Ribonucleoside triphosphate

The reduction of ribonucleoside triphosphates by various dithiols which are capable of intramolecular cyclization on oxidation (dihydrolipoate, dithioerythritol, dithiothreitol) yields 2 -deoxyribonucleoside triphosphates. These reactions also require 5-deoxyadenosylcorrinoids. 

Template binding RNA polymerase (RNAP) binds to DNA and locates a promoter (P) melts the two DNA strands to form a preinitiation complex (PIQ. (2) Chain initiation RNAP holoenzyme (core + one of multiple sigma factors) catalyzes the coupling of the first base (usually ATP or GTP) to a second ribonucleoside triphosphate to form a dinucleotide. (3) Chain elongation Successive residues are added to the 3 -OH terminus of the nascent RNA molecule. (4) Chain termination and release The completed RNA chain and RNAP are released from the template. The RNAP holoenzyme re-forms, finds a promoter, and the cycle is repeated. 

Synthetic capped mRNAs are useful tools to study all of the processes mentioned previously. To create capped mRNAs, DNA templates are transcribed with either a bacterial (Contreras etal., 1982) or bacteriophage (Konarska et al., 1984 Yisraeli and Melton, 1989) RNA polymerase in the presence of all four ribonucleoside triphosphates and a synthetic cap dinucleotide, m7Gp3G. The polymerase initiates transcription with a nucleophilic attack by the 3 -OH of the Guo moiety ofm7Gp3G on the a-phosphate... 

Figure 20.13 Summary of the reactions by which all four deoxy-ribonucleoside triphosphates can be synthesised from the nucleosides, adenosine and uridine. The reactions are summaries of the processes presented in Figures 20.8, 20.9 and 20.12. AMP is converted to IMP by a deaminase (Chapter 6). The conversion of UTP to CTP is catalysed by CTP synthetase.

Mercaptopurine ribonucleoside triphosphate is a potent inhibitor of adenylyl transfer to nicotinamide mononucleotide and is itself converted to the NAD analogue in vitro [355, 356]. Since it has not been established that 6-mercaptopurine is converted to its triphosphate in vivo, the significance of these observations is uncertain. It has also been suggested, but not established, that 6-mercaptopurirfe may interfere with co-factor A [357,358]. 

I. 17.4.2], also known as ribonucleoside-triphosphate reductase, catalyzes the reaction of a 2 -deoxyribonucleo-side triphosphate with oxidized thioredoxin and water to produce a ribonucleoside triphosphate and reduced thioredoxin. In this case, cobalt and ATP are cofactors. 

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. 

There are three major types of RNA that participate in the process of protein synthesis ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA). They are unbranched polymers of nucleotides, but differ from DNA by containing ribose instead of deoxyribose and uracil instead of thymine. rRNA is a component of the ribosomes. tRNA serves as an adaptor molecule that carries a spe dfic amino acid to the site of protein synthesis. mRNA carries genetic information from the nuclear DNA to the cytosol, where it is used as the template for protein synthesis. The process of RNA synthesis is called transcription, and its substrates are ribonucleoside triphosphates. The enzyme that synthesizes RNA is RNA polymerase, which is a multisub-irit enzyme. In prokaryotic cells, the core enzyme has four subunits—... 

Initiation of transcription involves binding of the RNA polymerase to the promoter region. This sequence contains characteristic consensus nucleotide sequences that are highly conserved. These include the Pribnow box and the -35 sequence. Elongation involves RNA polymerase copying one strand of the DNA double helix, pairing C s with G s and A s (on the DNA template) with U s on the RNA transcript. Substrates are ribonucleoside triphosphates. Termination may be accomplished by the RNA polymerase alone, or may require p factor. 

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. 

The first successful attempts to study RNA evolution in vitro were carried out in the late sixties by Sol Spiegelman9 and his group at Columbia University (Spiegel-man, 1971). They made use of an RNA replicase isolated from Escherichia coli cells infected by the RNA bacteriophage QP and prepared a medium for replication by adding the four ribonucleoside triphosphates (GTP, ATP, CTP, and UTP) in a suitable buffer solution. QP RNA, when transferred into this medium, instantaneously started to replicate. Evolutionary experiments were carried out by means of the serial transfer technique (Figure 4). Materials consumed in RNA replication... 

