Olke C. Uhlenbeck

Synthesis of small RNAs using T7 RNA polymerase.

Publisher Summary This chapter discusses how transcription templates are prepared by cleaving the plasmid DNA with a restriction enzyme of choice. The individual nucleoside triphosphates (NTPs) are diluted to desired concentrations and are mixed in equimolar ratios to make a stock solution. If they are supplied in ethanol and will be used in large amounts, the ethanol should be removed prior to use. The addition of serum albumin to the reaction is optional and is generally omitted in large-scale reactions. Differences in template sequence and length can result in substantial differences in the optimal enzyme and template concentrations required for the production of RNA. In addition, one or more of the components may be more valuable than the others. It is, therefore, worthwhile to carry out trial reactions to find optimal reaction conditions for a given synthesis. Although performing trial reactions to optimize reaction conditions can be tedious, reaction yields can be substantially higher than those obtained using the general conditions and the reactions scale up readily.

Stability of ribonucleic acid double-stranded helices

Abstract The hypochromicity, as a function of temperature for 19 oligoribonucleotides capable of forming perfectly base-paired double helices, is used to extract thermodynamic parameters of helix formation. The data are analyzed by an all or none model of helix melting which permits assignment of Δ G , Δ H , and Δ S of formation to each of the ten possible Watson-Crick base-paired nearest-neighbor sequences. Helix stability is found to have a striking dependence on sequence, and formulae are provided to predict the T m of any RNA double helix of known sequence.

Hammerhead ribozyme kinetics.

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).

Specific interaction between RNA phage coat proteins and RNA.

Publisher Summary This chapter describes the biochemistry of the interaction of phage coat protein with RNA and attempt to provide a molecular understanding of its high specificity. Coat protein binding is believed to serve two functions in the life cycle of the phage: 1) it acts as a translational repressor of the replicase gene early in infection, and 2) as an initiation site of phage assembly late in infection. This interaction has been extensively a prototype of sequence specific RNA-protein interactions. It is now clear that the phage coat proteins can be considered an example of a class of RNA hairpin binding proteins that are quite common in prokaryotes and eukaryotes. In case of bacteriophage coat protein, the coat protein assembles into phage-like capsids that can be purified by differential centrifugation and ion-exchange chromatography. Most coat proteins can be successfully renatured by the transfer from storage buffer directly into a variety of neutral buffers of moderate ionic strength. In many cases, these renatured proteins are fully active in both RNA binding and capsid assembly.

Escherichia coli DbpA is an RNA helicase that requires hairpin 92 of 23S rRNA

Escherichia coli DbpA is a member of the DEAD/H family of proteins which has been shown to have robust ATPase activity only in the presence of a specific region of 23S rRNA. A series of bimolecular RNA substrates were designed based on this activating region of rRNA and used to demonstrate that DbpA is also a non‐processive, sequence‐specific RNA helicase. The high affinity of DbpA for the RNA substrates allowed both single and multiple turnover helicase assays to be performed. Helicase activity of DbpA is dependent on the presence of ATP or dATP, the sequence of the loop of hairpin 92 of 23S rRNA and the position of the substrate helix with respect to hairpin 92. This work indicates that certain RNA helicases require particular RNA structures in order for optimal unwinding activity to be observed.

Comparison of the hammerhead cleavage reactions stimulated by monovalent and divalent cations.

Although the hammerhead reaction proceeds most efficiently in divalent cations, cleavage in 4 M LiCl is only approximately 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.

Modulation of tRNAAla identity by inorganic pyrophosphatase

A highly sensitive assay of tRNA aminoacylation was developed that directly measures the fraction of aminoacylated tRNA by following amino acid attachment to the 3′-32P-labeled tRNA. When applied to Escherichia coli alanyl-tRNA synthetase, the assay allowed accurate measurement of aminoacylation of the most deleterious mutants of tRNAAla. The effect of tRNAAla identity mutations on both aminoacylation efficiency (kcat/KM) and steady-state level of aminoacyl-tRNA was evaluated in the absence and presence of inorganic pyrophosphatase and elongation factor Tu. Significant levels of aminoacylation were achieved for tRNA mutants even when the kcat/KM value is reduced by as much as several thousandfold. These results partially reconcile the discrepancy between in vivo and in vitro analysis of tRNAAla identity.

