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RNA

The RNA is generally single-stranded (exceptions, e.g. certain viruses, like reovirus, wound tumour virus, etc., where it is double-stranded), whereas the DNA is double-stranded (exceptions, e.g. certain viruses, like X174). In the nucleic acid strands, the backbone is made up of alternating units of sugar and phosphate, whereas the bases lie at right angle to the backbone and are attached to the sugar. The adjacent nucleotides are linked by phosphodiester bonds that are formed by the elimination of water molecules (Fig. 7.6 and 7.7). Fig.7.6                                                                                                     Fig. 7.6 A single DNA strand. Fig.7.7

Fig. 7.7 Comparative structures of DNA and RNA.

  Thus, whenever the nucleic acid is found as double-stranded, the two strands run anti-parallel (one in 5’→ 3′ direction and the other in 3′ → 5′ direction where 5′ and 3′ are either free PO43- or OH groups) and the pyrimidine base in one strand always pairs with a purine base in another strand through hydrogen bonds. The bases uracil, thymine and guanine are keto forms, whereas cytosine and adenine are amino bases. In base pairing, an enol base pairs with the amino base, thus, cytosine (amino form of pyrimidine) in one strand always pairs with guanine (keto form of purine) through three hydrogen bonds. and thymine or uracil (keto form of pyrimidine) with adenine (amino form of purine) through two hydrogen bonds. Thus, according to Chargaff, in double-stranded DNA, A = T and C = G (complementary base pairing) and the sequence of bases in one chain determine the sequence of bases in other chain (Fig. 7.8). Fig.7.8 Fig. 7.8 Pairing between complementary bases   J.D. Watson and F.H.C. Crick (1953) (Fig. 7.9) gave the model for double helical structure of DNA that was published in the journal, Nature. According to their model, the two polynucleotide chains connected by hydrogen bonds run in opposite directions and they are coiled around a central axis in a right-handed manner, so that they can be separated only by uncoiling and not by lateral separation. Thus, the backbones of the two strands, consisting of alternating sugar and phosphate units, Fig.-7.9

Fig. 7.9 James D. Watson (left) and Francis Crick (right).

are like the railing of a staircase whereas, the hydrogen-bonded base pairs, found at right angle to the long axis, lie at the steps. The diameter of the DNA double helix is 2 nm (20 Å) with a pitch (1 round) of 3.4 nm (34 Å), so that the distance between two adjacent bases becomes 0.34 nm (3.4 Å), as 10 bases constitute one turn. This is known as Bform of DNA (Fig. 7.10 and 7.11). Fig.-7.10

Fig 7.10

Fig.-7.11

                                                                                 Fig. 7.11 Base pairing in double-stranded DNA molecules. A- and Z-forms (1979) of DNA have been obtained in crystalline form. The occurrence of A-form of DNA (right-handed coiled) in the cells is uncertain, but evidences indicate the presence of short fragments of Z-form in both prokaryotes and eukaryotes that may play some role in regulation of gene expression or in genetic recombination. The pitch in Z-form DNA is 4.5 nm with 12 base pairs/turn, diameter 1.8 run, distance between two adjacent bass 0.38 nm, sugar-phosphate backbone zig-zag, repeating units being dinucleotides, and coiling of the two strands left-handed (Fig. 7.12). Fig.-7.12

Fig.7.12 Different types of DNA molecules.

Fig.-7.13

Fig.7.13 Structure of DNA

Cells, having both DNA as well as RNA, DNA constitutes the genetic material containing the information for development and existence of an organism, whereas RNA constitutes the genetic material only in certain RNA viruses, e.g. retroviruses, reoviruses, etc. In eukaryotes, the DNA is mainly found in the nucleus, but is also present in the organelles, chloroplasts and mitochondria. In certain eukaryotes it is also found in the cytoplasm as plasmids that are double-stranded circular molecules of DNA. In prokaryotes, the nucleoid contains a circular double-stranded DNA attached to mesosome. Many prokaryotes also contain one to many plasmids in the-cytoplasm.   RNA in the cells is of three types: (i) mRNA (ii) tRNA (iii) rRNA (Details given in the chapter “Protein Synthesis”)   Hammerling’s grafting experiments (1953) on the green alga Acetabularia demonstrated that nucleus contains the hereditary information. The body of alga is made up of a rhizoid that contains the nucleus, a stalk and a cap that is generally umbrella-shaped reproductive body. He used two species, Acetabularia mediterranea and A. crenulata that differ with respect to the shape of Fig.7.14