The second type of terminator lacks the U region and requires the p termination factor for RNA chain release. The p factor is a hexamer of 45 kD subunits that binds a stretch of 72 nucleotides of single-stranded RNA. The p factor hydrolyzes ribonucleoside triphosphates to nucleoside diphosphates in the presence of single-stranded RNA and moves unidirectionally along nascent mRNA toward the transcription bubble and breaks the RNA-DNA hybrid, pulling away the RNA. 

The gene encoding ppk2 (ppk2) was identified from the amino acid sequence of the purified protein. It encodes a protein of 357 amino acids with a molecular mass of 40.8 kDa. Both of the polyphosphate kinases ppkl and ppk2 may be involved in regulation of the level of ribonucleoside triphosphates and deoxyribonucleoside triphosphates that modulate cell division and survival in the stationary phase (Ishige et al., 2002). 

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. 

No. All RNA polymerases use duplex DNA as a template and copy one of the strands they synthesize RNA in the 5 — 3 direction and use ribonucleoside triphosphates as substrates. 

Despite these similarities, there are some very important differences between the reactions carried out by DNA polymerase and RNA polymerase. Remember that DNA is synthesized with the use of four deoxyn bon ucl cosidc triphosphates (dATP, dGTP, dTTP, and dCTP). In the double-stranded DNA helix, adenine bases hydrogen-bond with thymine bases, and guanine bases hydrogen-bond with cytosine bases. RNA, however, is synthesized with the use of four ribonucleoside triphosphates (ATP, GTP, CTP, and UTP). Adenine and guanine bases in the DNA template will direct the addition of uracil and cytosine bases to the growing RNA molecule, respectively, while... 

The best-studied enzymes to date that contain glycyl radicals are pyruvate formate-lyase (PFL) and a ribonucleoside triphosphate reductase (ARR), both isolated from anaerobically growing E. coli. These enzymes play central roles in the anaerobic metabolism of the bacterium. The first catalyzes the reversible formation of acetyl-CoA and formate from pyruvate and coenzyme A, while the second is responsible for synthesizing the deoxyribonucleotide monomers of the polymer DNA. It is intriguing to note that formate, a product of the PFL reaction, is a substrate for the ARR. It supplies the reducing equivalents needed for each round of deoxynucleotide synthesis. ... 

Lawrence, C. C., and Stubbe, J., 1998, The function of adenosylcobalamin in the mechanism of ribonucleoside triphosphate reductase from Lactobacillus leichmanrdi. Curr. Opin. Chem. Biol. 2 650n655. 

Licht, S. S., Booker, S., and Stubbe, J., 1999a, Studies on the catalysis of carbon-cobalt bond homolysis by ribonucleoside triphosphate reductase evidence for concerted carbon-cobalt bond homolysis and thiyl radical formation. Biochemistry 38 12219 1233. 

Lawrence CC, Stubbe J. The function of adenosylcobalamin in the mechanism of ribonucleoside triphosphate reductase from... 

Chen D, Abend A, Stubbe J, Frey PA. Epimerization at carbon-5 of (5 R)-(5 - H)adenosylcobalamin by ribonucleoside triphosphate reductase cysteine 408-independent cleavage of the Co-C5 bond. Biochemistry 2003 42 4578 584. 

Activated precursors. All iour ribonucleoside triphosphates —ATP, GTP, UTP, and CTP—are required. 

The flow of genetic information in normal cells is from DNA to RNA to protein. The synthesis of RNA from a DNA template is called transcription, whereas the synthesis of a protein from an RNA template is termed translation. Cells contain several kinds of RNA messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA), which vary in size from 75 to more than 5000 nucleotides. All cellular RNA is synthesized by RNA polymerase according to instructions given by DNA templates. The activated intermediates are ribonucleoside triphosphates and the direction of... 

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