Quantitative in situ hybridization of ribosomal RNA species to polytene chromosomes of Drosophila melanogaster

Abstract In situ hybridization of 125 I-labelled 5 S and 18 + 28 S ribosomal RNAs to the salivary polytene chromosomes of Drosophila melanogaster was successfully quantitated. Although the precision of the data is low, it is possible to compare the hybridization reaction between an RNA sample and chromosomes in situ with the reaction between the same RNA sample and Drosophila DNA immobilized on nitrocellulose filters. The in situ hybrid dissociates over a narrow temperature range with a midpoint similar to the value expected for the filter hybrid. The kinetics of the in situ hybridization reaction can be fit with a single first-order rate constant that has a value from three to five times smaller than the corresponding filter hybridization reaction. Although the reaction saturates at longer times or higher RNA concentrations, the saturation value does not correspond to an RNA molecule bound to every available DNA sequence. With the acid denaturation procedure most commonly used to preserve cytological quality, only 5 to 10% of the complementary DNA in the chromosomes is available to form hybrids in situ . This hybridization efficiency is a function of how the slides are prepared and the conditions of annealing, but is approximately constant with a given procedure for both 5 S RNA and 18 + 28 S RNA over a number of different cell types with different DNA contents. The results provide further evidence that the formation of RNA-DNA hybrids is the sole basis of in situ hybridization, and show that the properties of the in situ hybrids are remarkably similar to those of filter hybrids. It is also suggested that for reliable chromosomal localization using the in situ hybridization technique, the kinetics of the reaction should be followed to ensure that the correct rate constant is obtained for the major RNA species in the sample and an impurity in the sample is not localized instead.

Involvement of a specific metal ion in the transition of the hammerhead ribozyme to its catalytic conformation

Previous crystallographic and biochemical studies of the hammerhead ribozyme suggest that a metal ion is ligated by thepro-R p oxygen of phosphate 9 and by N7 of G10.1 and has a functional role in the cleavage reaction. We have tested this model by examining the cleavage properties of a hammerhead containing a unique phosphorothioate at position 9. The R p-, but notS p-, phosphorothioate reduces the cleavage rate by 103-fold, and the rate can be fully restored by addition of low concentrations of Cd2+, a thiophilic metal ion. These results strongly suggest that this bound metal ion is critical for catalysis, despite its location ∼20 Å from the cleavage site in the crystal structure. Analysis of the concentration dependence suggests that Cd2+ binds with a K d of 25 μm in the ground state and a K d of 2.5 nm in the transition state. The much stronger transition state binding suggests that the P9 metal ion adopts at least one additional ligand in the transition state and that this metal ion may participate in a large scale conformational change that precedes hammerhead cleavage.

Self-cleaving catalytic RNA.

We describe the structures and catalytic properties of several naturally occurring self‐cleaving RNA motifs that give 2′, 3′ cyclic phosphate products. The hammerhead and hairpin motifs are derived from plant pathogenic RNAs and the delta motif is part of the human hepatitis delta element. A fourth motif from Neurospora is less well characterized. By assembling the self‐cleaving RNAs from more than one oligoribonucleotide, the cleavage reaction can be examined under a variety of conditions and catalytic turnover can be demonstrated. Mutagenesis and chemical methods to introduce modified nucleotides allowed the structural requirements to be deduced. The role of divalent cations in the catalytic mechanism is discussed.— Long, D. M., and Uhlenbeck, O. C. Self‐cleaving catalytic RNA. FASEB J. 7: 25‐30; 1993.