Copyright © 2000-2011 Mark Brandt, Ph.D. 71 Nucleotides and Nucleic Acids Nucleotides have a wide variety of functions. One major function is to provide the thermodynamic driving force for a number of chemical reactions. This is especially well-known for ATP, but GTP is also used for a variety of reactions, UTP is used in glycogen and complex carbohydrate biosynthesis, and CTP is used in complex lipid synthesis. Nucleotides are used to form intracellular signaling molecules such as cAMP and cGMP. In addition, ATP, ADP, and AMP act as signals to modulate energy metabolism. Nucleotides form parts of some cofactors, including NAD, FAD, and coenzyme A. Finally, nucleotides are the monomer units that comprise the nucleic acids RNA and DNA. Cells maintain pools of free nucleotides for a variety of purposes. Adenosine derivatives are the most common free nucleotides, because ATP is used in the largest number of reactions. In addition, ATP is converted into S-adenosyl-methionine, and a number of other molecules involved in metabolic reactions. As mentioned above, pools of other free nucleotides are also important in some types of reactions, although these pools tend to be much smaller than those of ATP. Synthesis of nucleic acids (and especially synthesis of DNA) requires synthesis of nucleotides, because the cellular pools of the required free nucleotides are insufficient to provide all of the monomer units required. Nucleotide nomenclature and structure Nucleotides are comprised of a nitrogen-containing molecule, called a base, attached to a ribose ring. The bases are derivatives of two possible ring structures, purine and pyrimidine, and are numbered according to their parent compound. The bases all contain significant conjugated π-systems, which absorb ultraviolet light.16 The base and ribose ring together are termed a nucleoside (the suffix “-oside” means a compound covalently bonded to carbohydrate). The base and the ribose with one or more phosphate attached are termed a nucleotide. The ribose ring numbering is 16 DNA and RNA exhibit a maximum absorption at about 260 nm, due to the absorbance of the nucleotide bases. Most proteins have a maximum absorption at 280 nm, due to the absorbance of tryptophan and tyrosine. One method for assessing the relative amount of protein and nucleic acid in a sample is to measure the A260/A280 ratio; a ratio of above 1.8 indicates that the solution contains nucleic acid with little protein. NNNNNN123456789123456Purine PyrimidineOOHHOCH2HOBase1´2´3´4´5´Nucleoside1´2´3´4´5´NucleotideMonophosphate1´2´3´4´5´2´-DeoxyribonucleotideMonophosphatePOOOOOHOCH2BasePOOOOOOHHOCH2BaseCopyright © 2000-2011 Mark Brandt, Ph.D. 72 from 1´ to 5´. The “prime” modifier to the ribose ring number designates the ribose carbons as distinct from the atoms of the purine or pyrimidine ring. Nucleotides all have the base attached to the 1´ carbon. Deoxyribonucleotides, the nucleotides present in DNA, lack the 2´-hydroxyl. The base in the nucleoside can have one of two possible conformations: syn and anti. Both are present, although the anti conformation is more common physiologically, due both to the lower steric strain and to that fact that this conformation is required for nucleic acid structure. The rotation about the base-ribose bond is restricted by steric hindrance, so the nucleotide must be synthesized in one form or the other. (For purines, the syn structure is drawn most frequently; this is not a reflection of the common conformation, but instead merely allows the drawing to occupy less space on the page!) The most common bases are the two purine and three pyrimidine derivatives shown below. OOHHOCH2ONNNNNH2POOONNNNNH2OOHHOCH2OPOOOsyn antiNNNHNNH2HNNNHNOH2NNHNONH2AdenineGuanineCytosineThymineUracilBase NucleotidePurines PyrimidinesBase NucleotideAdenosineGuanosineCytidineUridineThymidineNHNHOONHNHOOH3CCopyright © 2000-2011 Mark Brandt, Ph.D. 73 These are not the only bases used in physiology. Others include xanthine and hypoxanthine (intermediates in purine metabolism), a methylated version of adenine (with the methyl group attached to the nitrogen attached to C6), a methylated version of cytosine (5-methyl-cytosine), pseudouridine (which has the ribose attached to C5 instead of N1 of uracil), and 1,3,7-trimethylxanthine, better known as caffeine. Uric acid is a major excretion product following purine degradation; accumulation of uric acid in tissues results in gout. The phosphates in nucleotide triphosphates are most commonly attached to the 5´ carbon. If the site of phosphate attachment is not given, it is assumed to be at the 5´-position, while other sites of attachment must be stated explicitly. Thus, AMP is 5´-AMP, while 3´-AMP has to be written out. RNA contains adenosine, cytidine, guanosine, and uridine (commonly abbreviated as A, C, G, and U); DNA contains deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine (commonly abbreviated as A, C, G, and T). Note that in nucleic acids, thymine is therefore only present in DNA, and uracil is only present in RNA. In humans, most purines and pyrimidines present as nucleotides (the major exceptions are xanthine and uric acid). Some organisms use purines for other purposes, and maintain the purines in the free form. An example of this is provided by the series of methylated xanthine derivatives produced in plants that have biological activity in humans: 1,3 dimethylxanthine (theophylline, an active ingredient in chocolate), 3,7 dimethylxanthine (theobromine, found in tea), and 1,3,7 trimethylxanthine (caffeine). NNNHNOHNNHNHNOOHNNHNHHNOOOHNNHOONNNNOOCH3H3CCH3Hypoxanthine Xanthine Uric acid Pseudouracil 1,3,7-TrimethylxanthineNNNNNH2OOHHOCH2OPOOONNNNNH2OOHOCH2HOPOOOAMP 3´-AMPCopyright © 2000-2011 Mark Brandt, Ph.D. 74 The structure and role of nucleic acids Nucleic acids are polymers of nucleotides, in which the phosphate from the 5´ position of one nucleotide is attached to the 3´ hydroxyl of the preceding nucleotide. This phosphodiester link is created using the energy from the triphosphate form of the nucleotide being