Hydrolysis of RNA

FIGURE 11.29 The vicinal—OH groups of RNA are susceptible to nucleophilic attack leading to hydrolysis of the phosphodiester bond and fracture of the polynucleotide chain DNA lacks a 2 -OH vicinal to its 3 -0-phosphodiester backbone. Alkaline hydrolysis of RNA results in the formation of a mixture of 2 - and 3 -nucleoside monophosphates. 

Complete hydrolysis of RNA by alkali yields a random mixture of 2 -NMPs and 3 -NMPs. 

Hydrolysis of RNA by alkali or pancreatic RNase leads initially to fragments which terminate in 2, 3 -cyclic phosphodiesters. Micrococcal nuclease, on the other hand, gives rise to fragments terminating in 3 -phos-phomonoester groups which facilitate their isolation, and this enzymic hydrolysis has been used to prepare 3 -ribodinucleotides. ... 

Wall et al. built a binuclear copper(II) complex 43 in order to see acceleration of phosphodiester cleavage (52). With the substrate (50 p.M) shown, the reaction might be considered as a model for the first step of the hydrolysis of RNA, in which the alcohol function of the side chain intramolecularly attacks the Cun-activated phosphate as a nucleophile for a ring closure reaction. Compared to an analogous mononuclear complex 44 (at 1 mM), a rate constant ca. 50 times larger for 43 (at 1 mM) was observed at 25°C and pH 7, implying that the two metal ions probably cooperate. An analogous zinc(II) complex 45 was reported only as a structural model for the active site of phospholi-... 

Ribonuclease EC 3.1.27.5 Hydrolysis of RNA Milk is a very rich source similar to pancreatic RNase... 

Ribonucleoside 2, 3 -cyclic monophosphates are isolatable intermediates, and ribonucleoside 3 -monophosphates are end products of the hydrolysis of RNA by certain ribonucleases. Other variations are adenosine 3, 5 -cyclic monophosphate (cAMP) and guanosine 3, 5 -cyclic monophosphate (cGMP), considered at the end of this chapter. 

FIGURE 8-6 Some adenosine monophosphates Adenosine 2 -monophosphate, 3 -monophosphate, and 2, 3 -cyclic monophosphate are formed by enzymatic and alkaline hydrolysis of RNA. 

RGURE 8-8 Hydrolysis of RNA under alkaline conditions The 2 hydroxyl acts as a nucleophile in an intramolecular displacement. The 2, 3 -cyclic monophosphate derivative is further hydrolyzed to a mixture of 2 - and 3 -monophosphates DNA, which lacks 2 hydroxyls, is stable under similar conditions... 

Draw the structure of a dinucleotide that might be obtained by the partial hydrolysis of RNA. Indicate the following ... 

A recent discovery in biochemistry is that RNA can act as an enzyme in chemical reactions, usually reactions involving RNA hydrolysis. Discuss the features of RNA structure that might favor evolution of enzymes composed entirely of a single polyribonucleotide chain, and describe a proposed mechanism for RNA-catalyzed hydrolysis of RNA molecules. 

These reactions are the easiest to tackle, since they require only one phosphoryl oxygen to be substituted in both the substrate and the product. The classic example of this experiment is the first step in the hydrolysis of RNA catalyzed by bovine pancreatic ribonuclease. As discussed in detail in Chapter 16, ribonucle-ase catalyzes the hydrolysis of RNA by a two-step reaction in which a cyclic intermediate is formed. The stereochemistry of the first step (cyclization) (equation 8.35),... 

Bovine pancreatic ribonuclease catalyzes the hydrolysis of RNA by a two-step process in which a cyclic phosphate intermediate is formed (equation 16.35). The cyclization step is usually much faster than the subsequent hydrolysis, so the intermediate may be readily isolated. DNA is not hydrolyzed, as it lacks the 2 -hydroxyl group that is essential for this reaction. There is a strong specificity for the base B on the 3 side of the substrate to be a pyrimidine—uracil or cytosine. 

The hydrolysis of RNA catalyzed by RNase A occurs in two distinct steps, with a 2, 3 -phosphate cyclic diester intermediate (see fig. 8.12). The intermediate can be identified relatively easily, because its breakdown is much slower than its formation. Ribonucleases do not hydrolyze DNA, which lacks the 2 -hydroxyl group needed for the formation of the cyclic intermediate. 

The hydrolysis of adenosine 3. 5 -cyclic monophosphate (cAMP) by the cobalt complexes (215) was considered here earlier,187 as was the Ce(IV)-catalysed hydrolysis of phospho monoesters in nucleotides.189 A review (ca 100 references) on current data on the mechanism of cleavage-transesterification of RNA has appeared.258 In this review special attention was focused on the two crucial steps in the hydrolysis of RNA, i.e. cleavage-transesterification and hydrolysis of the cyclic phosphodiester (Scheme 14). The catalysis of various amines for the hydrolysis of RNA has been looked at and ethylenediamine and propane-1,3-diamine are highly active under physiological conditions because they exist as the catalytically active monocation forms.259... 