Fig. 7.14 Hammerling’s grafting experiment on Acetabularia.

caps. He cut the body of the organisms of the two species in 3 pieces – cap, stalk and the rhizoid. Then, he grafted the stalk of one species over the rhizoid of the other, thus, the cap regenerated resembled the species that donated the rhizoidal portion containing the nucleus (Fig. 7.13). (For details see the chapter ‘Introduction to Cell Structure and Viruses’).   Many experiments have demonstrated that nucleic acids contain the genetic information:   Transformation Experiment of Griffith   Griffith (1928) worked on two strains of the bacterium, Streptococcus pneumoniae (Pneumococcus pneumoniae or Diplococcus pneumoniaei where the cells are found in aggregates of two (diplococci):   (a)    S III strain that is virulent (pneumonia disease causing) and forms smooth colonies on solidified nutrient medium. The cells are enveloped within a polysaccharide capsule that prevents the bacterial cells from being engulfed by the WBC in host’s (mouse) blood, and thus is responsible for the virulence. (b)   R II strainthat is non-virulent (did not cause pneumonia) and forms rough colonies on solidified nutrient medium. The cells are not enveloped within any capsule and, thus, are easily engulfed by the WBC in host blood.   He performed a series of experiments:

  1. S III strain when injected into the mice, it caused pneumonia and death of the host. Cells of the S III strain of the bacterium were recovered from the blood of dead mice.

 

  1. R II strain when injected into the mice, it did not cause pneumonia and, thus, no death of the host occurred,

 

  1. Heat-killed S III strain when injected into the mice, it did not cause pneumonia and no death of the host.

 

  1. Heat-killed S III strain and living R II strain when injected into the mice, it caused pneumonia and death of the host. Again, the cells of S III strain of the bacterium were recovered from the blood of dead mice (Fig. 7.15).

These experiments demonstrated that in experiment No.4, something from the heat-killed S III strain transformed the non-virulent R II into the virulent S III strain.   Later, O.T. Avery, C.M. Macleod and Me Carthy (1944) through their in vitro (outside the intact cells) experiments demonstrated that the virulence-transforming agent from heat-killed S III strain into non-virulentR II strain was the DNAs.

  1. Extract of heat-killed S III strain and living R II strain when injected into the mice, it caused death of the host. Again, a few cells of S III strain along with the RII strain of the bacterium were recovered from the blood of dead mice.

Fig.-7.15                                                         Fig. 7.15 Griffith’s experiment of transformation in Pneumococcus pneumoniae. 

  1. To know the transforming agent in the heat killed S III strain, extract of heat-killed S III
    strain, living R
    II strain along with the DNase enzyme, was injected into the mice, and
    it did not cause death of the host. Thus, the DNase enzyme, here, destroyed the transforming agent DNA from the heat-killed S III strain that transformed a few cells of non-
    virulent R n strain into the virulent S ill strain in experiment No.1.

 

  1. But, when the injection of extract of heat-killed S ill strain, living R II strain along
    with the protease enzyme, was given to the mice, it caused death. Thus, the protease
    enzyme, here, destroyed the proteins in the heat-killed S III strain and the actual
    transforming agent, DNA, remained intact. A portion of this DNA entered into a few
    non-virulent R II strain cells and got incorporated into their genetic material, thus,
    transforming them into the virulent S ill strain that were recovered from the blood of the
    dead mice (Fig. 7.16).

Fig.-7.16

                                                 Fig.7.16 Experiment of Avery, Macleod and Me earthy on Streptococcus pneumoniae. 

Experiment of Hershy & Chase

A.D. Hershy and M.J. Chase (1952) performed experiments with T2 bacteriophage that
infects E. coli bacterium. The T2 phage particle consists of a proteinaceous head and a tail, where
the head contains a double-stranded DNA molecule. Since proteins contain S but no P and the DNA
contains P but no S, in order to know whether the protein or the DNA contains the information for
the formation of intact progeny phages during the infection of a host cell, Hershey & Chase separately radio labeled the protein and DNA of T2 phage.

