1. Nucleic Acids – Essentials of Molecular Biology

1

Nucleic Acids

CONCEPT OUTLINE
  • Introduction
  • DNA and RNA as Genetic Materials
    • DNA
    • RNA
  • Structure of Nucleic Acids
    • The chemical nature of DNA and RNA
    • Keto-Enol tautomerism of the nitrogenous bases
    • Nucleosides
    • Pyrimidines exist entirely in the anti-conformation
    • Chargaff rule
    • Nucleotides
    • The primary structure of DNA
    • Watson and Crick model of DNA – secondary structure of DNA
    • The DNA grooves
    • DNA conformations
    • B-DNA
    • A-DNA
    • Z-DNA
    • Triple standard DNA
    • Cruciforms or Holliday junction
    • Tertiary structure
  • Properties of the DNA
    • Physical properties of the DNA
    • Chemical stabibility of nucleic acids
  • DNA Topology
    • Linking, twisting and writhing
    • Topoisomerases
  • Types of RNA
  • Types of DNA
    • The chromosomal DNA
    • The autosomal DNA
    • Y-DNA
    • Mitochondrial DNA (mtDNA)
    • Chloroplast DNA
    • Plasmid DNA
  • DNA as Vehicle of Inheritance
    • Griffith transformation
    • Oswald Avery, Colin MacLeod and Maclyn McCarty experiment
    • Disadvantages of the experiment
    • Hershey and Chase experiment
  • Summary
  • References
INTRODUCTION

The complex systems of living organisms encompass hundreds to thousands of proteins that exist help us to carry out our daily functions. The information required to manage this complex system is stored in a set of molecules called ‘nucleic acids’. The three most abundant biological macromolecules are ‘proteins’, ‘nucleic acids’ and ‘polysaccharides’. The information-carrying molecules ‘DNA (deoxyribonucleic acid)’ and ‘RNA (ribonucleic acid)’ are the nucleic acids in biological systems; in addition, RNA molecules act as catalysts.

DNA AND RNA AS GENETIC MATERIALS

DNA

In most living organisms (except viruses), the genetic information is stored in the molecule called deoxyribonucleic acid or DNA. DNA is made and it resides in the nucleus of the living cells. The name DNA was derived from the sugar molecule contained in its backbone—deoxyribose. The high molecular weight nucleic acid, DNA, is found chiefly in the nuclei of complex cells, known as eukaryotic cells, or in the nucleoid regions of prokaryotic cells, such as bacteria.

The first isolation of DNA was accomplished by Johann Friedrich Miescher in 1870. He reported a weakly acidic substance of unknown function and named it as nuclein. A few years later, Miescher separated nuclein into protein and nucleic acid components. In the 1920s, nucleic acids were found to be major components of chromosomes. Nucleic acids contain C, H, N, O and P. Unlike proteins, nucleic acids contain no sulphur. Nucleic acids are acidic in nature due to the phosphoric acid moiety.

RNA

‘RNA’ is distributed throughout the cell, most commonly in small numerous organelles called ‘ribosomes’. It is a lower molecular weight, but much more abundant nucleic acid. The RNAs play a vital role in the transfer of genetic information (transcription) from the DNA library to the protein factories called ribosomes, and in the interpretation of that information (translation) for the synthesis of specific polypeptides. These functions are described in later chapters. Some RNAs, called ‘ribozymes’, have catalytic activity.

STRUCTURE OF NUCLEIC ACIDS

The Chemical Nature of DNA and RNA

The backbone of a nucleic acid is made of alternating pentose ‘sugar’ (deoxyribose sugar in DNA and ribose sugar in RNA) and ‘phosphate’ molecules bonded together in a long chain. Each of the sugar groups in the backbone is attached to ‘nitrogenous base’. The ‘2′-deoxy-’ notation means that there is no -OH group on the 2′ carbon atom

 

 

The nitrogenous bases of DNA are adenine, guanine, cytosine and thymine whereas the nitrogenous bases of RNA are adenine, guanine, cytosine and uracil.

 

 

The nitrogenous bases of nucleic are pyrimidines, which are monocyclic, and purines, which are bicyclic. Each has at least one N-H site at which an organic substituent may be attached. They are all polyfunctional bases and exist in tautomeric forms called keto-enol tautomerism. Adenine is 6-amino purine; guanine is 2-amino-6-oxypurine; thymine is 5-methy 1, 2, 4-dioxypyrimidine; cytosine is 4-amino-2-oxypyrimidine and uracil is 2, 4-dioxypyrimidine.

Keto-Enol Tautomerism of the Nitrogenous Bases

Three of the purine and pyrimidine base components of the nucleic acids could exist as hydroxypyrimidine or purine tautomers, having an aromatic heterocyclic ring. Despite the added stabilization of the aromatic ring, these compounds prefer to adopt amide-like structures. The following diagram explains this.

 

 

Nucleosides

Nucleosides are N-glycosides of 2′-deoxyribose or ribose, combined with the heterocyclic amines through a β-glycosidic linkage. They are formed by the loss of water from a sugar plus a purine or pyrimidine, OH from the anomeric position of the sugar, and H from a nitrogen of the base. Purines bond to the C1′ of the sugar at their N9 atoms. Pyrimidines bond to the sugar C1′ atom at their N1 atoms.

Nucleosides are the basic building blocks of the nucleic acids. In medicine, several nucleoside analogues are used as antiviral or anticancer agents. The nucleosides are adenosine, guanosine, thymidine, methyl uridine, uridine and cytidine respectively named after their bases. The purine nucleosides end with the suffix ‘-sine’: adenosine and guanosine. The pyrimidine nucleosides end with the suffix ‘-dine’: cytidine, uridine and deoxythymidine.

 

 

Pyrimidines Exist Entirely in the Anti Conformation

Two conformational variations are possible for nucleosides based on the rotation around the base-to-sugar bond, and puckering of the sugar ring, called ‘Syn’ and ‘Anti’ conformations. Consider the following two structures for adenosine.

 

 

Pyrimidines exist entirely in the Anti-conformation. The puckering of the sugar ring usually involves having either C2′ or C3′ out of the plane formed by C1′, O and C4′.

If C2′ or C3′ is on the same side of the ring as the glycosidic bond, the conformation is described as endo-; if on the other side, it is exo.

Chargaff Rule

On carefully analysing the DNA from many sources, Erwin Chargaff found its composition to be species-specific. In addition, he found that the amount of adenine (A) always equalled the amount of thymine (T) and the amount of guanine (G) always equalled the amount of cytosine (C), regardless of the DNA source. The ratio of (A+T) to (C+G) varied from 2.70 to 0.35 (Table 1.1).

 

Table 1.1 Nucleoside base distribution in DNA

 

Nucleosides are significantly important constituents that are essential for many vital functions. There are two genetic defects related to nucleosides; one is adenosine deaminase (ADA) deficiency and the other is purine nucleoside phosphorylase (PNP) deficiency, account for two immunodeficiencies that result in severe combined immunodeficiency (SCID).

Adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency are autosomal recessive disorders. Adenosine deaminase and purine nucleoside phosphorylase are ubiquitous ‘housekeeping genes’. In both disorders, the enzyme-deficiency results in toxic metabolites accumulation especially in lymphocytes. In adenosine deaminase deficiency, the toxic metabolites block the development of T-cells, B-cells and natural killer (NK)-cells; while in purine nucleoside phosphorylase deficiency, the metabolites are toxic to the development of T-cells.

