Functions of dna

The DNA plays a many-fold role in organisms:

1. It is the genetic material and carries hereditary characters from parents to the young ones. This is achieved through its unique property fo replication.

2. It enables the cell to maintain, grow and divide by directing the synthesis of structural proteins.

3. It controls metabolism in the cell by direct­ing the formation of necessary enzymatic proteins.

4. It produces RNAs by transcription for use in protein synthesis.

5. It creates variety in population by causing recombinations through crossing over.

6. It contributes to the evolution of the or­ganism by undergoing gene mutations (changes in the sequence of base pairs).

7. It brings about differentiation of cells during development. Only certain genes remain functional in particular cells. This enables the cells having similar genes to assume different structure and function.

8. It controls postnatal development through adulthood to death by its "internal clock".

Thus, DNA is the very basis of life.

Use of DNA and Proteins in Determination of Evolutionary Kinship

Genes (DNA) and their products show the hereditary background of an organism. The linearsequences of nucleotides pass from parents to off­spring through gametes, and these DNA sequences determine the amino acid sequences in the proteins formed in the offspring. Siblings have more similarity in their DNA and protein sequences than the unrelated individuals of the same species have. This concept of "molecular genealogy" indicates evolutionary relationship between species. For ex­ample, 146 amino acids-long/3 polypeptide chain of human haemoglobin differs in just one amino acid from the corresponding haemoglobin polypeptide of gorilla. More distantly related species have chains that are less similar (Table 25.4). VII. Summary of DNA Characteristics

The important features of DNA are briefly listed below:

1. DNA molecule is a double chain of deoxyribonucleotide units.

2. The successive units are joined by phos phodiester bonds in each chain (strand).

3. The sugar-phosphate-sugar-phosphate backbones are located on the outside of the molecule.

4. The bases project inward at planes ap­ proximately perpendicular to the long axis of the molecule, and are, therefore, stacked one above the other.

5. The two chains are spirally coiled around a common axis to form a right-handed double helix.

6. The helix has a major groove and a minor groove alternatively.

7. The helix is 20 A wide; its one complete turn is 34 A long, and has 10 base pairs; the successive base pairs are 3 • 4 A apart.

7. The two chains are complementary to each other with respect to base sequence.

7. The two chains are hydrogen bonded; A on one chain is joined to T on the other chain by 2 hydrogen bonds; C on one chain is linked to G on the other chain by 3 hydrogen bonds.

10. The two chains are antiparallel, one aligned in 5'-»3' direction, the other in 3'->5' direc­ tion.

11. The amount of A + G = the amount of T + C; the amount of A = the amount of T; and the amount of G = the amount of C. Sugar and phos­phate groups occur in equal proportions.

12. The DNA molecule is remarkably stable due to hydrogen bonding and hydrophobic and electronic interactions.

13. The DNA moelcule undergoes denatura-tion and renaturation easily.

14. The denatured DNA strands are hyper-chromic.

15. The DNA molecule can replicate and repair itself, and can also transcribe RNAs.

16. The base sequence of one chain (sense chain) serves as the genetic code.

17. The DNA can function in vitro.

18. The amount of DNA per nucleus is con­stant in all the body cells of a given species.

19. Solutions of DNA are highly viscous due to long stiff molecules.

20. DNA double helix has polyanionic surface that forms complexes with positively charged proteins.

20. DNA is dextrorotatory.

21. The DNA code is discontinuous, having noncoding segments (introns) between coding seg­ments (exons).

20. Eukaryotic DNA has many repetitive se­quences.

24. Each strand in a DNA molecule has polarity with 3' and 5' ends.

25. Only a small fraction of DNA is functional in eukaryotes.

26. Some repetitive sequences, may be "mobile".

27. DNA has five forms A, B, C, D and Z. The first four are right handed helices, the last is left- handed helix.

B. REPLICATION : SYNTHESIS OF DNA

Meaning. A unique property of DNA is that it governs its own synthesis. The copying process of DNA to produce additional DNA molecules is called replication.

Time and Site of Occurrence. Replication in eukaryotes occurs in the nucleus during the S phase of the cell cycle when chromosomes are in their extended form and are not readily visible. The stimulus which starts the process at this time and stops it at other times is not fully known. In prokaryotes, replication takes place in the cytoplasm and is almost a continuous process.

Mode of Replication. Watson and Crick sug- \ gested that the two strands of DNA molecule uncoil ^;; and separate, and each strand serves as a template for the synthesis of a new (complementary) strand \ alongside it. The template and its complement then I form a new DNA double strand, identical to the original DNA molecule. The sequence of bases which should be present in the new strands can be \ easily predicted because these would be com- ; plementary to the bases present in the old strands. A will pair with T, T with A, C with G, and G with \ C. Thus, two daughter DNA molecules are formed from the parent DNA molecule and these are iden- | tical to the parent molecule. Each daughter DNA molecule consists of one old (parent) strand and one new strand. Since only one parent strand is conserved in each daughter molecule, this mode of replication is said to be semiconservative (Fig. i 25.11).

