The structure of DNA and RNA

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A more radical departure from B helix is called Z-DNA (Z helix), so-named because the backbone has a zigzag shape. Although double stranded, the Z helix is left-handed, and has 12 base pairs per turn (Fig.1.12). Thus the length of a turn is 4.5 nm rather than 3.5 nm (B helix), which make Z helix appear longer and slimmer-looking than B helix. Different conformations of DNA molecules can coexist within the same molecule when they exist as the very long DNA molecules inside cells. Regions of the Z conformation can be found interspersed among regions of essentially B conformation. Z form predominantly occurs in DNA regions with a regular alternation of purines and pyrimidines (e.g. CGCGCG). Compared to A- and B-DNA, in Z-DNA the nucleotide bases are flipped upside down, relative to the phosphate backbone. Some of the helical parameters of different helical configurations (A-, B- and Z-DNA) are presented in Table 1.2.

Initially significance of occurrence of Z-DNA was not realized. Investigations into the biological role of Z-DNA eventually discovered that Z-DNA is largely found in transcribing genes and that the cessation of transcription results in rapid conversion of Z-DNA to B-DNA through the action of topoisomerases. It has been proposed that the negative torsional strain induced by the movement of RNA polymerase stabilize Z-DNA formation near the transcription start site. Thus, Z-DNA was a metastable conformation, forming and disappearing depending upon physiological activities. A protein responsible for mRNA editing has been reported to be activated upon binding to Z-DNA upstream of a gene. Although the sequences which can form Z-DNA are essentially not found in the bacteria E. coli, more than 10% of the eukaryotic genomes contain such sequences.

Although most of DNA in nature is double stranded, single-stranded DNA molecules may also exist in some viruses. In such cases the ratio of purines and pyrimidines does not remains equal to one. The structure of this DNA is much more irregular, the single strand folds back on itself and short double-stranded helical regions may form between complimentary regions of the molecule. These duplex structures are separated by loop structures, and residual single-stranded regions. As single-stranded DNA molecules have more contours and compact, and they are denser than double-stranded DNA.

Markin and Frank-Kamenetskii in 1994 reported occurrence of triple- or even four-stranded DNA molecules which are known as H form of DNA molecule. In DNA sections where one strand comprises only purine and the other only pyrimidine bases, triple-stranded helices may form (Fig1.13). The main element of the H form is a triple helix stabilized by Watson-Crick and Hoogsteen base pairs. Under these circumstances, one strand of a repeating unit folds back into the major groove of the preceding repeating unit. This conformation leaves one strand of the repeating unit unpaired. The four-stranded helix is characterized by G-rich region of DNA (Fig 1.14). Such structures are often found at the telomeres of chromosomes, and apparently take part in the process of meiosis. Occurrence of the H form is stimulated by the reduction of pH of the solution.

In 1963, it was discovered that double stranded DNA of the polyoma virus exist in a closed circular form. Thereafter, it became clear that many DNAs, especially prokaryotic chromosomes and mitochondrial and chloroplast DNA, are circular, with the ends of the helix covalently joined. This finding has raised topological questions regarding the effect of the circular state on the helical structure. Due to polarity of the strands, the 5’-terminus of one strand can only join its own 3’-end to close the circle. Therefore, circular, double stranded DNA is actually two circles of single stranded DNA twisted around each other (Fig.1.15).

Two forms of circular DNA molecules are extracted from the cell, designated as form I and form II. The more compact form I was found to form into form II after a single stranded nick was introduced into one chain of the double helix. In a situation where the double helix has to twist 3600 in the same direction before joining the ends, it would have the effect of further tightening the double helix, as the double helix already has a complete turn every 10-11 bases. If the twist is in the opposite direction of the helix, it would loosen the helix. Circular DNA that has incorporated one or more twist to increase the number of times one strand crosses the other is said to be positively supercoiled or superhelical. A negative superhelix results from decreasing the number of times the strands twist. The ‘positive’ and ‘negative’ supercoiling refers to different directions of twisting under different DNA conformations. In the right- handed helix (B and A conformation), ‘positive’ supercoiling would add additional right-handed twist. On the other hand, in the left handed helix (Z conformation), ‘positive’ supercoiling would entail left-handed twist. DNA compensates for the higher energy state of supercoiling by modifying the dimensions (e.g., pitch) of the preferred conformations of the double helix.

In the event of positive and negative supercoiling the axis of the double helix curves slightly to maintain the 10-base periodicity of the B helix. Other effect of highly negative supercoil DNA may be conformational changes in the DNA molecule, leading to switch to a Z structure, thereby compensating for two negative turns per left-handed turn. Highly negative supercoil DNA can also be stabilized by the formation of short bubbles of unpaired, single-stranded DNA, called cruciform structure (Fig.1.16). Cruciform are conformations that can be adopted by highly symmetrical regions of DNA. Because of the 5’ to 3’ polarity of the strands and the complimentary of bases, cruciform occur in the regions of palindromic (inverted repeat) DNA. Such cruciform structures in the DNA may serve as the protein binding sites, involved in the regulation of gene expression. DNA structures are known to remain dynamic during the life cycle of a cell. Thus many regions of DNA molecule may undergo conformational change between a normal B helix and other forms to perform its role effectively. It has been shown that the enzymes called topoisomerases can modify the superhelicity of closed circular DNA molecules. This is accomplished by breaking one or both strands and twisting the ends with respect to each other before linking them together again.