The inborn errors are characterized by neurodevelopmental delay and are especially prevalent in purine nucleoside phosphorylase deficiency with neurologic symptoms, including mental retardation and muscle spasticity, reported in 67 per cent of patients. Autoimmune disorders, such as autoimmune haemolytic anaemia, immune thrombocytopenia, neutropenia, thyroiditis and lupus also are associated with the disease.

Adenosine deaminase deficiency results in the absence of T-cells, B-cells and NK-cells, resulting in a form of SCID associated with marked lymphopenia. Purine nucleoside phosphorylase deficiency causes decreased numbers of T-cells and lymphopenia. Serum immunoglobulin (Ig) levels are normal to near-normal, but antibodies are deficient.

Nucleotides

Nucleotides are phosphate esters of nucleosides. The phosphoryl group is attached to the oxygen of the 5′-hydroxyl. Monophospates can be further phosphorylated to produce di- and tri-phosphates. The nucleic acid backbone is a polymer with an alternating sugar-phosphate sequence. The deoxyribose/ribose sugars are joined at both the 3′-hydroxyl and 5′-hydroxyl groups to the phosphate groups in ester links, which are also known as ‘phosphodiester’ bonds. Nucleic acids may be formulated as alternating copolymers of phosphoric acid (P) and nucleosides (N), as follows:

 

∼P–N–P–N′–P–N″–P–N‴–P–N

 

At physiological pH, the phosphates are ionized, as depicted in the picture.

 

 

The nucleotides are named by their nucleoside name, followed by the suffix ‘mono-’, ‘di-’ or ‘triphosphate’: for example, adenosine monophosphate, guanosine triphosphate and deoxythymidine monophosphate (Table 1.2).

 

Table 1.2 Nucleosides and their mono-, di- and triphosphates

 

Nucleotides have a number of roles. They are

  • The monomers for the nucleic acid polymers.
  • Nucleoside triphosphates, such as ATP and GTP, are energy carriers in metabolic pathways.
  • Nucleotides are also components of some important coenzymes, such as FAD, NAD+ and Coenzyme A.
  • Some nucleotides act as intracellular second messengers or signal transducers; for example, cAMP.

‘Single nucleotide polymorphisms’ (SNPs) are DNA sequence variations that occur when a single nucleotide (A, T, C or G) in the genome sequence is altered. For example, an SNP might change the DNA sequence AAGGCTAA to ATGGCTAA.

SNPs do not cause disease, but they can help determine the probability that someone will develop a particular illness. For example, apolipoprotein E (ApoE) is associated with Alzheimer’s disease, which contains two SNPs that result in three possible alleles for this gene: E2, E3 and E4. Each allele differs by one DNA base, and the protein product of each gene differs by one amino acid (genomics.energy.gov, Human genome project).

The Primary Structure of DNA

It is the sequence of nucleotide chains.

Watson and Crick Model of DNA – Secondary Structure of DNA

DNA has a number of special physical and chemical properties that are important to its structure and functioning. The modern era of molecular biology began in 1953 when James D. Watson and Francis H. C. Crick proposed correctly the double-helical structure of DNA (the article ‘Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid’ was released on 25 April 1953 in the journal Nature), based on the analysis of the x-ray diffraction patterns of DNA fibres taken by Rosalind Franklin and Maurice Wilkins.

The important features of their model of DNA are:

  • DNA exists as a pair of molecules and the two strands of DNA are twisted in the shape of a double helix. The two strands are held together by hydrogen bonds, which can be found between the bases attached to the two strands.
  • Two helical polynucleotide chains are coiled around a common axis. The chains run in ‘anti parallel direction’. That is, their 5′ → 3′ directions are oppositely oriented.
  • The purine and pyrimidine bases are positioned inside the helix, whereas the phosphate and deoxyribose units are on the outside. The planes of the bases are perpendicular to the helix axis. The planes of sugars are nearly at right angles to those of the bases.
  • The diameter of the helix is 20 Å. Adjacent bases are separated by 3.4 Å along the axis of the helix and related by a rotation of 36°. Hence, the helical structure repeats after ten residues on each chain; that is, at intervals of 34 Å.
  • Adenine always pairs with thymine and guanine always pairs with cytosine.
  • Any sequence of bases may occur along a polynucleotide chain. ‘The precise sequence of bases carries the genetic information.’
  • ‘The base pairing is highly specific. The precise Watson and Crick base pairing of adenine pairing with thymine, and guanine with cytosine, is because of steric and hydrogen-bonding features.’ A is paired with T through two hydrogen bonds and G is paired with C through three hydrogen bonds. This base-pair complementarity is a consequence of the size, shape and chemical composition of the bases. The presence of thousands of such hydrogen bonds in a DNA molecule contributes greatly to the stability of the double helix. Hydrophobic and van der Waals’ interactions between the stacked adjacent base pairs also contribute to the stability of the DNA structure.
  • Chemical analysis of DNA (Chargaff, 1950) showed that A equals T and G equals C. In vivo DNA exists predominantly as the B-DNA. Watson and Crick derived model also is based on the B-DNA.

 

 

Though many scientific interventions are included to the DNA structure, Watson and Crick model’s four major features remain the same yet today. These features are as follows:

  • DNA is a double-stranded helix. The two strands are connected by hydrogen bonds. The A bases are always paired with Ts and the C bases are always paired with Gs. This explains the Chargaff’s rule.
  • Most DNA double helices are right-handed; that is, if you were to hold your right hand out, with your thumb pointed up and your fingers curled around your thumb, your thumb would represent the axis of the helix and your fingers would represent the sugar-phosphate backbone. Only one type of DNA, called ‘Z-DNA’, is left-handed. The right-handed helix is the favoured conformation in aqueous systems and has been termed the ‘B-helix’.
  • The DNA double helix is anti-parallel, that is one strand runs in the 5′ → 3′ direction, while the other strand runs in the 3′ → 5′ direction. Nucleotides are linked to each other by their phosphate groups, in which the 3′-OH end of one sugar binds to the 5′-PO4 end of the next sugar.
  • The outer edges of the nitrogen-containing bases are exposed and available for potential hydrogen bonding as well apart from connecting the two strands. These hydrogen bonds provide easy access to the DNA for other molecules, including the proteins that play vital roles in the replication and expression of DNA.

 

 

The DNA Grooves

The twisting of the DNA strands around each other leaves gaps between each set of phosphate backbones. There are two gaps/grooves created because of such twisting around the surface of the double helix: one groove, called the ‘major groove’, is 22 Å wide and the other, called the ‘minor groove’, is 12 Å wide. The edges of the bases are more accessible in the major groove. As a result, DNA-binding proteins such as transcription factors usually make contacts to the sides of the bases exposed in the major groove.

DNA Conformations

The precise geometries and dimensions of the double helix can vary. DNA can exist in three different conformations namely A-, B- and Z-DNAs.

B-DNA

The most common conformation in most living cells (Wat-son and Crick Model) is known as B-DNA (Figure 1.1).

A-DNA

A-DNA is a shorter and wider form. It has been found in dehydrated samples of DNA and rarely under normal physiological circumstances. The A-form is more compact than the B-form. There are 11 bases per turn and the stacked bases are tilted (Figure 1.1).

