of the Azoarcus group I intron. Since a monovalent ion binding site is integral to the tetraloop receptor, a tertiary structural motif that occurs frequently in RNA, monovalent metal ions are likely to participate in the folding and activity of a wide diversity of RNAs.

Role of metal ions in the tetraloop–receptor complex as analyzed by NMR

“…These data are also in agreement with recent FRET results of a tethered tetraloop-receptor complex that docks in the presence of potassium (K 1/2 = 200 mM) without divalent cations (Downey et al 2006). Since crystal structures have revealed several ions closely associated with the tetraloop receptor Basu et al 1998;Golden et al 1998; FIGURE 1. Sequence and structure of the tetraloop-receptor RNA construct used in these studies.…”

“…The manganese associated below the AA platform (Fig. 7, site 2) is in the vicinity of a potassium binding site (Basu et al 1998), but is closer to the electron density observed by Juneau et al (2001). The NMR data suggest a divalent ion can localize to the AA platform, although the NMR observed manganese ions are on average 6.9 Å away from the potassium binding site.…”

“…The second area of manganese association is just below the AA platform in the receptor, in the same region previously shown to bind potassium (Fig. 5C, site 2; Basu et al 1998). The third region is also in the receptor at the tandem G-C pairs, and was observed in the crystal structure as a directly coordinated magnesium ion (Fig.…”

“…Previous NMR data were obtained in the presence of 9 mM magnesium, and no complex formation was detected at low concentration (<100 mM) monavalent ions. X-ray crystallography of group I introns has revealed three metal ions associated with the bound tetraloopreceptor complex, including: (1) a magnesium directly coordinated to the G250 phosphate oxygen within the receptor Juneau et al 2001;Adams et al 2004a,b); (2) a monovalent ion coordinated to the guanosine immediately below the adenosine platform nucleotides Basu et al 1998), and (3) a cobalt hexammine binding site at the tandem G-U wobble pairs in the major groove of the tetraloop stem Juneau et al 2001). EPR studies have revealed a magnesium dependence for tetraloop docking to the receptor (Qin et al 2005).…”

Characterization of Metal Ion-Nucleic Acid Interactions in Solution

“…These monovalent ions are far less apt to establish a close packing of the negative charges of the nucleic acid backbone. Usually a largely unspecific role as general charge screeners is attributed to K + and Na + , even if there are also examples of specific monovalent ion binding sites ( Figure 2) [58,59]. 2+ and K + Na + and Ca 2+ levels in the cell are lower than those of Mg 2+ and K + .…”