Nucleotides 5 —>3 direction

Niemeyer, C M, Sano, T, Smith, C L, and Cantor, C R (1994) Oligo-nucleotide-directed self-assembly of proteins semisynthetic DNA-strepta-vidin hybrid molecules as connectors for the generation of macroscopic arrays and the construction of supramolecular bioconjugates. Nucleic Acids Res. 22, 5330-5339. 

Like purine nucleotides, pyrimidine nucleotides can be synthesized either de novo or by the salvage pathways from nucleobases or nucleosides. However, salvage is less efficient because, except in the case of utilization of uracil by bacteria, and to some extent by mammalian cells, pyrimidine nucleobases are not converted to nucleotides directly but only via nucleosides. 

The kinetic properties (see Section II,E) and spectral characteristics (7, 9, 10, 17, 19) of the BB-specific transhydrogenases strongly suggest that these enzymes are fiavoproteins. Consistent with this suggestion, Louie and Kaplan (7) showed that, in the presence of 1 M urea, was taken up from the medium and was incorporated into the reduced nicotinamide nucleotide. Direct proof for the occurrence of FAD in both the... 

It is of interest that water-soluble carbodiimides have been used to couple nucleotides directly to proteins, presumably by formation of a P-N bond. ... 

Velyvis A, Yang YR, Schachman HK, Kay LE (2007) A solution NMR study showing that active site ligands and nucleotides directly perturb the allosteric equilibrium in aspartate transcarbamoylase. Proc Natl Acad Sci USA 104 8815-8820... 

MT-binding protein affinity does not appear to involve binding to the nucleotide directly, but rather suggests that the conformations of monomers within a polymer retain enough flexibility or stmctural plasticity that they present different surfaces on the filament exterior depending in part on the bound nucleotide. ... 

Llpolysls In Adipose Tissue - It now appears reasonably well established that hormone-induced llpolysls In adipose tissue Is mediated, at least In part, by cAMP. Attempts to Induce llpolysls by adding the cyclic nucleotide directly to Intact adipose tissue preparations, however, have not generally been successful except In a medium devoid of both Ca and... 

In conclusion, our results showed that counting labeled nucleotides directly on dried DEAE disks produces erroneous results. The failure to recognize this is responsible for the variable and low thymidine kinase activities in the cerebellum reported in previous studies (Weichsel, 1974, Weichsel and Dawson, 1976). The elution of the labeled nucleotides with 1 ml of 1 M HCl containing 0.5 M NaCl in the counting vial in improved counting efficiency and reproducibility. Furthermore, it is not necessary to dry the disks, thus the overall method became less time-consuming. 

The utilization of free purines is accomplished by reaction with PRPP to form nucleotides directly. 

The hypothesis that the P5C-initiated cascade is due to the activation of the oxidative arm of the pentose phosphate pathway requires direct confirmation because the nonoxidative arm can also produce ribose 5-phosphate. Using differentially labeled glucose and measuring the radioactivity incorporated as the ribose moiety of nucleotides, direct support for this hypothesis was obtained. The underlying rationale for these experiments is that glucose labeled in the C-1 carbon would lose its radioactivity as C02 in the oxidative arm whereas glucose labeled in the C-6 position would result in labeled ribose via both the oxidative and nonoxidative arms (45). In normal human red cells P5C had no effect on... 

Hydrophobic interactions between nonpolar amino-acid residues and the bases of DNA contribute to the function of DNA binding proteins through conformational effects and direct interactions with the DNA nucleotides. Direct evidence of the role of hydrophobic interactions in DNA-protein complexes has been found for T7 RNA polymerase [69, 70], Epstein-Barr virus replication factor EBNAl [71], and bovine papillomavirus helicase El [72, 73], for example. 

Because nucleotides are added m the 5 — 3 direction the processes by which the two new chains grow are different Cham growth can be continuous m the leading strand but not m the lagging strand... 

Among the 76 nucleotides of tRNA are two sets of three that are especially important The first is a group of three bases called the anticodon, which is comple mentary to the mRNA codon for the ammo acid being transferred Table 28 3 lists two mRNA codons for phenylalanine UUU and UUC (reading m the 5 3 direction) Because base pairing requires the mRNA and tRNA to be antiparallel the two anticodons are read m the 3 5 direction as AAA and AAG... 