 

Figure 1.1 Different forms of DNA

Z-DNA

The Z-DNA helix is left-handed and has a structure that repeats every two base pairs. Unlike A- and B-DNAs, there is a little difference in the width of the major and minor grooves. The formation of this Z-DNA conformation is generally unfavourable. However, certain conditions can promote it. Alternating purine–pyrimidine sequence (especially poly(dGC)2) or high salt and some cations (all at physiological temperature, 37°C, and pH 7.3–7.4) are some of the factors that favour Z-DNA conformation (Table 1.3).

Z-DNA is a transient form of DNA and it can exist only occasionally, in response to a certain types of biological activity. Z-DNA was first discovered in 1979, but its existence was largely ignored until recently. Certain proteins bind very strongly to Z-DNA, suggesting that Z-DNA plays an important biological role in protection against viral disease (Figure 1.1).

Triple Stranded DNA

A triple-stranded DNA structure can also exist in vitro and possibly during the recombination and DNA repair. For example, when synthetic polymers of poly(A) and polydeoxy(U) are mixed, a triple-stranded structure is formed. The synthetic oligonucleotide can insert as a third strand and binds in a sequence-specific manner called ‘Hoogsteen base pairs’ (Figure 1.2).

Cruciforms or Holliday Junction

‘Palindromes’ are words, phrases or sentences that are the same when read forward or backward, such as ‘radar’, ‘Madam, I’m Adam’ etc. DNA sequences that are ‘inverted repeats’, or palindromes, can form a tertiary structure known as a ‘cruciform’ (meaning ‘cross-shaped’) if the normal inter-strand base pairing is replaced by intra-strand pairing. In effect, each DNA strand folds back on itself in a hairpin structure to align the palindrome in the specific base-pairing form resulting in the formation of cruciforms. The unpaired DNA sequences are looped as the result they are never as stable as normal DNA duplexes. Cruciform structures have a twofold rotational symmetry about their centres and these regions act as the recognition sites for specific DNA-binding proteins. This structure is important for the critical biological processes of DNA recombination and repair that occur in the cell (Figure 1.3).

 

Table 1.3 Comparison of properties of different forms of DNA

 

Figure 1.2 Triple stranded DNA

 

Figure 1.3 Cruciforms or holliday junction

 

The formation of a cruciform structure from a palindromic sequence within DNA. The selfcomplementary inverted repeats can rearrange to form hydrogen-bonded cruciform loops.

Tertiary Structure

This refers to how a DNA is stored in a confined space to form the chromosomes. This varies in prokaryotes and eukaryotes. In prokaryotes, the DNA is folded like a super-helix, usually in circular shape and associated with a small amount of protein. The DNA of cellular organelles such as mitochondria and chloroplasts also takes a similar structure. In eukaryotes, since the amount of DNA from each chromosome is very large, it is compacted into the nucleus with the help of proteins such as histones and other proteins of non-histone nature.

PROPERTIES OF THE DNA

Physical Properties of the DNA

Size and shape

The DNA molecules range in length from about 2 microns (virus) to 2.1cm (Drosophila’s largest chromosome) to 1.6—8.2 cm (human chromosome) (1 kb DNA = 103 base pairs = 0.34 × 10-6 m). Some DNA molecules are circular (E. coli chromosome, mitochondrial DNA, plasmidDNA) and some are linear (human DNA, T7 DNA, lambda phage DNA). DNA molecules can be supercoiled or relaxed.

DNA denaturation and renaturation

The unwinding and separation of DNA strands is referred to as denaturation or ‘melting’. Denaturation can be induced experimentally. For example, if a solution of DNA is heated, the thermal energy increases molecular motion; this breaks the hydrogen bonds and other forces that stabilize the double helix and consequently the strands separate.

The denaturation of the double-stranded DNA can be followed spectroscopically. The purine and pyrimidine bases in the DNA absorb UV light maximally at a wavelength of approximately 260 nm. In the double-stranded DNA, this UV absorption is decreased due to base-stacking interactions. When a DNA is denatured, these interactions are disrupted and, as the result, an increase in absorbance is seen. This change is called the hyperchromic effect. The extent of the effect can be monitored as a function of temperature. Thus, as the DNA denatures its absorption of UV light increases. Near the denaturation temperature, a small increase in the temperature causes a simultaneous loss of the multiple, weak and co-operative interactions holding the two strands together, so that denaturation rapidly occurs throughout the entire length of the DNA (Figure 1.4).

 

 

The temperature at which the strands of 50 per cent of a DNA molecule will separate is called the melting temperature, Tm. Tm depends on several factors. Molecules that contain a greater proportion of G≡C pairs require higher temperatures to denature because the three hydrogen bonds in G≡C pairs make them more stable than A═T pairs with two hydrogen bonds. The percentage of G≡C base pairs in a DNA sample can be estimated from its Tm (Figure 1.4). In addition to heat, the solutions of low ion concentration also favour DNA denaturation, causing it to melt at lower temperatures. DNA is also denatured by exposure to other agents that destabilize hydrogen bonds, such as alkaline solutions and the concentrated solutions of formamide or urea.

 

Figure 1.4 DNA denaturation and renaturation and melting tempertaure of DNA

 

The single-stranded DNA molecules that result from denaturation form random coils without a regular structure. Lowering the temperature or increasing the ion concentration causes the two complementary strands to re-associate to form the double helix. The extent of such ‘renaturation’ is dependent on time, the DNA concentration and the ionic content of the solution. Two DNA strands that are not complementary in sequence will remain as random coils and will not renature. Further, they will not greatly inhibit complementary DNA partner strands from finding each other. The renaturation of melted DNA results in the decrease in UV absorption. This phenomenon is called ‘hypochromicity’.

The principles of DNA denaturation and renaturation form the basis of nucleic acid hybridization. The nucleic acid hybridization technique is used to study the relatedness of two DNA samples and to detect and isolate specific DNA molecules in a mixture containing numerous different DNA sequences.

Chemical Stability of Nucleic Acids

Hydrolysis by acids and alkali

DNA is generally quite stable and resists the action of acid and alkaline solutions. However, in mild acid solutions—at pH 4—the beta-glycosidic bonds to the purine bases are hydrolysed. Protonation of purine bases (N7 of guanine and N3 of adenine) occurs at this pH. The protonated purines undergo hydrolysis. Once depurinated, the sugar can be easily isomerized into the open-chain form and in this form the depurinated (or apurinic) DNA is susceptible to cleavage. DNA is thus acid labile.

In contrast to DNA, RNA is very unstable in alkali solutions due to the hydrolysis of the phophodiester backbone. The 2′-OH group in ribo-nucleotides renders RNA molecules susceptible to strand cleavage in alkali solutions. Thus, RNA is alkali labile.

The alkaline hydrolysis of RNA results in the equimolar mixture of 2′- and 3′-nucleoside mono-phosphates.

 

Hydrolysis by enzymes

Enzymatic hydrolysis of RNA: There are many enzymes that cleave RNA—‘ribonucleases’.

Enzymatic hydrolysis of DNA: DNA is hydrolysed by ‘deoxyribonucleases’. These enzymes may digest a DNA strand from the end(s)—exonucleases—or internally—endonucleases (Table 1.4).

Salt concentration

The negatively charged phosphate groups in the DNA double helix are close together and will tend to repel one another unless they are neutralized. Since the concentration of salt (cations) in solution will affect the degree of neutralization, the stability of the DNA double helices also depends on the salt concentration. Salt ions, polyamines and special DNA-binding proteins help in the neutralization, which is taking place inside the cell.

