Intermolecular domain docking in the hairpin ribozyme: metal dependence, binding kinetics and catalysis.

The hairpin ribozyme is a prototype small, self-cleaving RNA motif. It exists naturally as a four-way RNA junction containing two internal loops on adjoining arms. These two loops interact in a cation-driven docking step prior to chemical catalysis to form a tightly integrated structure, with dramatic changes occurring in the conformation of each loop upon docking. We investigate the thermodynamics and kinetics of the docking process using constructs in which loop A and loop B reside on separate molecules. Using a novel CD difference assay to isolate the effects of metal ions linked to domain docking, we find the intermolecular docking process to be driven by sub-millimolar concentrations of the exchange-inert Co(NH 3) 6 (3+). RNA self-cleavage requires binding of lower-affinity ions with greater apparent cooperativity than the docking process itself, implying that, even in the absence of direct coordination to RNA, metal ions play a catalytic role in hairpin ribozyme function beyond simply driving loop-loop docking. Surface plasmon resonance assays reveal remarkably slow molecular association, given the relatively tight loop-loop interaction. This observation is consistent with a "double conformational capture" model in which only collisions between loop A and loop B molecules that are simultaneously in minor, docking-competent conformations are productive for binding.

Conformationally restricted nucleotides as a probe of structure-function relationships in RNA.

We introduce the use of commercially available locked nucleic acids (LNAs) as a functional probe in RNA. LNA nucleotides contain a covalent linkage that restricts the pseudorotation phase of the ribose to C3'-endo (A-form). Introduction of an LNA at a single site thus allows the role of ribose structure and dynamics in RNA function to be assessed. We apply LNA probing at multiple sites to analyze self-cleavage in the lead-dependent ribozyme (leadzyme), thermodynamic stability in the UUCG tetraloop, and the kinetics of recognition of U1A protein by U1 snRNA hairpin II. In the leadzyme, locking a single guanosine residue into the C3'-endo pucker increases the catalytic rate by a factor of 20, despite the fact that X-ray crystallographic and NMR structures of the leadzyme ground state reported a C2'-endo conformation at this site. These results strongly suggest that a conformational change at this position is critical for catalytic function. Functional insights obtained in all three systems demonstrate the highly general applicability of LNA probing in analysis of the role of ribose orientation in RNA structure, dynamics, and function.

Alternate-site isotopic labeling of ribonucleotides for NMR studies of ribose conformational dynamics in RNA.

Heteronuclear NMR spin relaxation studies of conformational dynamics are coming into increasing use to help understand the functions of ribozymes and other RNAs. Due to strong 13C-13C magnetic interactions within the ribose ring, however, these studies have thus far largely been limited to (13)C and (15)N resonances on the nucleotide base side chains. We report here the application of the alternate-site (13)C isotopic labeling scheme, pioneered by LeMaster for relaxation studies of amino acid side chains, to nucleic acid systems. We have used different strains of E. coli to prepare mononucleotides containing (13)C label in one of two patterns: Either C1' or C2' in addition to C4', termed (1'/2',4') labeling, or nearly complete labeling at the C2' and C4' sites only, termed (2',4') labeling. These patterns provide isolated 13C-1H spin systems on the labeled carbon atoms and thus allow spin relaxation studies without interference from 13C-13C scalar or dipolar coupling. Using relaxation studies of AMP dissolved in glycerol at varying temperature to produce systems with correlation times characteristic of different size RNAs, we demonstrate the removal of errors due to 13C-13C interaction in T (1) measurements of larger nucleic acids and in T (1rho) measurements in RNA molecules. By extending the applicability of spin relaxation measurements to backbone ribose groups, this technology should greatly improve the flexibility and completeness of NMR analyses of conformational dynamics in RNA.