Both the sugar and phosphate which constitute the backbone of the DNA molecule are soluble in water. On the other hand bases present in the middle of the helix are relatively hydrophobic and insoluble in water. Since the bases are flat, they stack on top of each other in order to form a hydrophobic micro-environment. The bases twist slightly in order to maximize their hydrophobic interactions with each other and due to this twisting process the helical structure is developed. Thus the reason for development of a helix in DNA is primarily due to the hydrophobic stacking interactions of the bases.

The terms major and minor grooves are based on the two grooves of the Watson-Crick B-DNA structure. The major groove occurs where the backbones are far apart, and the minor groove occurs where they are close together. Although the dimensions of the major and minor grooves are different for the three different types of helix, from the point of view of the bases, the major groove is always on the same side for a given base pair. In B-DNA the width of major groove is 22 Å and that of minor groove is 12 Å, where as in A- and Z-DNA the widths of both the grooves are much smaller. After base pairing between A-T or G-C, it can be observed that sugars are closer in one side of the base pair than the other. There is less space between the sugars on the lower side of the base pair. Thus the convention is that the side closest to the sugars is called the minor groove side and the reverse is the major groove side.

Certain proteins bind to DNA to alter its structure or to regulate transcription (copying DNA to RNA) or replication (copying DNA to DNA). It is easier for these DNA binding proteins to interact with the bases (the internal part of the DNA molecule) on the major groove side, as the interference of the backbones are not there. Due to narrowness of the minor grooves, edges of the bases shall not be available to the regulatory proteins. In B-DNA helices, binding of regulatory proteins to specific base sequences take place through insertion of alpha-helix into the major groove. Proteins that bind DNA nonspecifically (e.g. chromatin proteins) usually bind DNA in the minor groove, through interaction with their beta-strand.

There are three primary types of RNA present within the cells; ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA). Each of these general classes of RNA is actually composed of several unique types; 3 or 4 rRNAs, up to 50 tRNAs, and over 1000 mRNAs. RNA molecules like DNA are linear polynucleotide chain. However, RNA has the following distinction; ribose sugar replaces deoxyribose, and thymine (T) is replaced by uracil (U). Further, with the exception of certain viruses, RNA is single-stranded. The RNA chains can fold back on themselves, creating loops and small base-paired stretches between complimentary regions, thereby forming stem-loop or hairpin structures (Fig.1.17). RNA is defined with respect to its primary (the order of the nucleotides in the chain), secondary (the pattern of base pairing in the fold back structure), and tertiary (the conformation of the molecules in three dimensions) structures.

The structure of RNA has direct relationship to its functions. Therefore, to develop a mechanism to predict the pattern of base pairing in RNA molecules, considerable research has been carried out. Basically the secondary structures are considered to develop a model as the techniques involved in prediction are empirical. All the predictions have been made through computer programming based on either of the following considerations: (i) the number of base pairs in the molecules, or (ii) the overall free energy of the molecule using assigned energy values for different base pairs. Since G-C base pairs are held together by three hydrogen bonds, they are expected to pair more consistently for bonding in the model compared to A-U pairs. Second approach is to look for available information on sequence of RNA molecules from several other organisms.

The structures of nucleic acids are usually so large in size that they are difficult to characterize, with few exceptions like tRNA. Thus the strategy adopted is to breakdown the polymer into smaller fragments. The phosphodiester bonds of both DNA and RNA can be broken by hydrolysis (addition of a H2O molecule) either chemically or enzymatically.

Hydrolysis of both DNA and RNA occur at very low pH (1 or less). At higher pH of 13, the DNA remains very resistant to hydrolysis, whereas RNA remains sensitive to alkaline hydrolysis due to presence of 2’-OH group. A variety of enzymes called nucleases hydrolyse nucleic acids readily. They usually show chemical specificity and are classified as either deoxyribonucleases (DNase) or ribonucleases (RNase). Some DNase act only on single-stranded molecules, while others act only on double-stranded molecules, although DNase which acts on both kinds are also exists. Nucleases which act at the end of a nucleic acid, removing a single nucleotide at a time, are called exonucleases. They can also be specific for the 3’ to 5’ end of the strand. Nucleases which act within the strand are called endonucleases. Some of endonucleases can also be very specific in that they cleave only between particular bases.

Primary structures of many RNAs have been determined with the use of base-specific endonucleases. For example, endonuclease RNase T1 cleaves an RNA chain 3’ to a G, and pancreatic RNase cleaves 3’ to a U or C. In contrast, base-specific DNase is not available.

Recently, it has been discovered that some RNAs have a transphosphorylation catalytic activity. Previously, the dogma of biochemistry was that only proteins could have the catalytic activity for making or breaking bonds. The RNA enzymes, called ribozymes, are able to cleave and form specific phosphodiester bonds in a manner analogous to protein enzymes.