 

Table 1.4 Nucleases

Exonucleases
5′ → 3′ exonucleases Exonuclease VII, Bal31 nuclease
3′ → 5′ exonucleases DNA polymerase I, Exonuclease l
Endonucleases
Non-specific endonucleases DNase I, Micrococcal nuclease, Mung bean nuclease
Specific endonucleases Restriction enzymes

pH

At a very high pH, the DNA will denature as single-stranded DNA. This is a commonly employed technique and is called alkaline gel electrophoresis. Low pH depurinates DNA (see hydrolysis by acids and alkali), which denatures the double helix. At very low pH, the phosophodiester back-bone of DNA hydrolyses forming nucleotides and nucleosides.

Ionic interactions

Proteins interact with DNA through ionic interactions. For example, the proteins called histones interact with the DNA. Arg and Lys can bind to any of the bases, other than C, by H-bond. However, these two residues almost always bind to G even through a single contact. This is probably due to ionic effects on the contacts. The G base has two acceptors and a partial negative charge, while T base is less negative, as it has only one acceptor. The A base has an acceptor and a donor and is nearly neutral; therefore, Arg and Lys bind to G much more often than to T, and to T more often than to A. His is less charged than Arg and Lys and thus its binding preference is expected to be weaker. The DNA charges may be neutralized by polypeptide chains that are bound to the grooves of the DNA double helix.

Mutagenic capacity of the bases

In general, the bases are stable because they are sequestered inside the double helix. However, notably two reactions can occur.

  1. Oxidative deamination of amino groups

    e.g. Cytosine → uracil
    Adenine → hypoxanthine

     

     

  2. Tautomerization: Sometimes the bases tautomerize to less common forms, (ex.) imino form of adenine, enol form of guanine.

Both reactions (i) and (ii) can affect the base-pairing potential of the bases.

 

 

DNA TOPOLOGY

The double-stranded structure of a DNA determines its biological function. Metabolic events involving unwinding impose great stress on the DNA because of the constraints inherent in the double helix. Molecular processes such as replication and transcription require the unwinding of the DNA double helix. While unwinding, correct topological tension in the DNA (super-helical density) should be maintained in order for genes to be regulated and expressed normally.

In 1965, Vinograd et al. discovered that the circular DNA chromosomes isolated from small viruses such as SV40 or polyoma virus were in a highly compact or folded conformation.

This supercoiling or writhing of the circular DNAs was a result of the DNAs being under wound with respect to the relaxed form of DNA.

When a linear DNA is free in solution, it assumes a pitch that contains 10.4 base pairs per turn. As the result, the DNA is less tightly wound than 10.0 base pairs per turn in the Watson and Crick B-form DNA.

In order to understand the origin of supercoiling; imagine a linear DNA of 4,200 base pairs in length. If the DNA were in the B-form, one would expect the two strands of the helix to be wrapped around each other 400 times (4,200 bp/10.4 bp/turn). Imagine a linear DNA in which the two ends become connected to form an open circle. This is referred to as a relaxed circular DNA. On the other hand, if the linear DNA were unwound 10 per cent, say 40 turns, before its ends were joined, then the DNA molecule would be under stress. When the molecule is free in solution, it will coil about itself in space, as the two strands simultaneously twist about each other in order to return to equilibrium value of 10.4 base pairs per turn.

The DNA that is ‘underwound is referred to as negatively supercoiled’. The DNA in this case forms the right-handed double helices. The DNA that is ‘overwound is referred to as a positively supercoiled DNA helix and is a left-handed helix’ (Figure 1.5).

 

Figure 1.5 The structure of super-coils. (a) Positive supercoils – the front segment of a DNA molecule cross over the back segment from left to right (b) Negative supercoils (c) The positive supercoil in bacteria during DNA replication

 

Linking, Twisting and Writhing

The total number of times one strand of the DNA helix is linked with the other in a covalently closed circular molecule is known as the ‘linking number (Lk)’ (Figure 1.6).

 

Figure 1.6 Linking, twisting and writhing

 

Salient features about linking number

  • The Lk is only defined for a covalently closed DNA and its value is fixed provided the molecule remains covalently closed. The Lk does not change whether the covalently closed circle is forced to lie in a plane in a stressed conformation or it is allowed to supercoil about itself freely in space.
  • The Lk of a circular DNA can only be changed by breaking a phosphodiester bond in one of the two strands, allowing the intact strand to pass through the broken strand and then rejoining the broken strand.
  • Lk is always an integer since two strands must always be wound about each other an integral number of times upon closure.
  • The Lk of a covalently closed circular DNA can be resolved into two components called ‘the twists (Tws) and the writhes (Wrs)’.

     

    Lk = Tw + Wr

     

  • In a relaxed circular DNA duplex of 500 bp, L is 50 (assuming that 10 bp per turn in the B-DNA).

     

     

  • The Lk for a relaxed DNA is usually taken as the reference parameter and is written as L0.
  • By convention, the Lk is defined as positive for right-handed helix and negative for left-handed helix. Since the left-handed Z-DNA occurs very rarely negative Lks are not encountered in all DNA studies for practical purpose.

Twists and writhes

‘The number of times the two strands of DNA are twisted about each other is called the twists (Tws). The writhes (Wrs) are the number of times that the DNA helix is coiled about itself in three-dimensional space.’ Tw and Wr are geometric rather than topological properties. The Tw and the Wr are not necessarily integers. It is just their sum, the Lk that is an integer.

If we use an SV40 DNA molecule, for example, which is precisely 5,243 base pairs in length, we would find that:

 

Lk = Tw + Wr
Lk = 5,243/10.4 = 504.13.

 

The DNA length and its pitch in solution determine the Tw of DNA.

 

Tw = length (bp)/pitch (bp/turn).

 

The Tw and the Lk determine the value of the Wr.

 

Wr = Lk − Tw
                 = 504.13 − 24.13
= 480.

 

Unlike the Tw and the Lk, the Wr of a DNA only depends on the path the helix axis takes in space. If the path of the DNA is in a plane, the Wr is always zero. In addition, if the path of the DNA helix were on the surface of a sphere, then the total Wr can also be shown to be zero.

Wrs can come in different forms. If a DNA molecule wrap around itself, then the Wrs are known as supertwists. If a DNA molecule wrap around something else (another molecule for instance), then the Wrs are known as ‘solenoidial’ Wrs. In solution, the Wrs can isomerize between the supertwist and solenoidal forms.

Measuring supercoils

The topoisomerases change the Lk (some directly and some indirectly). The change in the Lk, ΔLk, is a measure of the supercoiling. If the Lk in a supercoiled DNA and the Lk in the relaxed state (both of which must be integers) is compared, then the ratio would be:

 

ΔLk/Lk = σ = the superhelical density.

 

‘Super helix density or specific linking difference’ is the difference between the Lk of a DNA in the supercoiled form and the Lk in the relaxed form.

A σ of 0.1 means that 10 per cent of the helical turns in a sample of DNA (in its B configuration) have been removed. This underwinding results in negative supercoiling. In a cell, σ is usually of 5–7 per cent.

The superhelical density of a circular DNA can be observed and measured in several ways; for example, electron microscopy, sedimentation velocity or electrophoresis. Supercoiling can be measured by sedimentation procedure. Since supercoiled molecules are more compact, they sediment faster in a centrifuge than when they are relaxed. Supercoiling can also be determined by electrophoresis in an agarose gel. A supercoiled DNA migrates much more rapidly than does a relaxed molecule of the same length. The DNA separates into discrete bands depending on the Lk. Since the DNAs resolved in this way differ from each other only in their topology, they are referred to as ‘topological isomers or topoisomers’. Molecules that differ by one unit in Lk can be separated by electrophoresis in agarose due to the difference in their Wr (that is due to difference in folding).

