Ribonucleotide triphosphate

Na salts of ribonucleotide triphosphates (Roche or Sigma) bovine serum albumin RNase-free, 20 mg/ml (Roche) RNasin ribonuclease inhibitor, 40 U/ml (Promega) both bacteriophage T7 RNA polymerase and RNA Cap structure analog m7G(5/)ppp(5/)G are from BioLabs DNase-RNase-free (Roche) complete EDTA-free proteinase inhibitors cocktail (Roche) pyruvate kinase (PK) (Roche). 

Because the yield of transcription can vary depending upon a large number of factors (type and quality of the DNA template, T7 RNA polymerase, ribonucleotide triphosphates, etc.), it is recommended to optimize the reaction conditions on a small scale before embarking on a large-scale mRNA prep. 

Both the 26S proteasome and the RC hydrolyze all four nucleotide triphosphates, with ATP and CTP preferred over GTP and UTP [58]. Although ATP hydrolysis is required for conjugate degradation, the two processes are not strictly coupled. Complete inhibition of the peptidase activity of the 26S proteasome by calpain inhibitor I has little effect on the ATPase activity of the enzyme. The nucleotidase activities of the RC and the 26S proteasome closely resemble those of E. coli Lon protease, which is composed of identical subunits that possess both proteolytic and nucleotidase activities in the same polypeptide chain. Like the regulatory complex and 26S proteasome, Lon hydrolyzes all four ribonucleotide triphosphates, but not ADP or AMP [18]. 

The acylalanines are characterised by metalaxyl, the most studied member of the group. Metalaxyl exists as two enantiomers, metalaxyl M being the most active. Metalaxyl is known to interact with the RNA polymerase-I-template complex,8 inhibiting the incorporation of ribonucleotide triphosphates into ribosomal RNA. 

Convert mRNAs to cDNAs by reverse transcriptase, using fluorescently labeled deoxy ribonucleotide triphosphates. 

Three types of enzymatic reactions depend upon alkyl corrin coenzymes. The first is the reduction of ribonucleotide triphosphates by cobalamin-dependent ribonucleotide reductase, a process involving intermo-lecular hydrogen transfer (Eq. 16-21). The second type of reaction encompasses the series of isomerizations shown in Table 16-1. These can all be depicted as in Eq. 16-34. Some group X, which may be attached by a C-C, C-O, or C-N bond, is transferred to an adjacent carbon atom bearing a hydrogen. At the same time,... 

Figure 28-4 (A) Hypothetical structure of a "transcription bubble" formed by an RNA polymerase. Shown is a double-stranded length of DNA with the unwound bubble in the center. This contains a short DNA-RNA hybrid helix formed by the growing mRNA. The DNA double helix is undergoing separation at point A as is the hybrid helix at point B. NTP is the ribonucleotide triphosphate substrate. See Yager and von Hippel.71 (B) Stereoscopic view of the structure of RNA polymerase from Thermus aquaticus in a complex with a promoter DNA. Included are the al, all, (0, (3, P, and a subunits. However, the a C-terminal domains have been omitted. The template (t) strand passes through a tunnel, which is formed by the P and P subunits and two of the structural domains of the a subunit. The nontemplate (nt) strand follows a different path. The position of the -10, -35, and UP elements of the DNA are marked. From Murakami et al.33d Courtesy of Seth A. Darst.

Initiation of an RNA chain begins by reaction within the transcription bubble of either ATP or GTP with a second ribonucleotide triphosphate (Eq. 28-2) to form a dinucleotide still bearing a triphosphate at the 5 end. Further addition of nucleotide units at the 3 end by the same type... 

Although this picture seems clear and simple, many uncertainties remain. Transcription does not proceed evenly but by pauses and spurts. This has suggested the possibility of an "inchworm" type of movement of RNA polymerase.72-75 However, the observations may also be explained by variations in the sequence. There are both pausing or stalling sites76 and terminator sequences. The concentrations of the needed ribonucleotide triphosphate precursors will also affect the kinetics. In addition, defects in the... 

Ribonucleotide reductase and the thioredoxin system. In some lactobacilli, vitamin B12 is involved in the reduction of ribonucleotide triphosphate to deoxyribonucleotide. 

RNA polymerase. An enzyme that catalyzes the formation of RNA from ribonucleotide triphosphates, using DNA as a template. 

Enzymes immobilized on PAN gel, unless stated otherwise. The actual phosphorylating agent, ATP, is regenerated either by acetyl phosphate and acetoldnase, or enolpyruvate phosphate and pyruvate kinase. Phosphorylations by a mixture of the common ribonucleotide triphosphates. Not isolated.4 Soluble enzymes. Enzymes immobilized on agarose.1 Enzymes enclosed in a dialysis bag. 

Fig. 2.11. In (a), 3 -end labelling is achieved using deoxynucleotidyl transferase and an ff-[32P] ribonucleotide triphosphate followed by elimination of the ribonucleotide residues. In (b), 5 -end labelling is carried out using polynucleotide kinase and y-[32P]ATP. Partial digestion with spleen phosphodiesterase removes nucleotides sequentially from the 5 -end of the oligonucleotide giving a mixed population of partially degraded molecules each labelled at the 3 -end. Venom phosphodiesterase removes nucleotides from the 3 -end similarly yielding a mixed population of shortened fragments. The products of the reaction are resolved by two-dimensional electrophoresis-homochromatography and the sequence deduced by the characteristic...

FIGURE 6. The role of free radicals in the reduction of ribonucleotides to 2 -deoxyribonu-cleotides catalyzed by AdoCbl-dependent ribonucleotide triphosphate reductase. 

Licht, S. S., Lawrence, C. C., and Stubbe, J., 1999b, Thermodynamic and kinetic studies on cobalt-carbon bond homolysis by ribonucleotide triphosphate reductase The importance of entropy in catalysis. Biochemistry 34 123491242. 

Vitamin B12 (2 a) participates in the aqueous-phase biosynthesis of purine and pyrimidine bases, the reduction of ribonucleotide triphosphates, the conversion of methylmalonyl-coenzyme A to succinyl-coenzyme A, the biosynthesis of methionine from homocysteine, and the formation of myelin sheath in the nervous systems. 

Scheme 3. Adenosylcobaiamin cofactor of ribonucleotide-triphosphate reductase (RTPR).

In addition to the RNA polymerase holoenzyme and a double-strand DNA template containing a promoter, RNA synthesis also requires all four ribonucleotide triphosphates (ATP, GTP, CTP, and UTP) and Mg2+ as a cofactor. 

Most of the carbon that flows through nucleotide synthetic pathways goes into ribonucleotide triphosphates (rNTPs - ATP, CTP, GTP, and UTP). A relatively small fraction is diverted to the synthesis of deoxyribonucleoside triphosphates (dNTPs). rNTPs are synthesized in excess of dNTPs because most cells contain 5-10 times as much RNA as DNA and because rNTPs have multiple metabolic roles, whereas dNTPs are used only to make DNA. 

DNA bands are visualized either by staining with silver ions or fluorescent DNA-binding dyes, or by autoradiography using (or P)-labeled deoxy-ribonucleotide triphosphates. 

Reduction of ATP to the 2 -deoxy-compound has been carried out on a preparative scale using recombinant Lactobacillus leichmannii ribonucleotide triphosphate reductase. ... 

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