Ribonucleoside phosphodiesters

Transfer RNA precursors may undergo further posttranscriptional processing. The 3 -terminal trinucleotide CCA(3 ) to which an amino acid will be attached during protein synthesis (Chapter 27) is absent from some bacterial and all eukaryotic tRNA precursors and is added during processing (Fig. 26-23). This addition is carried out by tRNA nucleotidyltransferase, an unusual enzyme that binds the three ribonucleoside triphosphate precursors in separate active sites and catalyzes formation of the phosphodiester bonds to produce the CCA(3 ) sequence. The creation of this defined sequence of nucleotides is therefore not dependent on a DNA or RNA template—the template is the binding site of the enzyme. 

The reaction catalyzed by polynucleotide phosphorylase differs fundamentally from the polymerase activities discussed so far in that it is not template-dependent. The enzyme uses the 5 -diphosphates of ribonucleosides as substrates and cannot act on the homologous 5 -triphos-phates or on deoxyribonucleoside 5 -diphosphates. The RNA polymer formed by polynucleotide phosphorylase contains the usual 3, 5 -phosphodiester linkages, which can be hydrolyzed by ribonuclease. The reaction is readily reversible and can be pushed in the direction of breakdown of the polyribonucleotide by increasing the phosphate concentration. The probable function of this enzyme in the cell is the degradation of mRNAs to nucleoside diphosphates. 

Enzymes are available from a variety of sources which split the phosphodiester bond of nucleoside 2, 3 - and 3, 5 -cyclic phosphates. The ability of the ribonucleases to hydrolyze ribonucleoside 2, 3,-cyclic phosphates to the corresponding 3 -phosphates is well known. During... 

Shaw (115) reported a 300-fold purification of enzyme from tobacco leaves. Activity of the enzyme was optimal at pH 5.5-5.7, and divalent cations were not required for activity. The enzyme possessed high activity toward ribonucleoside 2 - and 5 -monophosphates and glucose 1-phosphate. There was no activity toward RNA or phosphodiesters. Fluoride acts as a noncompetitive inhibitor for this enzyme. This behavior of fluoride is in contrast to the behavior with prostatic acid phosphatase where the inhibition is strictly competitive. 

RNA is synthesized in the 5 - 3 direction by the formation of 3 -5 -phosphodiester linkages between four ribonucleoside triphosphate substrates, analogous to the process of DNA synthesis. The sequence of bases in RNA transcripts catalyzed by DNA-depen-dent RNA polymerases is specified by the complementary sequences of the DNA template strand. 

Thus, it uses the four ribonucleoside triphosphates (ATP, GTP, UTP, and CTP) to assemble an RNA chain, the sequence of which is determined by the template strand of DNA. Nucleotide addition occurs sequentially, the phosphodiester bond being formed through the same mechanism as described for DNA polymerase (see Chap. 16, Fig. 16-9). RNA chain growth is in the 5 — 3 direction. An important distinction between RNA polymerase and DNA polymerase, however, is the ability of the former to start a new chain de novo i.e., it does not have an obligatory requirement for a primer. The first nucleotide to be incorporated into the chain of RNA contains either adenine or guanine and retains its 5 triphosphate. 

It selects the correct ribonucleoside triphosphate and catalyzes the formation of a phosphodiester bond. This process is repeated many times as the enzyme moves unidirectionally along the DNA template. RNA polymerase is completely processive a transcript is synthesized from start to end by a single RNA polymerase molecule. 

A ribonucleoside triphosphate pairs with the complementary base in the DNA template and forms a phosphodiester bond with the 3 -hydroxyl of the ribose at the end of the growing chain. -Pyrophosphate is produced and cleaved to two inorganic phosphates, releasing energy that drives the reaction. 

It selects the correct ribonucleoside triphosphate and catalyzes the formation of a phosphodiester bond. This process is repeated many times as... 

The THP ether product is stable to base. The THP function (see below) has been used for the synthesis of 2 -protected ribonucleosides. As was noted (see above) the 2 -protected hydroxyl is vital for the synthesis of the 3, 5 -phosphodiester linkage. However, a complicating factor can be the presence of an asymmetric center in the THP protected product. 

Acetal links have been used to make dinucleotide analogues of type 154 (B Thy, Cyt) in which the phosphodiester has been replaced by a neutral, conformationally restricted unit the acetals were formed conventionally by condensation of a S protected thymidine ribonucleoside with the dimethyl acetal of a chain-extended lower section.236... 

---