Topoisomerases

‘Topoisomerases’ are enzymes that change the Lk of a circularly wound double-stranded DNA. The change in Lk changes the Wr. The variation in Wr subsequently changes the state of the compaction of the DNA molecule. The naturally occurring DNA is underwound or negatively supercoiled. This is advantageous because it permits the DNA to be transiently and locally melted to permit the enzymes of the DNA replication and transcription to copy and synthesize new DNA or RNA.

There are two classes of topoisomerase: Type I and Type II.

Type I topoisomerases

These enzymes remove supercoils by breaking only one of the two strands of the DNA. As a result, these enzymes change the Lk by 1 each time. The best-characterized member of this class in E. coli is Topoisomerase I. This enzyme is 864 amino acids in length and is monomeric; it is encoded by the topA gene.

The mechanism of catalysis involves the formation of a covalent intermediate between a tyrosine residue and the phosphodiester backbone. Specifically, nucleophilic attack from the hydroxyl group of tyrosine to a phosphorus atom creates a phosphodiester link between the enzyme and the DNA and generates a free 5′-hydroxyl group. Formation of this bond is energetically neutral as the reaction involves the replacement of one phosphomonoester bond with another—such reactions are called ‘transesterification’ reactions. The other strand of the DNA is held in place by binding non-covalently to a domain of the enzyme. The cleaved strand is resealed. Thus, one supercoil is removed. Topo I from E. coli acts only on negative supercoils; while eukaryotic Topo I can remove both negative and positive supercoils (Figure 1.7).

 

Figure 1.7 Topoisomerase I

Type II topoisomerases

These enzymes act through a mechanism, in which both the phosphodiester backbone chains are broken simultaneously. As a result, the Lk changes by two. Some Type 2 enzymes can use ATP to introduce the superhelical turns into the DNA. The best-characterized member of this class is E. coli Topoisomerase II—better known as DNA gyrase. E. coli DNA gyrase is a tetrameric protein consisting of two A subunits (875 aas) and two B subunits (804 aas). Depending on the DNA substrates, these enzymes can change positive supercoils into negative supercoils or increase the number of negative supercoils by 2. Type II topoisomerases catalyse catenation and decatenation, i.e., linking and unlinking of two different DNA duplexes. The enzyme also introduces negative supercoils at or near the Ori C site in the DNA template. DNA gyrase also removes the positive supercoils that are formed ahead of the growing fork during replication (discussed in Chapter 3).

DNA gyrase is composed of two identical subunits. The hydrolysis of ATP by gyrase’s inherent ATPase activity powers the conformational changes that are critical to the enzyme’s operation. The enzyme is a dimer, which has two identical subunits. Initially, the enzyme binds one part of a DNA strand, the G segment, inducing a conformational change in the B, B′, A and A′ domains of the enzyme 2. After the binding of ATP (indicated by the asterisks) and another part of the DNA strand, the T segment, a series of reactions occur in which the G segment is cut by the A and A′ domains of the enzyme and the ends of the G-DNA become covalently linked to tyrosine residues in these domains 3 and 3a. Simultaneously, the ATP-binding domains move towards each other, transporting the T segment through the break and into the central hole 4. The cut G segment is then resealed and the T segment is released by a conformational change that separates the A and A′ domains at the bottom of the enzyme 5. The interface between the A and A′ domains then re-forms, a reaction that requires ATP hydrolysis and regenerates the starting state 2. At this point, the G segment can dissociate from the enzyme by the conversion of 2 into 1. Alternatively, the enzyme can proceed through another cycle, again passing the T segment through the G segment and thus removing two more supercoils (Berger et al., 1996) (Figure 1.8).

 

Figure 1.8 Topoisomerase II

 

Topoisomerases are essential enzymes. The mutations of any of the genes coding for topoisom-erases are usually lethal. They are, therefore, the targets for the antibiotics and other drugs. Bacteria can be killed by novobiocin or nalidix acid. Both of these inhibit DNA gyrase. Novobiocin blocks ATP binding and nalidixic acid blocks the breakage and rejoining mechanism. These antibiotics do not inhibit eukaryotic topoisomerases and can be used to eradicate bacterial infections. Some bacteria, however, are now resistant to novobiocin. Eukaryotic topoisomerase inhibitors, such as doxorubicin and etoposide, are used as chemotherapeutic agents.

TYPES OF RNA

There are four types of RNA, each encoded by its own type of gene:

  1. mRNA—Messenger RNA: Encodes amino acid sequence of a polypeptide.
  2. tRNA—Transfer RNA: Brings amino acids to ribosomes during the translation.
  3. rRNA—Ribosomal RNA: With ribosomal proteins makes up the ribosomes, the organelles that translate the mRNA.
  4. snRNA—Small nuclear RNA: With proteins forms complexes that are used in RNA processing in eukaryotes (not found in prokaryotes).

 

Figure 1.9 Structure of a eukayotic mRNA

Messenger RNA

‘The genetic information stored in the DNA, inside the nucleus, is conveyed to the ribosomes in the cytosol for the synthesis of proteins through the mRNA. Thus, the mRNA carries the genetic message from the nucleus to the cytosol.’ The genetic code is translated for mRNA, not for DNA. The structure of mRNA includes more than simply a copy of the gene from the DNA. On one end of the mRNA is a cap. This is a structure that allows the mRNA to bind to the ribosome and is very important in the protein synthesis. mRNA contains a section of RNA that is non-coding. This section can vary in length. Next is an initiation codon, which signals the beginning of the coding sequence, and ends with the stop codon. Finally, there is the coding region that contains the copy of the genes. The mRNA also bears a series of adenine residue at its 3′ end called the poly-A tail (Figure 1.9).

Ribosomal RNA

Ribosomal RNA (rRNA) or insoluble RNA constitutes the largest part (up to 80%) of the total cellular RNA. It is found primarily in the ribosomes although; it is synthesized in the nucleus. It is also detected in the nucleus. It contains the four major RNA bases with a slight degree of methylation and shows differences in the relative proportions of the bases between species. rRNA molecules are single polynucleotide strands that are unbranched and flexible. At low ionic strength, rRNA behaves as a random coil; however, with increasing ionic strength, the molecule takes secondary structures showing helical regions produced by base pairing between self-complementary sequences, adenine and uracil, guanine and cytosine (Figure 1.10).

 

Figure 1.10 Structure rRNA

Types of rRNA

The eukaryotic cells have three kinds of rRNA molecules, namely 28S rRNA (the sedimentation constant varies between 25S and 30S depending up on the species), 18S and 5S rRNAs. The 28S and 5S rRNAs occur in 60S ribosomal submit, while 18S rRNA occurs in 40S ribosomal submit of the 80S ribosomes of eukaryotes. The prokaryotes contain 5S and 23S rRNAs in the 50S subunit and 16S in the 30S subunit (Table 1.5).

The sequences of the small and large rRNAs from several thousand organisms are now known. Though their primary nucleotide sequences vary considerably, the same parts of each type of rRNA theoretically can form base-paired stem-loops, generating a similar three-dimensional structure for each rRNA in all organisms.

