RNA phosphodiesters

Another class of cofactor-dependent deoxyribozymes that use the amino acid histidine to promote phosphodiester cleavage was isolated recently by Roth and Breaker [113]. One of the isolated DNA requires L-histidine or a closely related analog to support RNA phosphodiester cleavage with a rate enhancement of nearly a million-fold over the basal substrate cleavage rate (Fig. 14). 

Sun W, Pertzev A, Nicholson AW. Catalytic mechanism of Escherichia coli ribonuclease 111 kinetic and inhibitor evidence for the involvement of two magnesium ions in RNA phosphodiester hydrolysis. Nucleic Acids Res. 2005 33(3) 807-815. 

The known ribozymes in biology may be divided into three different classes dehned by differences in stmctural complexity as well as by the chemical reactions they catalyze (Fig. 1). Small ribozymes cleave an RNA phosphodiester backbone by catalyzing the intramolecular attack of a ribose 2 -OH on the... 

Figure 9 A binuclear site predicted from the 1.5 A resolution X-ray crystal structure of the loop E sub-domain from 5S ribosomal RNA. Phosphodiester-ligated ions bridged by aqueous ligands are shown as spheres labeled B and C (PDB 354D) (reproduced with permission from Cell, 1997,91, 705-712 1997, Cell Press).

Mikkola S, Stenman E, Nurmi K, Yousefi-Salakdeh E, Stromberg R, Lormberg H The mechanism of the metal ion promoted cleavage of RNA phosphodiester bonds involves a general acid catalysis by the metal aquo ion on the departure of... 

Another type of bond that is ubiquitous in nature is phosphodiester bond making up the backbone of DNA or RNA. Enzymes can use more than one amino acid side chain in their active side for simultaneous bifimctional or multifunctional catalysis. Often an add-base catalyst is formed as in the enzyme ribonuclease A. The natural enzyme consists of 124 amino acids and catalyzes the hydrolysis of RNA phosphodiester bonds between the phosphorous atom and the 5 -oxygen atom. The mechanism of ester cleavage proceeds via a 2, 3 cyclo-phosphate intermediate. The histidine 119 and 12 function as acid and base to catalyze the formation of the cyclic intermediate while the lysine stabilizes the pentacoordinated transition state. The hydrolysis of the cyclic intermediate is then again catalyzed by both histidine residues (Figure 26). ... 

Geyer, C. R. Sen, D. (1997) Evidence for the metal-cofactor independence of an RNA phosphodiester-cleaving DNA enzyme. Chemistry and Biology, 4, 579-593. 

The discovery of nbozymes (Section 28 11) in the late 1970s and early 1980s by Sidney Altman of Yale University and Thomas Cech of the University of Colorado placed the RNA World idea on a more solid footing Altman and Cech independently discovered that RNA can catalyze the formation and cleavage of phosphodiester bonds—exactly the kinds of bonds that unite individual ribonucleotides in RNA That plus the recent discovery that ribosomal RNA cat alyzes the addition of ammo acids to the growing peptide chain in protein biosynthesis takes care of the most serious deficiencies in the RNA World model by providing precedents for the catalysis of biologi cal processes by RNA... 

The other important feature of the primary stmcture of RNA is the presence of the 2 -hydroxyl group in ribose. Although this hydroxyl group is never involved in phosphodiester linkages, it does impose restrictions on the heHcal conformations accessible to double-stranded RNA. 

HammerheadRtbozyme. A small RNA molecule that catalyzes cleavage of the phosphodiester backbone of RNA is known as the hammerhead ribozyme. This ribozyme occurs namrally in certain vimses where it facihtates a site-specific self-cleavage at the phosphate and generates a 2 3 -cychc phosphate and a 5 -hydroxyl terminus. The reaction requires a divalent metal ion, such as or, as a cofactor. Whereas the... 

The methylphosphonates differ from the phosphodiesters and phosphorothiolates in that the methyl derivatives are uncharged and are thus less water soluble. Moreover, compared to the naturally occurring phosphodiesters, the methylphosphonates form slightly less stable duplexes with complementary DNA and RNA sequences. This effect has been ascribed to the iaevitable chiraUty problem that is, if one isomer biads less well, the overall binding is decreased. Methylphosphonates can enter cell membranes by a passive mechanism and are completely resistant to nucleases. 

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. 

