5.5 Nucleotide Composition

An important characteristic of nucleic acids is their nucleotide composition or, in other words, composition and ratio of the consituent monomer units. In the late forties and early fifties, when such research tools as paper chromatography and UV spectroscopy came into being, many analyses of the composition of nucleic acids were carried out (Chargaff, Belozersky). Their results provided a decisive argument for rejecting the older notions of nucleic acids as polymers containing recurring tetranucleotide sequences (so-called tetranucleotide theory of nucleic acid structure reigned supreme in the thirties and forties) and paved the way toward modern concepts not only of the primary structure of DNA and RNA but also their macromolecular structure and functions.

The method for determining the composition of nucleic acids is based on analysis of the products of their enzymatic or chemical degradation. Three chemical methods are usually employed. Acid hydrolysis under vigorous conditions (70 % perchloric acid, 100⁰C, one hour or 100 % formic acid, 175⁰ C, two hours), conducted for analysis of both DNA and RNA, leads to cleavage of all N-glycosidic bonds and formation of a mixture of purine and pyrimidine bases. In the case of RNA, use can be made of mild acid hydrolysis (1 N hydrochloric acid, 100⁰C, one hour), resulting in purine bases and pyrimidine nucleoside 2'(3')-phosphates, as well as alkaline hydrolysis (0.3 N KOH, 37⁰ C, 20 hours), yielding a mixture of nucleoside 2'(3')-phosphates.

Since the number of nucleotides of each type in nucleic acids is equal to that of the corresponding bases, to establish the nucleotide composition of a given nucleic acid one can simply determine the quantitative ratio of the bases. To this end, individual compounds are separated from the hydrolysates by paper chromatography or electrophoresis (when the hydrolysis yields nucleotides). Irrespective of whether it is associated with the carbohydrate moiety or not, each base displays a characteristic absorption maximum in the UV spectrum, whose intensity depends on concentration. Therefore, the UV spectra of the separated compounds can be used to find the quantitative ratio of the bases and, consequently, determine the nucleotide composition of the starting nucleic acid.

Quantitative analysis of minor nucleotides, especially such unstable ones as dihydrouridylic acid, is based on enzymatic hydrolysis (snake venom and spleen PDE).

Experience with the above analytic techniques has shown that nucleic acids of different origin consist, with rate exceptions, of four- major nucleotides, whereas minor nucleotides may vary widely.

As will be shown later, analyses of the nucleotide composition of DNA have made it possible to establish its three-dimensional structure.

While studying the nucleotide composition of native DNAs differing in origin, Chargaff noticed the following regularities.

1. All DNAS, regardless of their origin, contain equal numbers of purine and pyrimidine bases. Hence, in any DNA there is a pyrimidine nucleotide for each purine one.

2. Any DNA always contains equal amounts of adenine and thymine, guanine and cytosine, in pairs usually denoted A=T and G=C. These two regularities give rise to a third one.

3. The number of bases containing amino groups in positions 4 of the pyrimidine ring and 6 of the purine ring (cytosine and adenine) equals that of bases containing an oxo group in each of the same positions (guanine and thymine); that is, A + C = G + T. These regularities have become known as Chargaffs rules. It was also established that for each type of DNA the sum of guanine and cytosine is not equal to the sum of adenine and thymine, which is to say that the ratio (G + C)/(A + T) usually differs from unity (it may be greater or less than unity). This serves as an indicator to distinguish between the two main types of DNA: the A:T type with adenine and thymine being predominant and the G :C type with prevalence of guanine and cytosine.

The ratio of the sum of guanine and cytosine to that of adenine and thymine, characterizing the nucleotide composition of a given DNA, is known as the coefficient of specificity. Each DNA has a characteristic coefficient of specificity, which may vary from 0.3 to 2.8. In calculating this coefficient, one must take into account the content of minor bases and replacement of the major bases by their derivatives. For example, when the coefficient of specificity is calculated for DNA from wheat embryos, containing six per cent of 5-methy]cytosine, the latter forms part of the sum of guanine (22.7 %) and cytosine (16.8). The meaning of Chargaffs rules for DNA became understood after its three-dimensional structure had been established.

5.5.2 Composition of RNA

The first indications of the nucleotide composition of RNA came from the analysis of RNA preparations representing mixtures of cellular RNAs (ribosomal, messenger and transfer RNA, known as total RNA fraction). Chargaff's rules are not obeyed in this case, although there is a certain relationship between the content of guanine and cytosine on the one hand and that of adenine and uracil on the other.

The results of RNA analyses conducted in recent years indicate that individual RNAs do not obey Chargaff's rules either. However, the differences in the contents of each pair of bases are insignificant for most RNAs so that we can say that in general Chargaffs rules hold for RNA as well. This is due to the macrostructural features of RNA.

As has already been mentioned, minor bases are characteristic structural features of some RNAS. The corresponding nucleotides are usually present in transfer and some other RNAs in very small amounts, which is why determination of the nucleotide composition of such RNAs in its entirety is sometimes rather difficult.

In conclusion of this section it should be emphasized once again that knowledge of the nucleotide composition of DNA and RNA has been fundamental in elucidating the primary structure of these biopolymers. What is more, it has prepared the groundwork for concepts of the macromolecular structure of DNA and RNA as well as their functions. Yet the nucleotide composition ist not fully representative even of the primary biopolymer structure. To establish the primary structure of nucleic acids one must know the sequence in which nucleotides occur in individual nucleic acid molecules.