 

Table 1.5 Prokaryotic and eukaryotic rRNAs

Transfer RNA

These are small RNA molecules and consist of about 73 to 94 nucleotides in a single chain. They are also known as soluble RNAs and help in transferring amino acids from the ‘amino acid pool’ to the site of protein synthesis. tRNAs account for 15 per cent of the total RNA of the cell. Eukaryotic cells are estimated to have approximately 60 different types of tRNAs. tRNA acts as an ‘adapter molecule’ linking the information in mRNA codons with the specific amino acids in proteins. For each of the 20 amino acids, there is at least one specific type of tRNA molecule.

Robert Holley et al. worked out the complete sequence of yeast alanine tRNA molecule. All tRNA molecules have a two-dimensional cloverleaf structure. This is because the majority of the bases are hydrogen-bonded to one another; the complementary stretches of bases in the chain form stem and loop structures and hence the overall pattern of H-bonding can be represented as a cloverleaf. Each cloverleaf consists of four H-bonded segments—three loops and the stem where the 3′- and 5′-ends of the molecule meet. These four segments are designated ‘the acceptor stem, the dihydrouridine loop, the anticodon loop and the ribothymidine pseudouracil cytosine loop’. The four stems are short double helices stabilized by Watson–Crick base pairing; three of the four stems have loops containing seven or eight bases at their ends, while the unlooped stem contains the free 3′ and 5′ ends of the chain (Figure 1.11).

 

Figure 1.11 tRNA cloverleaf structure

The acceptor stem

Specific aminoacyl-tRNA synthetases recognize the surface structure of each tRNA for a specific amino acid and covalently attach the proper amino acid to the unlooped ‘amino acid acceptor stem’. The 3′ end of all tRNAs has the sequence CCA, which is added after the synthesis and processing of the tRNA are completed. The 3′-OH of the adenine residue of the CCA terminus is esterified with the carboxyl group of the amino acid.

The anticodon loop

Three nucleotides termed the anticodon are located at the centre of the ‘anticodon loop’. These can form base pairs with the three complementary nucleotides forming a codon in mRNA. Viewed in three dimensions, the folded tRNA molecule has an L shape with the anticodon loop and acceptor stem forming the ends of the two arms. Base pairing of anticodon with the codon on mRNA allows a particular tRNA species to deliver its amino acid to the protein-synthesizing apparatus. It represents the key event in translating the information in the nucleic acid sequence, so that the appropriate amino acid is inserted at the right place in the amino acid sequence of the protein being synthesized.

The dihydrouridine loop

Ribosomes bind tRNAs through the recognition of this loop. The ‘D loop’ is so named because this tRNA loop often contains dihydrouridine (D) residues. In addition to dihydrouridine, tRNAs characteristically contain a number of unusual bases, including inosine, thiouridine, pseudouridine and hypermethylated purines.

The ribothymidine pseudouracil cytosine loop

Most tRNAs are synthesized with a four-base sequence of UUCG near the middle of the molecule. The first uridylate is methylated to become a thymidylate; the second is rearranged into a pseudouridylate (abbreviated ψ), in which the ribose is attached to carbon 5 instead of to nitrogen 1 of the uracil. These modifications produce a characteristic ‘TψCG loop’.

Extra arm or variable loop

The next loop in tRNA sequence in the 5′ → 3′ direction is a loop that varies from tRNA to tRNA in the number of residues that it has and is called ‘extra arm or variable loop’. Some tRNAs lack this loop.

tRNA tertiary structure

Tertiary structure in tRNA arises from hydrogen-bonding interactions between bases in the D loop with bases in the variable and ‘TψCG loops’, as shown for yeast phenylalanine tRNA (Figure 1.12).

These H bonds fold the D loop and ‘TψCG loop’ together and bend the cloverleaf into the stable L-shaped tertiary form. Many of these H bonds involve base pairs that are not canonical A=T or G≡C pairings. The amino acid acceptor stem is at one end of the L, separated by approximately 7 nm from the anticodon at the opposite end of the L. The D loop and ‘TψCG loop’ form the corner of the L. In the L-conformation, the bases are oriented to maximize hydrophobic stacking interactions between their flat faces. Such stacking interactions stabilize L-form tertiary structure of the tRNA.

 

Figure 1.12 (a) The three–dimensional structure of yeast phenylalanine tRNA as deducted from X-ray diffraction studies of its crystals. The tertiary folding is illustrated in the center of the diagram with the ribose—phosphate backbone presented as a continuous ribbon; H bonds are indicated by crossbars. Unpaired bases are shown as short, unconnected rods. The anticodon loop is at the bottom and the —CCA 3′ —OH acceptor end is at the top right. The various types of noncanonical hydrogen-bonding interactions observed between bases surround the central molecule. Three of these structures show examples of unusual H-bonded interactions involving three bases; these interactions aid in establishing tRNA tertiary structure. (b) A space-filling model of the molecule. (After Kim, S. H., in Schimmel, P., Söll, D., and Abelson, J. N., eds., 1979. Transfer RNA: Structure, Properties, and Recognition. New York: Cold Spring Harbor Laboratory)

TYPES OF DNA

The Chromosomal DNA

The DNA molecule may be circular or linear and can be composed of 100,000–10,000,000,000 nucleotides in a long chain. Typically, eukaryotic cells (cells with nuclei) have large linear chromosomes and prokaryotic cells (cells without defined nuclei) have smaller circular chromosomes, although there are many exceptions to this rule. In addition, cells may contain more than one type of chromosome; for example, mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes.

The Autosomal DNA

Most of the DNAs are autosomal/chromosomal DNAs. Half of the autosomal/chromosomal DNAs are from each of the parents. This is the DNA that can uniquely identify a specific individual. The autosomal DNA contains almost all of our health/medical information. The autosomal DNA is used in maternity/paternity tests and for forensic/crime purposes.

Y-DNA

Only males have the Y chromosome, Y-DNA, which rarely changes (mutates slowly) and is passed down to sons from the father’s direct paternal/male line.

Mitochondrial DNA (mtDNA)

Mitochondrial DNA (mtDNA) is the DNA located in mitochondria and is circular in shape. In mammals, each double-stranded circular mtDNA molecule consists of 15,000–17,000 base pairs. The nuclear and mitochondrial DNAs are believed to be of separate evolutionary origin. The mtDNAs are thought to be derived from the circular genomes of the bacteria that were engulfed by the early ancestors of today’s eukaryotic cells. Each mitochondrion is estimated to contain 2–10 mtDNA copies. About 100–10,000 separate copies of mtDNA are usually present per cell (egg and sperm cells are exceptions) in humans (and probably in metazoans in general).

In most multi-cellular organisms, the mtDNA is inherited from the mother (maternally inherited). Both males and females have the mtDNA.

The two strands of the mtDNA are differentiated by their nucleotide content. The guanine-rich strand referred to as the heavy strand and the cytosine-rich strand is referred to as the light strand. The heavy strand encodes 28 genes and the light strand encodes nine genes for a total of 37 genes. The 37 genes of the mtDNA encode various molecules. In total, 13 genes encode for proteins (polypeptides), 22 genes are for tRNA and two genes are for the small and large subunits of rRNA. The mtDNA is replicated by the DNA polymerase gamma complex.

Chloroplast DNA

Chloroplast genomes are relatively large, usually ∽140 kb in higher plants and <200 kb in lower eukaryotes. This is comparable to the size of a large bacteriophage, e.g., T4 of ∽65 kb. There are multiple copies of the genome per organelle, typically 20–40 in a higher plant.