The protein coat of T2 bacteriophage was labeled with radioactive 35S by growing T2-
infected E. coli cells in 35SO4-2 containing nutrient medium, whereas the DNA of a separate lot of
T2 bacteriophage was labeled with radioactive 32P by allowing growth of T2-infected E. coli cells in
32PO43- containing nutrient medium.

When the fresh cells of E. coli were infected with 35S-labeled T2 phage, the protein coat
remained outside the host cell and no radioactivity was observed in any of the progeny phages inside
the host cells (after agitation the protein coat detached from the infected host cells and the pellet
obtained after centrifugation contained the infected host cells). Here, radioactivity was detected in
the extracellular medium obtained after the centrifugation that contained the lighter T2 protein coat.

On the other hand, when fresh cells of E. coli were infected with 32P-labeled T2 phage,

radioactivity was detected in the pellet containing infected host cells with progeny phages inside the
obtained after the centrifugation, whereas the supernatant containing lighter T2 protein coat did
show any radioactivity.

This experiment confirms that at the time of infection, 32P-labeled DNA enters the host cell

directs the formation of progeny phage particles. Thus, the DNA contains the information for the
assembly of phage particles and not the proteins (Fig 7.18).

Fig.-7.17

Fig. 7.17 Experiment of Hershy and Chase.

Experiment of Fraenkel-Conrat

 H. Fraenkel-Conrat worked on a plant virus, Tobacco Mosaic Virus (TMV), where the genetic material is RNA and it infects thetobacco plant to cause mosaic on leaves. The virus is rod-shaped; consisting of a protein coat (made up of spirally-arranged capsomeres), enclosing a spirally-coiled single-stranded RNA molecule. They performed various experiments that showed that the genetic material RNA, here, contains the information for the formation of progeny phages: .

Fig.7.18

Fig. 7.18 Fraenkel-Conrat’s experiment on TMY.

  1. When tobacco leaves were infected with TMV, it caused mosaic on leaves and progeny viruses were recovered from the infected parts of the leaves.
  2. The isolated RNA of TMV also successfully infected the tobacco leaves and progeny virus particles could be obtained from the infected leaves.
  3. The isolated protein of TMV could not infect the tobacco leaves.
  4. Reconstructed virus from the isolated RNA and protein again caused infection (Fig. 7.18)

DNA Replication

DNA (and genetic RNA in RNA viruses) at the time of cell division (multiplication in case of viruses) undergo self replication. In case of double-stranded DNA. the replication is semi-conservative in nature, where the two strands separate and each parental strand synthesizes its complementary strand. Thus, in each of the two newly synthesised double-stranded DNA molecules, one strand is the older parental DNA molecule, whereas the other strand is newly assembled.

M. Meselson and F.W. Stahl (1958) were ~he first who gave evidence for semi-conservative replication of DNA in E. coli. They labeled the DNA of E. coli by growing the cells in nutrient medium containing 15N (heavy isotope of nitrogen in the form of 15NH4CI) for 14 generations. After harvesting the cells from 15N-containing medium, they were grown in 14N-containing medium, so that the newly synthesized DNA, now, contained the normal 14N isotope of nitrogen. The isolated DNA from the cells in successive generations accordingly showed variable heaviness in CsCl density gradient ultracentrifugation:

 

1. 0 generation → 100 % heavy DNA

2. 1st generation → 100 % intermediate DNA

3. 2nd generation → 50 % light + 50 % intermediate DNA

4. 3rd generation → 75 % light + 25 % intermediate DNA

5. This shows that DNA replicates in a semi-conservative manner (Fig. 7.19)

Fig.7.19

Fig. 7.19 Semi-conservative replication of DNA (L = light DNA, I = intermediate DNA).

If DNA replicated in the conservative manner to produce two double-stranded DNA molecules, where both the strands of one daughter DNA molecule remaining conserved and consisted of the older strands, whereas the other DNA molecule consisted of entirely newly synthesized DNA strands, they would show the result:

1. 0 generation → 100 % heavy DNA

2. 1st generation → 50 % heavy DNA + 50 % light DNA

3. 2nd generation → 25 % heavy + 75 % light DNA

4. 3rd generation → 12.5 % heavy + 87.5 % light DNA (Fig. 7.20).

As these results were not obtained, this confirms that DNA replicates in a semi-conservative manner.

Fig.7.20

Fig. 7.20 Various possible modes of replication of DNA.

At the time of DNA replication, the two strands of the DNA are required to be separated, so that each of the two single strands can be used to synthesize new complementary strands.