Metal-free catalysts for the hydrolysis of RNA derived from 2-aminobenzimidazoles were reported. The most active catalysts, tris derivatives (99 R = H, CC Me) built upon a framework of tris(2-aminoethyl)amine, were shown by fluorescence correlation spectroscopy to aggregate with oligonucleotides. However, at very low concentrations the compounds were still active in the non-aggregated state. Conjugates of the ester (99 CC Me) with antisense oligonucleotides or RNA binding peptides will, it was claimed, be promising candidates as site specific artificial ribonucleases.98... 

Inoue, H., et al. (1987). Sequence-dependent hydrolysis of RNA using modified oligonucleotide splints and RNase H. FEBS Lett. 215, 327—330. 

Unlike DNA, RNA is very susceptible to rapid degradation due to ribonu-cleases (RNases), which are highly stable RNA degrading enzymes. In addition, RNA is more labile than DNA, especially at higher temperatures (>65°C) and at alkaline pH (>9). The sensitivity of RNA toward alkaline hydrolysis can be used for selective hydrolysis of RNA in a mixture of RNA and DNA [5], Isolation of intact RNA is crucial to the success of many applications, such as the measurement of qualitative and quantitative changes in gene expression, preparation of cDNA or cDNA libraries, and in the synthesis of a probe for various molecular hybridization experiments. 

With the existence of this new cyclodextrin lock, it was again important to select a key to fit it and to serve as substrate. For this we wanted a cyclic phosphate ester that this cyclodextrin bisimidazole could hydrolyze. The enzyme ribonuclease hydrolyzes cyclic phosphates as the second step in the hydrolysis of RNA, and cyclic phosphates are used as assay substrates for the enzyme. The advantage to us of such a substrate was that the geometry of the transition state would be relatively well-defined, so that it should be possible to design congruence between the catalyst and the transition state. Molecular model building indicated that a possible substrate was the cyclic phosphate derived from 4-f-butylcatechol (VIII). Indeed, the cyclodextrin bisimidazole (VII) is a catalyst for the cleavage of cyclic phosphate (VIII) (14). 

Figure 10.17 Alkaline hydrolysis of RNA, showing only a single diester bond. The intermediate cyclic triester can be hydrolyzed at any two of the three locations indicated by dotted lines and labeled a, b, and c. If cleavage occurs at positions a and b, the first set of products is obtained. Cleavage at a and c produces the second set. A third set may be obtained by cleaving at b and c. P designates esterified phosphate.

There are 48 structural and cross-linking amino acids concerned with the shape of the protein but over half of the amino adds have functional groups sticking out of the chain—amino, hydroxy, acid groups, and the like. In fact, the enzyme uses only a few of these functional groups in the reaction it catalyses (the hydrolysis of RNA)—probably only two histidines and one lysine—but it is typical of enzymes that they have a vast array of functional groups available for chemical reactions. 

Hydrolytic catalysis by metal ions is also important in the hydrolysis of nucleic acids, especially RNA (36). Molecules of RNA that catalyze hydrolytic reactions, termed ribozymes, require divalent metal ions to effect hydrolysis efficiently. Thus, all ribozymes are metalloenzymes (6). There is speculation that ribozymes may have been the first enzymes to evolve (37), so the very first enzymes may have been metalloenzymes Recently, substitution of sulfur for the 3 -oxygen atom in a substrate of the tetrahymena ribozyme has been shown to give a 1000-fold reduction in rate of hydrolysis with Mg2+ but no attenuation of the hydrolysis rate with Mn2+ and Zn2+ (38). Because Mn2+ and Zn2+ have stronger affinities for sulfur than Mg2+ has, this feature provides strong evidence for a true catalytic role of the divalent cation in the hydrolytic mechanism, involving coordination of the metal to the 3 -oxygen atom. Other examples of metal-ion catalyzed hydrolysis of RNA involve lanthanide complexes, which are discussed in this volume. 

K. Matsuura, M. Endo, M. Komiyama, Lanthanide Complex-Oligo-DNA Hybrid for Sequence-Selective Hydrolysis of RNA , J. Chem. Soc., Chem. Commun., 2019 (1994)... 

D.-M. Zhou, K. Taira, The Hydrolysis of RNA From Theoretical Calculations to the Hammerhead Ribozyme-Mediated Cleavage of RNA , Chem. Rev., 98,991 (1998)... 

The 2 -OH group in RNA acts as an intramolecular nucleophil. In the alkaline hydrolysis of RNA, it forms a 2 -3 cyclic intermediate. 

The ribonuclease H (RNase H) family of enzymes are ubiquitous nucleases that catalyze the hydrolysis of RNA in RNA DNA hybrids (for review, see 1). In contrast to the well-studied ribonucleases A and Tl, RNase H does not employ the 2 -OH in RNA as a nucleophile but instead activates water as the nucleophile for hydrolysis in a metal-dependent reaction. The number and role(s) of divalent metal in the RNase H reaction mechanism are still unclear. 

The reaction is subject to general base catalysis because the OH group is a poor nucleophile anu removal of its proton during the ring closure accelerates the reaction. We now have a mechanisrr very similar to that on p. 1352 for the hydrolysis of RNA. In full this is general base catalysis jf... 

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