The chloroplast genome codes for all the rRNA and tRNA species needed for protein synthesis. The ribosomes include two small rRNAs in addition to the major species. The tRNA set resembles that of mitochondria. The chloroplast genome codes for ∽50 proteins, including RNA polymerase and some ribosomal proteins. The chloroplast genome of the higher plants varies in length (Table 1.6).

 

Table 1.6 Chloroplast DNA: The chloroplast genes codes for 4 rRNAs, 30 tRNAs and ∽40 proteins

Genes (RNA coding) Genes (Thylakoid membrane)
16S rRNA PS-I
23S rRNA PS-II
4.5S rRNA Cyt b/f
5S rRNA H+-ATPase
tRNA Others
Gene Expression NADH dehydrogenase
R Proteins Ferridoxin
RNA polymerase Ribulose BisPhosphate cyclooxygenase

 

All these gene products are used within the chloroplast, but all the chloroplast structures also depend on proteins encoded by nuclear genes, translated in the cytosol, and imported into the chloroplast.

Plasmid DNA

A ‘plasmid’ is a small DNA molecule that is separate from and can replicate independently of the chromosomal DNA. They are double-stranded and, in many cases, circular. Plasmids usually occur naturally in bacteria; however, sometimes, they are found in eukaryotes (e.g., the 2-µm-ring in Saccharomyces cerevisiae). Plasmid sizes vary from 1 to over 1,000 kb pairs. Plasmids are capable of autonomous replication within a suitable host.

DNA AS VEHICLE OF INHERITANCE

Griffith Transformation

In 1928, Frederick Griffith performed the first experiment to prove that DNA was the hereditary material. Griffith selected the bacterium that causes pneumonia, Diplococcus pneumoniae. Two strains of the bacterium namely virulent strain and avirulent strain were used for the study.

Virulence required the presence of a polysaccharide capsule around the bacterium. The avirulent mutants lacked this capsule. The colonies of avirulent bacteria did not have capsule and appeared rough. They were designated R. In contrast, the virulent form produced colonies that appeared smooth, so it was designated S. Virulent forms were of different types and each had a characteristic polysaccharide capsule (called I S, II S, III S, etc.), which is genetically inherited and is immunologically distinct from other forms.

Virulent bacterium of a particular capsule type (say II S) can mutate to a non-encapsulated, nonvirulent form (II R, because it derives from a Type II cell) and vice versa. This happens at a very low frequency (in less than one in a million cells), but it is inherited when it does occur. However, the II R cell line cannot mutate to a III S virulent form (Figure 1.13 (a)).

 

Figure 1.13 (a) Diplococcus pneumoniae–virulent and avirulent strains (b) Griffith experiment

 

When attenuated encapsulated Type III S cells were injected into mice, the mice did not develop pneumonia. Similarly, when II R cells were injected into mice, the mice did not develop the illness. When live Type III S cells were injected, it resulted in the death of the animal.

Griffith mixed Pneumococcus Type II R with attenuated II S cells. Both the strains mentioned when injected alone did not produce the disease. Therefore, no disease was expected from the mixed injections, as neither strain was virulent. However, many of the mice given mixed injections developed pneumonia and died. When the blood of the animal was analysed, they all contained living virulent Type III S cells. These cells could not have arisen from the Type II R cells by mutations (they would have produced Type II S cells), and the Type III S cells were attenuated. This proves that some factors must have passed from the dead III S cells to the live II R ones, which would have enabled them to make a capsule of the Type III transforming them to Type III S. Griffith called the factor as ‘transforming principle’ and the process as genetic transformation (Figure 1.13 (b)).

Oswald Avery, Colin MacLeod and Maclyn McCarty Experiment

The nature of the Griffith’s transforming principle was studied by Oswald Avery, together with his colleagues Colin MacLeod and Maclyn McCarty, of Columbia University, New York. These scientists followed Griffith’s experiment but with some changes. In order to identify the transforming principle, they isolated the R-II transformed S-III cells from the dead mice. The S-III cells were then lysed and the cellular contents of the cell were subjected to different enzyme treatments. To five tubes containing the cellular lysate, one of the following enzymes was added: RNase, an enzyme that destroys RNA; protease, an enzyme that destroys protein; DNase, an enzyme that destroys DNA; lipase, an enzyme that destroys Lipids; or a combination of enzymes that break down carbohydrates. To the sixth tube, no enzyme was added and used as the control tube.

If the ‘transforming agent’ was, for example, protein—the transforming agent would be destroyed in the test tube containing protease, but not the others. Thus, whatever the transforming agents was, the liquid in one of the tubes would no longer be able to transform the S. pneumoniae strains. The scientists observed that the liquid from the tubes that received RNase, protease, lipase and the carbohydrate-digesting enzymes was still able to transform the R strain of pneumonia into the S strain. However, the liquid that was treated with DNase completely lost the ability to transform the bacteria. Thus, it was apparent that the ‘transforming agent’ in the liquid was DNA (Figure 1.14).

To further prove their finding, the scientists took a liquid extracted from attenuated S. pneumoniae (S strain) and subjected it to extensive preparation and purification, isolating only the pure DNA from the mixture. This pure DNA was also able to transform the R strain into the S strain and generate pathogenic S. pneumoniae. These results provided powerful evidence that DNA, and not protein, was actually the genetic material inside of living cells.

Disadvantages of the Experiment

Avery’s experiments had several complicating factors.

  • It was not clear in the minds of all microbiologists that transformation really was a genetic phenomenon.
  • There were doubts about the specificity of the deoxyribonuclease enzyme that he used to inactivate the transforming principle.
  • It was believed that the added enzyme contained trace amounts of a contaminating protease and hence was also able to degrade protein.

 

Figure 1.14 Oswald Avery, Colin MacLeod and Maclyn McCarty experiment

 

These uncertainties necessitated the need for a second experiment to provide more information on the chemical nature of the genetic material.

Hershey and Chase Experiment

In 1952, two scientists named Alfred Hershey and Martha Chase performed an entirely different type of genetic experiment using bacteriophages to prove that DNA is the carrier of genetic information. Bacteriophages (or just phage, an extremely small virus) are viruses that infect bacterial cells. They use the host cell machinery and transform it into a factory for producing more phages. Scientist of the time knew that the phage itself does not enter the bacterium during an infection. Rather, a small amount of material is injected into the bacteria and this material must contain all of the information necessary to build more phages. Thus, this injected substance is the genetic material of the phage.

Hershey and Chase devised a very simple experiment using a technique called radioactive labelling to determine which molecule, whether DNA, RNA or protein, acted as the genetic material in phages.

 

Figure 1.15 Hershey and chase experiment

 

The chemical make-up of protein and of DNA is quite different. DNA contains phosphorus but proteins do not; on the other hand, proteins usually contain sulphur but DNA does not. By specifically labelling the phosphorus and sulphur atoms with radioisotopes, Hershey and Chase were able to distinguish between the protein and the DNA of the phage and determine whether either or both were injected into the bacterial cell during the course of infection.