Thus, various steps of DNA replication are:

1. Unwinding of double-stranded parental DNA molecule by the unwinding proteins, e.g., helicases.

2. Since, the two strands of DNA are right-handed coiled around each other that cannot be
separated laterally, the unwinding creates a coiling tension ahead of moving replication
fork. This coiling tension is reduced by topoisomerases that help in the separation of
two strands by relaxing super-coils by making cuts in one (e.g. co protein in E. coli) or
both the DNA strands (e.g. DNA gyrase in E. coli) and then after the release of tension,
causing rejoining of the broken strands.

3. Since the two strands of a double-stranded DNA molecule are complementary, the single-
strand binding proteins
(SSB) stabilize the single strands thus produced and prevent their rejoining by complementary base pairing.

4. The DNA replication requires a short segment of RNA primer that is synthesized by a RNA polymerase, primase.

5. Now, the polymerization process occurs over the RNA primer by the enzyme DNA polymerase III. In E. coli three types of DNA polymerases are found:

(i) DNA polymerase I: It was reported for the first time by A. Kornberg (1960) and has both exonuclease (5′ → 3′ ‘and 3′ → 5′) as well as polymerase (5′ → 3’) activities. The rate of polymerization of this enzyme is about 1,000/min and each cell contains about 400 molecules. Using its exonuclease and polymerase activities, it is mainly involved in:

(a) DNA repairing by removing mismatch bases (by 3′ → 75′ exonuclease activity) and then filling the gaps thus produced ( by 5′ → 3′ polymerase activity) (Fig. 7.21).

Fig.7.21

Fig. 7.21 Removal of wrong nucleotides during DNA replication,

(b) removal of RNA primer (by 3′ → 5′ exonuclease activity) and filling up of the gap by adding dNTPs (by 5′ → 3′ polymerase activity) (Fig. 7.22)


 Fig.7.22

Fig. 7.22 Removal of RNA primer during DNA replication.

(c) removing obstructions (e.g. wrong DNA segments) encountered during DNA synthesis ( by 5’→3′ exonuclease activity) (Fig. 7.23), etc.

Fig.7.23

Fig.7.23 Removal of the obstruction during DNA synthesis by DNA polymerase I.

(ii) DNA polymerase II: It has 3′ → 5′ exonuclease (no 5′ → 3′ exonuclease activity) and 5’→3′ polymerase activities. The rate of polymerization of this enzyme is about 50/min and each cell contains about 100 molecules. Its exact role is not known.

(iii) DNA polymerase III: It has 5′ 3′ and 3′ 5′ exonuclease and 5′ 3′ polymerase
activities. It is made up of 7, subunits (ex, α, β, δ, ε, γ, θ, τ) and is the main enzyme involved in DNA replication. The rate’ of polymerization of this enzyme ‘is about I5,000/min and each ceil contains about 10 molecules. (τ = tau)

In eukaryotes, the three types of DNA polymerases are:

1. DNA polymerase α: It is found in nucleus but leaks into the cytoplasm and is mainly responsible for DNA replication. It does not contain any exonuclease activity.

2. DNA polymerase β: It is found in the nucleus and is mainly involved in DNA repair and recombination. It does not have any exonuclease activity.

3. DNA polymerase γ: It is found in the nucleus and mitochondria and resembles bacterial
polymerases. It is mainly involved in DNA repair and recombination. It has 5′ 3′ and 3′ 5′ exonuclease activities.

After the separation of two strands of a double-stranded DNA molecule, each strand acts as

a template and DNA polymerase III reads its nucleotides and adds complementary nucleotides in the newly synthesizing DNA strand. Since, the two strands of a double-stranded DNA molecule are anti-parallel () and the same DNA polymerase III synthesizes the two new strands simultaneously using the two template DNA strands and since, DNA polymerization always occurs in 5′ 3′ direction, DNA replication is semi-discontinuous, where the synthesis of one of the two newly synthesizing DNA strands occurs continuously and that of the other discontinuously. The DNA fragments synthesized discontinuously are called Okazaki fragments (a Japanese scientist Okazaki discovered them). The template where the DNA synthesis occurs continuously is known as the leading strand, whereas the other template where the DNA synthesis occurs discontinuously is known as the lagging strand (Fig. 724).

 [Exception: In adenovirus, both the new DNA strands are synthesized continuously.]

