Primary Structure of Nucleic Acids

Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are macromolecular structures composed of regular repeating polymers formed from nucleotides. These are the basic building blocks of nucleic acids and are derived from nucleosides, which are composed of two elements: a five-membered pentose carbon sugar (2-deoxyribose in DNA and ribose in RNA) and a nitrogenous base. The carbon atoms of the sugar are designated ‘prime’ (l′, 2′, 3′, etc.) to distinguish them from the carbons of the nitrogenous bases of which there are two types, purine and pyrimidine. A nucleotide, or nucleoside phosphate, is formed by the attachment of a phosphate to the 5′ position of a nucleoside by an ester linkage (Figure 1). Such nucleotides can be joined together by the formation of a second ester bond by reaction between the phosphate of one nucleotide and the 3′-hydroxyl of another, thus generating a 5′ to 3′ phosphodiester bond between adjacent sugars; this process can be repeated indefinitely to give long polynucleotide molecules (Figure 2). DNA has two such polynucleotide strands; however, since each strand has both a free 5′-hydroxyl group at one end and a free 3′-hydroxyl at the other, each strand has a polarity or directionality. The polarities of the two strands of the molecule are in opposite directions, and thus DNA is described as an anti-parallel structure (Figure3).

Fig1. Structure of the building blocks of nucleic acids.

Fig2. Polynucleotide structure.

Fig3. The anti-parallel nature of DNA. Two complementary strands of DNA in a double helix run in the opposite directions. The two strands are held together by hydrogen bonds between the bases.

The purine bases (composed of fused five- and six-membered rings), adenine (A) and guanine (G), are found in both RNA and DNA, as is the pyrimidine base (a single six-membered ring) cytosine (C). The other pyrimidine bases are each restricted to one type of nucleic acid: uracil (U) occurs exclusively in RNA, whilst thymine (T) is limited to DNA. Thus it is possible to distinguish between RNA and DNA on the basis of the presence of ribose and uracil in RNA, and deoxyribose and thymine in DNA. However, it is the sequence of bases along a molecule that distinguishes one DNA (or RNA) from another. It is conventional to write a nucleic acid sequence starting at the 5′ end of the molecule, using single capital letters to represent each of the bases, e.g. CGGATCT. Note that there is usually no point in indicating the sugar or phosphate groups, since these are identical throughout the length of the molecule. Terminal phosphate groups can, when necessary, be indicated by use of a ‘p’; thus 5′-pCGGATCT-3′ indicates the presence of a phosphate on the 5′ end of the molecule.

Secondary Structure of Nucleic Acids

The two polynucleotide chains in DNA are usually found in the shape of a right handed double helix , in which the bases of the two strands lie in the centre of the molecule, with the sugar–phosphate backbones on the outside. A crucial feature of this double-stranded structure is that it depends on the sequence of bases in one strand being complementary to that in the other. A purine base attached to a sugar residue on one strand is always linked by hydrogen bonds to a pyrimidine base attached to a sugar residue on the other strand. Moreover, adenine (A) always pairs with thymine (T) or uracil (U) in RNA, via two hydrogen bonds, and guanine (G) always pairs with cytosine (C) by three hydrogen bonds (Figure 4). When these conditions are met, a stable double helical structure results, in which the backbones of the two strands are, on average, a constant distance apart. Thus, if the sequence of one strand is known, that of the other strand can be deduced. The strands are designated as plus (+) and minus (−), and an RNA molecule complementary to the minus (−) strand is synthesised during transcription. The base sequence may cause significant local variations in the shape of the DNA molecule and these variations are vital for specific interactions between the DNA and its binding proteins to take place. Although the three-dimensional structure of DNA may vary, it generally adopts a double helical structure termed the B form or B-DNA in vivo. There are also other forms of right-handed DNA such as A, C and Z, which are formed when DNA fibres are experimentally subjected to different relative humidities (Table 1). Experimental evidence suggests these alternative forms occur in both prokaryotic and eukaryotic cells.

Fig4. Base-pairing in DNA. ‘C’ in a circle represents carbon at the 1′ position of deoxyribose.

Table1. The various forms of DNA

The major distinguishing feature of B-DNA is that it has approximately 10 bases for one turn of the double helix (Figure 5); furthermore a distinctive major and minor groove may be identified. In the B form of DNA, the major groove is wider than the minor groove and therefore DNA-binding proteins such as transcription factors bind to the DNA molecule at the wider major groove. The minor groove is a binding site for the dye Hoechst 33258. In certain circumstances. In certain circumstances where repeated DNA sequences or motifs are found, the DNA may adopt a left-handed helical structure termed Z-DNA. This form of DNA was first synthesised in the laboratory and is thought not to exist in vivo. The various forms of DNA serve to show that it is not a static molecule, but dynamic and constantly in flux, and may be coiled, bent or distorted at certain times. Although RNA almost always exists as a single strand, it often contains sequences within the same strand that are self-complementary, and which can therefore base-pair if brought together by suitable folding of the molecule. A notable example is transfer RNA ( tRNA) which folds up to give a clover-leaf secondary structure (Figure 6).