Two batches of isotopically labelled bacteriophage particles were prepared. One was labelled with 32P in the phosphate groups of the DNA and the other with 35S in the sulphur-containing groups of the amino acids of the protein coat called capsid, of the virus. The two batches of labelled phages were allowed to infect the bacterial cells. After a short time interval, the suspension of phage-infected bacterial cells was agitated in a blender that sheared the viral capsids from the bacteria. The blended mixture was then centrifuged. This separated the bacterial cells from the empty viral ghost consisted only capsid. The cells infected with the 32P-labelled phage were found to contain 32P, indicating that the labelled viral DNA has entered the cell, while there was no radioactivity detected in the viral ghost (Figure 1.15).

The opposite occurred when 35S-labelled phage infected a bacterial culture. The cells infected with 35S-labelled phage were found to have no radioactivity after blender treatment but their viral ghosts remained suspended in the supernatant after centrifugation contained 35S. This proved that 35S-labelled phage protein did not enter the bacterial cell. This experiment proved that only the DNA from the phage entered the bacterial cell and dictated the information required for the production of progeny phages; in other words, the DNA is the carrier of genetic information (Figure 1.15).

A small amount of protein did enter the bacterial cell in the course of infection. However, this was not involved in the production of new bacteriophage. This fact was demonstrated by repeating the experiment with bacteria stripped of their cell walls (protoplasts). If protoplasts were infected with 32P phage DNA free of protein, virulent phages were produced. If the purified 32P was first treated with DNAase, no progeny phage was produced. This clearly confirmed that the labelled DNA contained all the information necessary to produce new virus particles.

  • Deoxyribonucleic acid (DNA), the genetic material, carries information to specify the amino acid sequences of proteins. It is transcribed into several types of ribonucleic acids (RNAs) including messenger RNAs (mRNAs), transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), which function in the protein synthesis.
  • Both DNA and RNA are the long and unbranched polymers of nucleotides. Each nucleotide consists of a heterocyclic base linked via a five-carbon sugar (deoxyribose or ribose) to a phosphate group.
  • DNA and RNA each contain four different bases. The purines adenine (A) and guanine (G) and the pyrimidine cytosine (C) are present in both DNA and RNA. The pyrimidine thymine (T) present in DNA, which is replaced by the pyrimidine uracil (U) in RNA.
  • The bases in nucleic acids can interact via hydrogen bonds. The standard Watson–Crick base pairs are G≡C and A=T in DNA and G≡C and A=U in RNA. Base pairing stabilizes the native three-dimensional structures of DNA and RNA.
  • Adjacent nucleotides in a polynucleotide are linked by phosphodiester bonds. The entire strand has a chemical directionality: the 5′ end with a free hydroxyl or phosphate group on the 5′ carbon of the sugar and the 3′ end with a free hydroxyl group on the 3′ carbon of the sugar. Polynucleotide sequences are always written in the 5′ → 3′ direction (left to right).
  • Natural DNA (B-DNA) contains two complementary polynucleotide strands wound together into a regular right-handed double helix with the bases on the inside and the two sugar-phosphate backbones on the outside. Base pairing (A=T and G≡C) and hydrophobic interactions between adjacent bases in the same strand stabilize this native structure.
  • Binding of protein to a DNA can deform its helical structure, causing local bending or unwinding of the DNA molecule.
  • Heat causes the DNA strands to separate (denature). The melting temperature of a DNA increases with the percentage of the G≡C base pairs. Under suitable conditions, the separated complementary nucleic acid strands will renature.
  • Local unwinding of the DNA helix induces stress, which is relieved by twisting of the molecule on itself, forming supercoils. This process is regulated by topoisomerases, which can add or remove supercoils.
  • Natural RNAs are single-stranded polynucleotides that form well-defined secondary and tertiary structures. Some RNAs, called ribozymes, have catalytic activity.
  1. Draw the chemical structures of the nitrogenous bases of DNA.

  2. What are purines and pyrimidines? Explain with their chemical structures.

  3. What is meant by the term SNPs?

  4. Describe in detail about the Watson and Crick model of DNA.

  5. Write short notes on the 3 conformations of DNA.

  6. Define Holliday junction.

  7. Discuss about the chemical stability of nucleic acids.

  8. What is known as linking number – Lk? Explain its significance.

  9. Define topoisomerase. Describe Topoisomerase-II in detail.

  10. Enumerate the main properties of RNA with its types and structures.

  11. What is a plasmid?

  12. Explain the steps involved in Hershey and Chase experiment.

MULTIPLE-CHOICE QUESTIONS
  1. RNA, is distributed throughout the cell, most commonly in small numerous organelles called ————.

    1. mitochondria
    2. nucleosome
    3. ribosomes
    4. lysosomes
  2. Nucleotides are ——— esters of nucleosides.

    1. sulphate
    2. methylate
    3. oxide
    4. phosphate
  3. The width of major groove in DNA is ———.

    1. 22 Å
    2. 20 Å
    3. 23 Å
    4. 24 Å
  4. The most common conformation of DNA in most living cells is ————.

    1. A-DNA
    2. B-DNA
    3. Z-DNA
    4. None of the above
  5. Renaturation of melted DNA results in decrease in uv absorption. This phenomenon is called ————.

    1. Hyperchromicity
    2. Hypochromicity
    3. denaturation
    4. Photo hydrolysis
  6. Which of the following carries the genetic message from the nucleus to the cytosol?

    1. tRNA
    2. rRNA
    3. siRNA
    4. mRNA
  7. Which of the following constitutes the largest part (up to 80%) of the total cellular RNA?

    1. rRNA
    2. mRNA
    3. siRNA
    4. tRNA
  8. The ———— helix is left-handed and has a structure that repeats every 2 base pairs.

    1. A-DNA
    2. B-DNA
    3. Z-DNA
    4. None of the above
  9. Which one of the following is not a pyrimidine nucleoside?

    1. Cytidine
    2. Uridine
    3. Deoxythymidine
    4. Guanosine

Berger, J. M., Gamblin, S. J., Harrison, S. C. and Wang, J. C. 1996 ‘Structure and Mechanism of DNA topoisomerase II’, Nature, 379: 225

Brown, Terence A. 2002 Genomes, 2nd edition. Oxford: Wiley-Liss.

Kim, S. H. 1979 Transfer RNA: Structure, Properties, and Recognition. In P.,Schimmel, D. Söll, and J. N. Abelson, (Eds.), New York, Cold Spring Harbor Laboratory.

Knutsen, Alan P, and Jyonouchi, Harumi. Purine Nucleoside Phosphorylase Deficiency. Med Scape Drugs, Diseases & procedures. http://www.emedicine.medscape.com/article/887823/overview.

Krebs, Jocelyn E., Lewin, Benjamin, Goldstein, Elliott S. and Kilpatrick, Stephen T. 2009 Lewin&’s GENES X. Jones and Bartlett.

Lehninger, Albert L., Nelson, David L. and Cox, Michael M. 2004 Lehninger Principles of Biochemistry. New York: W H Freeman & Co.

Lodish, Harvey, Berk, Arnold, Zipursky, S. Lawrence, Matsudaira, Paul, Baltimore, David. 2006 Molecular Cell Biology, 4th edition. New York: W H Freeman & Co.

Pray, Leslie A. 2008 &‘Discovery of DNA structure and function: Watson and Crick&’, Nature Education, 1(1).

Rich, A. and Zhang, S. 2003 &‘Z-DNA: The long road to biological function&’, Nature Reviews Genetics, 4, 566&–572

Stryer, Lubert, Biochemistry, 5th edition. W.H. Freeman & Co.

Voet, Donald and Voet, Judith G. 2004 Biochemistry, 3rd edition. J. Wiley & Sons, 1591