Fig.7.24

Fig.7.24

Fig. 7.24 DNA replication.

The DNA synthesis starts at a point called origin and can occur in two ways:

(a) Unidirectional, where a single enzyme complex (consisting of various enzymes required for the synthesis of DNA) binds at the origin and moves along the replication fork to synthesize the two new strands (Fig. 7.25).

Fig.7.25

(b) Bidirectional, where two enzyme complexes bind at the origin and move in opposite directions synthesizing the new DNA strands (Fig. 7.26).

Fig.7.26

Fig. 7.26 Bidirectional DNA replication.

In eukaryotes, the DNA replication starts at many sites and can be unidirectional or bidirectional.

6. The DNA ligase enzyme joins the Okazaki fragments by forming phosphodiester bonds.

7. In addition to these main enzymes, many other enzymes are also involved in DNA replication, e.g. exonucleases (removing nuc1eotides at the ends of DNA strand), endonucleases (cleaving DNA strand at internal positions), etc.

DNA Repairing

DNA replication occurs quite accurately and any mistake done during this process is proof read by a very strong proof reading enzyme, DNA polymerase 1. Despite this accuracy, if some mistake is done in DNA or during DNA replication, many repair systems are involved and some of the important ones are:

 

(i) Dark Repair: This repair mechanism is also known as excision repair mechanism. It does not need any light energy and can occur in dark as well as in light. Here, the wrong nucleotide segment is removed with the help of endonucleases and the correct DNA segment is synthesized by DNA polymerase I enzyme (Fig. 7.27).

Fig.7.27

Fig. 7.27 Mechanism of dark repairing.

(ii) Photoreactivation: The sunlight contains both visible light as well as UV radiation and whenever the DNA is exposed to UV, it induces the formation of intrastrand pyrimidine dimers (particularly thymine dimers). The enzyme DNA photolyase repairs the dimmers by binding at the dimers and utilizing the energy of white light (blue being most effective) it cleaves the abnormal bonds formed between the two adjacent pyrimidine dimers (Fig. 7.28).

Fig.7.28

Fig. 7.28 Mechanics of photoactivation

Important differences between DNA and RNA

S. No.

DNA

RNA

1.

Generally, DNA is the genetic material and it transcribes the genetic information to RNA. RNA is not the genetic material, except incertain viruses, e.g., TMV containing sin-gle-stranded RNA. Of the three types of

RNA molecules, mRNA is generally being translated to polypeptides, whereas, tRNA and rRNA that are not translated.

2.

Usually it is found in the nucleus and some cytoplasmic organelles (mitochondria and chloroplast), except in prokaryotes, where it occurs as nucleoid and in some cases as plasmids (also found in a few eukaryotes, like yeast) in the cytoplasm. Most of the RNA is found in cytoplasmand very small amount (newly synthesized) being present in the nucleus. There are mainly three types of RNA molecules, mRNA, tRNA and rRNA.

3.

Generally, it is double-stranded, except in some viruses, e.g., bacteriophage ɸX174 that contains a circular single-stranded DNA molecule as the genetic material. It is mostly single-stranded, except in some viruses like reo virus, wound tumour virus where it is double-stranded.

mRNA will newly synthesized polypeptides.

 

4.

The sugar found here is 2-deoxy-ribose

Here, the sugar is ribose

5.

The four types of bases present in DNA are thymine,cytosine, guanine and denine

Here, the base thymine is replaced by uracil, whereas the other three bases, cytosine, guanine and adenine, remaining the same.

6.

Unusual bases are very few or absent.

Generally contains many unusual bases.

7.

It is self-replicating and after replication, it gives rise to two daughter DNA molecules

It is not self-replicating and is produced by the transcription of DNA that produces

large number of RNA molecules.

 

QUESTIONS

  1.    Describe the experiment which initially demonstrated that DNA is the genetic material.
  2.    Discuss an experiment which showed that RNA carries the genetic information.
  3.    Discuss the structure of DNA.
  4.    What are the main differences between DNA and RNA.
  5.    Give a brief account of different kinds of RNAs known in the living system.
  6.    Discuss an experiment which demonstrated the semi-conservative mode of DNA replication.
  7.    Describe the DNA synthesis in living organisms; giving emphasis on Escherichia coli.
  8.     Describe the different mechanisms of DNA repair in prokaryotes.