Polysaccharides are polymers of monomeric sugars, most often glucose, or sugar derivatives. Usually they are very complex molecules as covalent bonds may occur between many pairs of carbon atom. This phenomenon allows one sugar unit to join to more than two other sugars, which results in the formation of highly branched macromolecules. These branched structures are sometimes so enormous that they are almost macroscopic. For example, the cell walls of many bacteria are single gigantic polysaccharide molecule.

Biological properties are acquired by the macromolecules through its unique three dimensional structures. These structures are formed due to noncovalent interactions, which are much weaker than typical covalent bonds. The noncovalent interactions that are responsible for determining three dimensional structures in macromolecules are described.

In a linear polypeptide and polynucleotide, each monomer would be free to rotate with respect to its adjacent monomers, due to presence of several bonds, and in the absence of intrastrand interactions. This is limited only by the fact that more than one atom cannot occupy the same space. The three dimensional structure of such a freely rotating chain is called a random coil. They are compact and globular in structure, and change their shape continually, due to constant bombardment by solvent molecules. In nature, presence of random coils is rare, as each molecule will be influenced by many interactions between regions of the chain. These interactions are hydrogen bonding, hydrophobic interactions, ionic bonds, and van der Waals interactions.

A hydrogen bond is formed by three atoms: one hydrogen atom and two electronegative atoms (often N or O). The hydrogen atom is covalently bonded to one of the electronegative atoms, called the hydrogen bond donor. The other electronegative atom is called the hydrogen bond acceptor. The two electronegative atoms may take up some electron density from the hydrogen atom. As a result, each electronegative atom carries partial negative charges and the hydrogen atom carries partial positive charge. The hydrogen atom and the hydrogen bond acceptor can then have attractive interactions.

The strength of the hydrogen bond depends on the donor and acceptor as well as their environment. The bond energy usually ranges from 1 kcl/mol to 5 kcl/mol. This energy is smaller than covalent bond energy, but greater than thermal energy (0.6 kcl/mol at room temperature). Therefore, hydrogen bond can provide a significant stabilising force in macromolecules such as proteins and nucleic acids.

In biological systems three types of hydrogen bonds are found (Fig.1.18). In proteins, intrastrand hydrogen-bonding occurs between a hydrogen atom on nitrogen adjacent to one peptide bond and an oxygen atom adjacent to a different peptide bond. This interaction produces several types of polypeptide chain configurations. In RNA, being single stranded, intrastrand hydrogen bonds induce the polynucleotide strand to fold. In DNA, the double stranded helical structure is possible due to interstrand hydrogen bonding.

A hydrophobic interaction is an interaction between two molecules that are poorly soluble in water. Usually the water molecules have a repulsion action against these molecules. Thus in response to such natural repulsion force, they tend to remain associated.

Hydrophobic property has been shown by many components in proteins and nucleic acids. In the bases of nucleic acids, organic rings carry localized weak charges. The localized charges are sufficient to maintain solubility. However, poorly soluble organic ring portions of the bases tend to cluster, due to hydrophobic interaction. This brings the faces of the rings in contact, an array known as base stacking (Fig.1.19). Stacking gives some rigidity to single polynucleotide chains, and help in determining nucleic acid structure. Many amino acid side chains are very poorly soluble and help in forming clusters or stacks. Hydrophobic interactions can also bring distant hydrophobic parts of a polypeptide chain together and form unstacked clusters. Presence of hydrophobic amino acids like alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine in a polypeptide chain may induce hydrophobic interactions.

Attraction between unlike charges can form ionic bonds. At physiological pH several amino acid chains are ionised. For example: aspartic and glutamic acids (negatively charged carboxyl groups) and lysine, histidine, and arginine (positively charged amino acids). These five amino acids can form ionic bonds.

Between two like charges, ionic interaction can also be repulsive. It would therefore be unlikely for a polypeptide chain to fold in such a way that two lysine are very near each other. Ionic bonds are the strongest of the noncovalent interactions. However, they are destroyed under extreme pH and high salt concentrations. Extreme pH can change the charge of the group thereby disrupting the ionic bond. At high salt concentration, the ions shield the charged groups from one another.

The van der Waals forces are produced due to both permanent dipoles and the circulation of electrons. The attractive force between two atoms is proportional to 1/r6 in which r is the distance between the two nuclei. Thus the attraction force is very weak and is effective only if two atoms are very close (about 1-2 Å) to one another.

Between two atoms, van der Waals forces are very weak, and can be easily disrupted by thermal motion. But, if interactions of several pairs of atoms are combined, the cumulative attractive force can be large enough to withstand being disrupted by thermal motion. However, two molecules can bind to one another by van der Waals force if their shapes are complimentary. Thus the regions can be hold together if their shapes match. van der Waals forces can significantly strengthen other weak interactions such as hydrophobic interaction, if they can complement each other.

Different regions of a linear polypeptide chain, which are lying far apart, may be brought together through the effect of non-covalent interactions. Four different kinds of non-covalent interactions (e.g. hydrophobic cluster, stacked rings, ionic bonds, and van der Waals bonds), may help in binding different regions of a linear molecule is shown in Fig.1.20.