For mixture.s the picture is different. Unless the mixture is to be examined by MS/MS methods, usually it will be necessary to separate it into its individual components. This separation is most often done by gas or liquid chromatography. In the latter, small quantities of emerging mixture components dissolved in elution solvent would be laborious to deal with if each component had to be first isolated by evaporation of solvent before its introduction into the mass spectrometer. In such circumstances, the direct introduction, removal of solvent, and ionization provided by electrospray is a boon and puts LC/MS on a level with GC/MS for mixture analysis. Further, GC is normally concerned with volatile, relatively low-molecular-weight compounds and is of little or no use for the many polar, water soluble, high-molecular-mass substances such as the peptides, proteins, carbohydrates, nucleotides, and similar substances found in biological systems. LC/MS with an electrospray interface is frequently used in biochemical research and medical analysis. 

A potentially general method of identifying a probe is, first, to purify a protein of interest by chromatography (qv) or electrophoresis. Then a partial amino acid sequence of the protein is deterrnined chemically (see Amino acids). The amino acid sequence is used to predict likely short DNA sequences which direct the synthesis of the protein sequence. Because the genetic code uses redundant codons to direct the synthesis of some amino acids, the predicted probe is unlikely to be unique. The least redundant sequence of 25—30 nucleotides is synthesized chemically as a mixture. The mixed probe is used to screen the Hbrary and the identified clones further screened, either with another probe reverse-translated from the known amino acid sequence or by directly sequencing the clones. Whereas not all recombinant clones encode the protein of interest, reiterative screening allows identification of the correct DNA recombinant. 

DNA polymerase enzymes all synthesize DNA by adding deoxynucleotides to the free 3 -OH group of an RNA or DNA primer sequence. The identity of the inserted nucleotide is deterrnined by its abiHty to base-pair with the template nucleic acid. The dependence of synthesis on a primer oligonucleotide means that synthesis of DNA proceeds only in a 5%o V direction if only one primer is available, all newly synthesized DNA sequences begin at the same point. 

Fig. 6. DNA sequence analysis, (a) Simplified methodology for dideoxy sequencing. A primer, 5 -TCTA, hybridized to the template, is used to initiate synthesis by DNA polymerase, (b) Stmcture of 2, 3 -dideoxy CTP. When no 3 -OH functionaUty is available to support addition of another nucleotide to the growing chain, synthesis terminates once this residue is incorporated into the synthetic reaction, (c) Representation of a DNA sequencing gel and the sequence, read from bottom to the top of the gel, gives sequence information in the conventional 5 to 3 direction.

Pig. 3. Representation of promoter sites on the pro-enkephalin gene. The numbers represent the distance in nucleotides from the pro-enkephalin initiation codon the arrow indicates the direction of transcription. The TATA promoter box occurs immediately before the pro-enkephalin initiation site the AP-2 site, which binds immediate-early gene products, is 70 nucleotides upstream, and the CRE site, which binds a regulatory protein involved in cAMP induction of mRNA synthesis, is 107 nucleotides upstream from the initiation codon. The expanded section shows that the CRE site actually consists of two elements, ENKCRE-1 and ENKCRE-2, which separately confer cAMP sensitivity to pro-enkephalin mRNA synthesis. 

Cellular protein biosynthesis involves the following steps. One strand of double-stranded DNA serves as a template strand for the synthesis of a complementary single-stranded messenger ribonucleic acid (mRNA) in a process called transcription. This mRNA in turn serves as a template to direct the synthesis of the protein in a process called translation. The codons of the mRNA are read sequentially by transfer RNA (tRNA) molecules, which bind specifically to the mRNA via triplets of nucleotides that are complementary to the particular codon, called an anticodon. Protein synthesis occurs on a ribosome, a complex consisting of more than 50 different proteins and several stmctural RNA molecules, which moves along the mRNA and mediates the binding of the tRNA molecules and the formation of the nascent peptide chain. The tRNA molecule carries an activated form of the specific amino acid to the ribosome where it is added to the end of the growing peptide chain. There is at least one tRNA for each amino acid. 

Fig. 6. Polymerase chain reaction (PCR) mediated site-directed mutagenesis. The 5 and 3 ends of the nucleotide strands are indicated. The four arrows surrounding the DNA template represent oligonucleotide primers 1—4. See text for discussion.

The nucleotide form of ribavirin does not manifest its antiviral activity simply by lowering the GTP levels, but may indeed participate directly in binding to specific G proteins (124). Ribavirin has recently been studied as an inhibitor of vesicular stomatitis vims and La Crosse vims (125). Of the phosphorylated forms of the dmg, ribavirin-5 -diphosphate was by far the most potent inhibitor of viral repHcation for these two vimses. 

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