Nanometal surface energy transfer (NSET), which describes an energy transfer event from optically excited organic fluorophores to small metal nanoparticles, may be used as a molecular beacon/ruler similar to FRET, but with advantages over this classical technique. Here we use NSET to measure Mg(2+)-induced conformational changes for a hammerhead ribozyme and confirm these measurements using FRET. These optical experiments enhance our understanding of the different kinetic pathways for this ribozyme.

No abstract

Pairing of a consensus sequence of the precursor (pre)-mRNA intron with a short region of the U2 small nuclear (sn)RNA during assembly of the eukaryotic spliceosome results in formation of a complementary helix of seven base pairs with a single unpaired adenosine residue. The 2' OH of this adenosine, called the branch site, brings about nucleophilic attack at the pre-mRNA 5' splice site in the first step of splicing. Another feature of this pairing is the phylogenetic conservation of a pseudouridine (psi) residue in U2 snRNA nearly opposite the branch site. We show that the presence of this psi in the pre-mRNA branch-site helix of Saccharomyces cerevisiae induces a dramatically altered architectural landscape compared with that of its unmodified counterpart. The psi-induced structure places the nucleophile in an accessible position for the first step of splicing.

The removal of noncoding sequences (introns) from eukaryotic precursor mRNA is catalyzed by the spliceosome, a dynamic assembly involving specific and sequential RNA-RNA and RNA-protein interactions. An essential RNA-RNA pairing between the U2 small nuclear (sn)RNA and a complementary consensus sequence of the intron, called the branch site, results in positioning of the 29OH of an unpaired intron adenosine residue to initiate nucleophilic attack in the first step of splicing. To understand the structural features that facilitate recognition and chemical activity of the branch site, duplexes representing the paired U2 snRNA and intron sequences from Saccharomyces cerevisiae were examined by solution NMR spectroscopy. Oligomers were synthesized with pseudouridine (c) at a conserved site on the U2 snRNA strand (opposite an A-A dinucleotide on the intron strand, one of which forms the branch site) and with uridine, the unmodified analog. Data from NMR spectra of nonexchangeable protons demonstrated A-form helical backbone geometry and continuous base stacking throughout the unmodified molecule. Incorporation of c at the conserved position, however, was accompanied by marked deviation from helical parameters and an extrahelical orientation for the unpaired adenosine. Incorporation of c also stabilized the branch-site interaction, contributing -0.7 kcal/mol to duplex DG8 37 . These findings suggest that the presence of this conserved U2 snRNA pseudouridine induces a change in the structure and stability of the branch-site sequence, and imply that the extrahelical orientation of the branch-site adenosine may facilitate recognition of this base during splicing.

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...