Fig5. The DNA double helix (B form).

Fig6. Clover-leaf secondary structure of yeast tRNA Phe . A single strand of 76 ribonucleotides forms four double-stranded ‘ stem’ regions by base-pairing between complementary sequences. The anticodon will base-pair with UUU or UUC (both are codons for phenylalanine); phenylalanine is attached to the 3′ end by a specific aminoacyl tRNA synthetase. Several ‘unusual’ bases are present: D, dihydrouridine; T, ribothymidine; ψ, pseudouridine; Y, very highly modified, unlike any ‘normal’ base. mX indicates methylation of base X (m 2 X shows dimethylation); Xm indicates methylation of ribose on the 2′ position; p, phosphate.

Separation of Double-Stranded DNA

The two anti-parallel strands of DNA are held together partly by the weak forces of hydrogen bonding between complementary bases and partly by hydrophobic inter actions between adjacent, stacked base pairs, termed base-stacking . Little energy is needed to separate a few base pairs, and so, at any instant, a few short stretches of DNA will be opened up to the single-stranded conformation. However, such stretches immediately pair up again at room temperature, so the molecule as a whole remains predominantly double-stranded.

If, however, a DNA solution is heated to approximately 90 °C or above, there will be enough kinetic energy to denature the DNA completely, causing it to separate into single strands. This is termed denaturation and can be followed spectrophotometrically by monitoring the absorbance of light at 260 nm. The stacked bases of double-stranded DNA are less able to absorb light than the less constrained bases of single-stranded molecules, and so the absorbance of DNA at 260 nm increases as the DNA becomes denatured, a phenomenon known as the hyperchromic effect.

The absorbance at 260 nm may be plotted against the temperature of a DNA solution, which will indicate that little denaturation occurs below approximately 70 °C, but further increases in temperature result in a marked increase in the extent of denaturation. Eventually a temperature is reached at which the sample is totally denatured, or melted. The temperature at which 50% of the DNA is melted is termed the melting temperature (Tm), and this depends on the nature of the DNA ( Figure 7). If several different samples of DNA are melted, it is found that the T m is highest for those DNA molecules that contain the highest proportion of cytosine and guanine, and T m can actually be used to estimate the percentage (C + G) in a DNA sample. This relationship between T m and (C + G) content arises because cytosine and guanine form three hydrogen bonds when base-paired, whereas thymine and adenine form only two. Because of the differential numbers of hydrogen bonds between A–T and C–G pairs those sequences with a predominance of C–G pairs will require greater energy to separate or denature them. The conditions required to separate a particular nucleotide sequence are also dependent on environmental conditions such as salt concentration.

Fig7. Melting curve of DNA.

If melted DNA is cooled, it is possible for the separated strands to reassociate, a process known as renaturation . However, a stable double-stranded molecule will only be formed if the complementary strands collide in such a way that their bases are paired precisely, and this is an unlikely event if the DNA is very long and complex (i.e. if it contains a large number of different genes). Measurements of the rate of renaturation can give information about the complexity of a DNA preparation.

Strands of RNA and DNA will associate with each other, if their sequences are complementary, to give double-stranded, hybrid molecules. Similarly, strands of radio actively labelled RNA or DNA, when added to a denatured DNA preparation, will act as probes for DNA molecules to which they are complementary. This hybridisation of complementary strands of nucleic acids is very useful for isolating a specific fragment of DNA from a complex mixture. It is also possible for small single-stranded fragments of DNA (up to 40 bases in length), termed oligonucleotides , to hybridise to a denatured sample of DNA. This type of hybridisation is termed annealing and again is dependent on the base sequence of the oligonucleotide and the salt concentration of the sample.

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مواضيع ذات صلة

Molecular Analysis of Nucleic Acid Sequences

Genes and Genome Complexity

Genomics: Genome-Wide Analysis of Gene Structure and Function

Chromosomal Organization of Genes and Noncoding DNA

RNA Interference Causes Gene Inactivation by Destroying the Corresponding mRNA

Linkage Studies Can Map Disease Genes with a Resolution of About 1 Centimorgan

DNA Polymorphisms Are Used as Markers for Linkage Mapping of Human Mutations

The Two- Hit Theory of Tumor Suppressor Gene Inactivation in Cancer

DNA Microarrays Can Be Used to Evaluate the Expression of Many Genes at One Time

Hybridization Techniques Permit Detection of Specific DNA Fragments and mRNAs

Cloned DNA Molecules Can Be Sequenced Rapidly by Methods Based on PCR

Cellular and Molecular Mechanisms in Development: Gene Regulation by Transcription Factors