Investigation of Overhauser effects between pseudouridine and water protons in RNA helices

Abstract

B y 1950, it was known that RNA extracted from cells contained a small percentage of a fifth base, thought to be 5-methylcytosine (1). Later, this fifth nucleoside, which accounted for Ϸ4% of nucleotides in yeast transfer (t)RNA, was identified as 5-ribosyluracil, an isomer of uridine (U) (2-5). Pseudouridine (abbreviated ) since has been found to be the most abundant modified base in tRNA, ribosomal RNA, and small nuclear (sn)RNA. Modification of U to , catalyzed by a site-specific pseudouridylase, involves scission of the nucleoside's glycosidic bond, followed by rotation of the base about its 3-6 axis, and reattachment through the carbon at the 5-position of the ring to form the only natural nucleoside with a COC base-sugar bond. The resulting modified base, which can form a Watson-Crick base pair with adenine, features an additional ring nitrogen atom that is protonated at physiological pH (NH1; Fig. 1; reviewed in ref. 6).The presence of this modified base has been associated with an increase in thermal stability of secondary structure (7) and has been shown to decrease the motion of neighboring bases in molecular dynamics simulations (8), which may explain its prevalence in structural RNAs. For example, are clustered within the peptidyl transferase center of the ribosome (9), are conserved within regions of snRNAs that are involved in RNA-RNA interactions (10), and have been implicated in spliceosome assembly (11).Although the presence of generally is not associated with major structural change (reviewed in ref. 6), we recently have found a major exception in the eukaryotic splicing apparatus: a highly conserved in the region of U2 snRNA that pairs with the intron to form the precursor (pre)-mRNA branch-site helix induces a dramatic change in conformation and modest increase in thermal stability, as compared with its unmodified analogue (12,36). The presence of results in extrusion of an adenosine, the 2ЈOH of which is the nucleophile in the first step of splicing, from the helix into a position that may facilitate its role in splicing catalysis. Such studies emphasize the biological importance of modified bases and may describe a means by which RNA-mediated chemical activity is enabled through sitespecific, posttranscriptional modification.The mechanism by which stabilizes (and, in the case of the pre-mRNA branch site, modifies) local RNA architecture remains unclear. Stabilization of RNA secondary structure by has been hypothesized to result from additional hydrogen bonds involving the NH1 (13) or from more favorable stacking interactions between and neighboring bases (14). Corroborating the first hypothesis is the observation of water-mediated hydrogen bonds between the NH1 proton and phosphate oxygen atoms in crystal structures of tRNA molecules (ref. 15; Fig. 2) and molecular dynamics simulations (8). Water-mediated hydrogen bonds involving NH1 have been postulated to stabilize -containing helices in solution (13, 16), but no direct evidence for their existence has been obtained. Experiments supportin...

Post-transcriptional modifications modulate conformational dynamics in human U2–U6 snRNA complex

“…2 ′ -O-methylations can alter the structural stabilization of U2 stem I by blocking sugar edge interactions and by reducing the reactivity of the RNA backbone (Auffinger and Westhof 1997;Helm 2006). In the case of pseudouridine, this may result from improved base stacking (Davis 1995) and from formation of a water-mediated hydrogen bond between the additional protonated imino group and adjacent phosphate residues, as reported in functionally important RNAs including tRNAs (Arnez and Steitz 1994;Newby et al 2002a).…”

A novel unanticipated type of pseudouridine synthase with homologs in bacteria, archaea, and eukarya

“…Nevertheless, considering the wide distribution of ⌿13 in tRNA (see below), its location at the end of a base-paired stem connected by a loop, and the known propensity of ⌿ for structural stabilization (Meroueh et al 2000;Newby and Greenbaum 2002, and references therein), it might be that certain tRNAs, like E. coli tRNA Glu , require this particular stabilization. Analysis of the database of sequenced tRNAs (Sprinzl and Vassilenko 2002) reveals a suggestive relationship between the distribution of U or ⌿ at position 13 and the nature of the putatively base-paired residue at position 22.…”

pH-dependent structural changes of helix 69 from Escherichia coli 23S ribosomal RNA

“…The crystal structure of tRNA Gln complexed with Gln-tRNA synthetase revealed a unique hydrogen bond between CN1H and a water molecule coordinated to the 59-phosphate group of C and residue on the 59 side (Arnez and Steitz 1994). The existence of a water-mediated hydrogen bond between CN1H and 59-phosphate oxygen was further revealed in solution through novel NMR methods (Newby and Greenbaum 2002).…”