DNA is not susceptible to alkaline hydrolysis. On the other hand, RNA is alkali labile and is readily hydrolyzed by dilute sodium hydroxide. Cleavage is random in RNA, and the ultimate products are a mixture of nucleoside 2 - and 3 -monophosphates. These products provide a clue to the reaction mechanism (Figure 11.29). Abstraction of the 2 -OH hydrogen by hydroxyl anion leaves a 2 -0 that carries out a nucleophilic attack on the phosphorus atom of the phosphate moiety, resulting in cleavage of the 5 -phosphodiester bond and formation of a cyclic 2, 3 -phosphate. This cyclic 2, 3 -phosphodiester is unstable and decomposes randomly to either a 2 - or 3 -phosphate ester. DNA has no 2 -OH therefore DNA is alkali stable. 

FIGURE 14.23 RNA splicing in TetraAjimejta rRNA matnradon (a) the gnanosine-mediated reaction involved in the antocatalytic excision of the Tetrahymena rRNA intron, and (b) the overall splicing process. The cyclized intron is formed via nncleophilic attack of the 3 -OH on the phosphodiester bond that is 15 nncleotides from the 5 -GA end of the spliced-ont intron. Cyclization frees a linear 15-mer with a 5 -GA end. 

Heterocycles as agents for the cleavage of phosphodiester bonds in RNA 98CRV961. 

Staphylococcal nuclease (SNase) is a single-peptide chain enzyme consisting of 149 amino acid residues. It catalyzes the hydrolysis of both DNA and RNA at the 5 position of the phosphodiester bond, yielding a free 5 -hydroxyl group and a 3 -phosphate monoester... 

The 5 -phosphoryl group of a mononucleotide can es-terify a second —OH group, forming a phosphodi-ester. Most commonly, this second —OH group is the 3 -OH of the pentose of a second nucleotide. This forms a dinucleotide in which the pentose moieties are linked by a 3 —> 5 phosphodiester bond to form the backbone of RNA and DNA. 

While formation of a dinucleotide may be represented as the elimination of water between two monomers, the reaction in fact strongly favors phosphodiester hydrolysis. Phosphodiesterases rapidly catalyze the hydrolysis of phosphodiester bonds whose spontaneous hydrolysis is an extremely slow process. Consequently, DNA persists for considerable periods and has been detected even in fossils. RNAs are far less stable than DNA since the 2khydroxyl group of RNA... 

Ribonucleic acid (RNA) is a polymer of purine and pyrimidine ribonucleotides finked together by 3, 5 -phosphodiester bridges analogous to those in DNA (Figure 35—6). Although sharing many features with DNA, RNA possesses several specific differences ... 

Figure 35-6. A segment of a ribonucleic acid (RNA) molecule in which the purine and pyrimidine bases— guanine (G), cytosine (C), uracii (U), and adenine (A)—are held together by phosphodiester bonds between ribo-syl moieties attached to the nucieobases by N-giycosidic bonds. Note that the polymer has a polarity as indicated by the iabeied 3 -and 5 -attached phosphates.

The process of RNA synthesis in bacteria—depicted in Figure 37-3—involves first the binding of the RNA holopolymerase molecule to the template at the promoter site to form a PIC. Binding is followed by a conformational change of the RNAP, and the first nucleotide (almost always a purine) then associates with the initiation site on the 3 subunit of the enzyme. In the presence of the appropriate nucleotide, the RNAP catalyzes the formation of a phosphodiester bond, and the nascent chain is now attached to the polymerization site on the P subunit of RNAP. (The analogy to the A and P sites on the ribosome should be noted see Figure... 

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. ... 

In contrast, the hairpin ribozyme (HPR) [107, 108], which catalyzes the reversible, site-specific phosphodiester bond cleavage of an RNA substrate, is unique in that the chemical steps of the reaction do not require involvement of a divalent metal ion [107-111]. This lack of an explicit metal ion requirement [112] makes the hairpin ribozyme an ideal target for theoretical studies aimed to characterize the contribution of generalized solvation provided by the solvated ribozyme on catalysis. 

The orientation of the cap in synthetic mRNA is the major contributor to overall translational efficiency. Cap analogs blocked at the 3 -O position of the first nucleoside moiety [m73/dGp3G (4) and m27,3 °Gp3G (2)] as well as modified in the 2 -0 position (which does not participate in phosphodiester formation) [m72 dGp3G (3) and m27 2 °Gp3G (1)], are incorporated into RNA exclusively in the correct orientation (Stepinski et al., 2001), which... 

The nucleotides forming RNA or DNA molecules are linked together in phosphodiester bonds with sugar-phosphate repeating units. The esters are directionally linked between the 3 -hydroxyl... 

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