Preparation of large quantities of RNA molecules of a defined sequence is a prerequisite for biophysical analysis, and is particularly important to the determination of high-resolution structure by X-ray crystallography. We describe improved methods for the production of multimilligram quantities of homogeneous tRNAs, using a combination of chemical synthesis and enzymatic approaches. Transfer RNA half-molecules with a break in the anticodon loop were chemically synthesized on a preparative scale, ligated enzymatically, and cocrystallized with an aminoacyl-tRNA synthetase, yielding crystals diffracting to 2.4 Å resolution. Multimilligram quantities of tRNAs with greatly reduced 3′ heterogeneity were also produced via transcription by T7 RNA polymerase, utilizing chemically modified DNA half-molecule templates. This latter approach eliminates the need for large-scale plasmid preparations, and yields synthetase cocrystals diffracting to 2.3 Å resolution at much lower RNA:protein stoichiometries than previously required. These two approaches developed for a tRNA–synthetase complex permit the detailed structural study of “atomic-group” mutants.

The Escherichia coli DEAD protein DbpA is an RNA-specific ATPase that is activated by a 153-nt fragment within domain V of 23S rRNA. A series of RNA subfragments and sequence changes were used to identify the recognition elements of this RNA–protein interaction. Reducing the size of the fully active 153-nt RNA yields compromised substrates in which both RNA and ATP binding are weakened considerably without affecting the maximal rate of ATP hydrolysis. All RNAs that stimulate ATPase activity contain hairpin 92 of 23S rRNA, which is known to interact with the 3′ end of tRNAs in the ribosomal A-site. RNAs with base mutations within this hairpin fail to activate ATP hydrolysis, suggesting that it is a critical recognition element for DbpA. Although the isolated hairpin fails to activate DbpA, RNAs with an extension of approximately 15 nt on either the 5′ or 3′ side of hairpin 92 elicit full ATPase activity. These results suggest that the binding of DbpA to RNA requires sequence-specific interactions with hairpin 92 as well as nonspecific interactions with the RNA extension. A model relating the RNA binding and ATPase activities of DbpA is presented.

The well-studied interaction between the MS2 coat protein and its cognate hairpin was used to test the utility of the methylphosphonate linkage as a phosphate analog. A nitrocellulose filter binding assay was used to measure the change in binding affinity upon introduction of a single methylphosphonate stereoisomer at 13 different positions in the RNA hairpin. Comparing these data to the available crystal structure of the complex shows that all phosphates that are in proximity to the protein show a weaker binding affinity when substituted with a phosphorothioate and control positions show no change. However, in two cases, a methylphosphonate isomer either increased or decreased the binding affinity where no interaction can be detected in the crystal structure. It is possible that methylphosphonate substitutions at these positions affect the structure or flexibility of the hairpin. The utility of the methylphosphonate substitution is compared to phosphate ethylation and phosphorothioate substitution experiments previously performed on the same system.

Although the hammerhead reaction proceeds most efficiently in divalent cations, cleavage in 4 M LiCl is only ∼10-fold slower than under standard conditions of 10 mM MgCl2 (Murray et al., Chem Biol, 1998, 5:587–595; Curtis & Bartel, RNA, 2001, this issue, pp. 546–552). To determine if the catalytic mechanism with high concentrations of monovalent cations is similar to that with divalent cations, we compared the activities of a series of modified hammerhead ribozymes in the two ionic conditions. Nearly all of the modifications have similar deleterious effects under both reaction conditions, suggesting that the hammerhead adopts the same general catalytic structure with both monovalent and divalent cations. However, modification of three ligands previously implicated in the binding of a functional divalent metal ion have substantially smaller effects on the cleavage rate in Li+ than in Mg2+. This result suggests that an interaction analogous to the interaction made by this divalent metal ion is absent in the monovalent reaction. Although the contribution of this divalent metal ion to the overall reaction rate is relatively modest, its presence is needed to achieve the full catalytic rate. The role of this ion appears to be in facilitating formation of the active structure, and any direct chemical role of metal ions in hammerhead catalysis is small.

The use of T7 RNA polymerase to prepare large quantities of RNA of a particular sequence has greatly facilitated the study of both the structure and function of RNA. Generally, it has been believed that the products of this technique are highly homogeneous in sequence, with only a few noted exceptions. We have carefully examined the transcriptional products of several tRNAs that vary in their 5′ end sequence and found that, for those molecules that begin with multiple, consecutive guanosines, the transcriptional products are far from homogenous. Although a template beginning with GCG showed no detectable 5′ end heterogeneity, two tRNA templates designed to have either four or five consecutive guanosines at their 5′ ends had more than 30% of their total transcriptional products extended by at least one untemplated nucleotide at their 5′ end. By simply reducing the number of consecutive guanosines, the heterogeneity was reduced significantly. The presence of this 5′ end heterogeneity in combination with the 3′ end heterogeneity common to T7 transcriptions results in a mixture of RNA molecules even after rigorous size purification.

The technique of cryoenzymology has been applied to the hammerhead ribozyme in an attempt to uncover a structural rearrangement step prior to cleavage. Several cryosolvents were tested and 40% (v/v) methanol in water was found to perturb the system only minimally. This solvent allowed the measurement of ribozyme activity between 30 and −33°C. Eyring plots are linear down to −27°C, but a drastic reduction in activity occurs below this temperature. However, even at extremely low temperatures, the rate is still quite pH dependent, suggesting that the chemical step rather than a structural rearrangement is still rate-limiting. The nonlinearity of the Eyring plot may be the result of a transition to a cold-denatured state or a glassed state.

The hammerhead ribozyme is a small RNA motif that self cleaves at a specific phosphodiester bond to produce 2′,3′ cyclic phosphate and 5′ hydroxyl termini (Hutchins et al., 1986; Forster & Symons, 1987a). The secondary structure of the hammerhead consists of three helices of arbitrary sequence and length (designated I, II, and III) that intersect at 15 nucleotides termed the catalytic core (Fig. 1A) (Forster & Symons, 1987b; Hertel et al., 1992). The X-ray crystal structures of two hammerhead ribozyme–inhibitor complexes revealed that the core residues fold into two separate domains and the helices are arranged in a Y-shape conformation with helix I and helix II forming the upper portion of the Y (Pley et al., 1994; Scott et al., 1995). Although the hammerhead is found as an intramolecular motif embedded in several RNAs in vivo (Symons, 1989), it can be assembled from two separate oligonucleotides (Fig. 1B) in three different arrangements (Uhlenbeck, 1987; Haseloff & Gerlach, 1988; Koizumi et al., 1988; Jeffries & Symons, 1989). In these bimolecular formats, the hammerhead effects RNA cleavage in a similar manner to a true “enzyme,” proceeding through multiple rounds of substrate binding, cleavage, and product release (Uhlenbeck, 1987).

An improved quantitative assay for tRNA aminoacylation is presented based on charging of a nicked tRNA followed by separation of an aminoacylated 3′-fragment on an acidic denaturing polyacrylamide gel. Kinetic parameters of tRNA aminoacylation by Escherichia coli AlaRS obtained by the new method are in excellent agreement with those measured by the conventional method. This assay provides several advantages over the traditional methods of measuring tRNA aminoacylation: (1) the fraction of aminoacyl-tRNA is measured directly; (2) data can be obtained at saturating amino acid concentrations; and (3) the assay is significantly more sensitive.