Discovery. Semiconservative DNA replica­tion in eukaryotic chromosomes was first demonstrated by Taylor in 1957.

Experimental Evidence for Semiconservative Replication. Meselson and Stahl in 1958 provided a strong experimental evidence which supported the semiconservative mode of DNA replication (Fig. | 25.12). They grew E. coli bacteria in a medium containing the heavy nitrogen isotope ¹⁵N for many generations. This produced a population of bac­terial cells that had uniformly ¹⁵N — labelled DNA, and this DNA was heavier than the DNA obtained from E. coli grown in ¹⁴N — containing medium. The bacterial cells with heavier DNA were then trans­ferred to a medium having the ordinary ¹⁴N isotope. From the daughter cells of first generation, DNA was extracted, purified and centrifuged. It was found that all the DNA molecules were hybrid ¹⁵N — ¹⁴N, i.e., all were half heavy. This is what is expected in case of semiconservative mode of replication. Daughter cells were allowed to divide again. The second generation cells were found to have two types of DNA molecules, 50% half heavy with ¹⁵N - ¹⁴N hybrid density and 50% light with ¹⁴N — ¹⁴N density. This again conforms to the prediction based on semiconserative replication.Procedure for DNA Replication 6

The semiconservative mode of DNA replica­tion is a complex, multistep process, requiring the services of many enzymes, protein factors and metal ions (Fig. 25.13). 1. Activation of Deoxyribonucleotides. Four types of deoxyribonucleoside monophosphates found floating free in the nuclear sap serve as the raw materials in DNA synthesis. These nucleotides include deoxyadenosine monophosphate or deAMP, deoxyguanosine monophosphate or deGMP. deoxycytidine mononhosohate or deCMPand deoxythymidine monophosphate or deTMP. For incorporation into DNA, nucleotides are ac­tivated by union with ATP. This reaction is called phosphorylation and is catalyzed by an enzyme phosphorylase. It produces deoxyribonucleoside triphosphates, namely, deoxyadenosine triphos­phate or deATP, deoxyguanosine triphosphate or deGTP, deoxycytidine triphosphate or deCTP and deoxythymidine triphosphate or deTTP.

2. Exposure of Parent DNA Bases. The parent DNA double helix uncoils and splits into single DNA strands by breakdown of hydrogen bonds. Naked A, T, C and G bases joined to the phos-

: phate-deoxyribose sugar back­bones project into the karyoplasm. The DNA molecule is intri­cately coiled in its chromosome and its unwinding is not an easy job. Enzymes called helicases (un-windases) help in unwinding the helix, using ATP hydrolysis as a \ source of energy. A nonenzymic single-stranded DNA-binding (SSB) protein, also called helix stabilizing protein, stabalizes the chain in single-stranded form to reduce the energy needed to un-l wind the DNA helix. Unwinding creates a coiling tension ahaed of the moving replication fork. Other enzymes named topoir;omerascs, may cut and rejoin one strand of DNA to facilitate uncoiling.

The entire DNA chain does J not split in one go. The DNA \ strands start separating at a specific point called origin of replication, from where separa- j tion slowly progresses to the other I end. Eukaryotic chromosomal

; DNA begins the process at many origins of replication. Unzipping j of the double stranded DNA

_; forms a Y- shaped structure called replication fork (Fig. 25.14). New DNA strands grow from and toward the fork.

Prokaryotic DNA generally forms a single origin of replication or Ori, and progresses in both directions.

3. Formation of RNA Primer. A short chain of RNA is formed on the DNA template at the 5' end. This is called RNA primer. The enzyme primase catalyzes the polymerization of RNA building blocks (A, U, G, C) into the primer. The

nucleoside monophosphates joined to each single DNA

: chain become linked

i together, forming a new DNA chain. The process is

: catalyzed by an enzyme DNA polymerase a and S, and is aided by metal ions Mn⁺⁺ or

I Mg⁺⁺.

: DNA polymerase III func­tions in prokaryotes. Eukaryotic polymerases a and 6 correspond to prokaryotic polymerases 1 and HI.

This produces two double DNA chains, which are identical to each other as well as to the origin-1 "mother" chain. It should be noted that each "daughter"

double chain inherits from the "mother' chain one single chain, which serves as the code specific template in the formation of the new single chain. In this manner, the genetic code is faithfully trans­mitted from one DNA "generation" to the next.

Leading and Lagging Strands. The DNA polymerase can polymerise the deoxyribonucleotides in the 5'—3' direction, i.e., from carbon 5' end to carbon 3' end of the sugar molecules in the DNA strands. Because the two DNA strands are antiparallel, the new strands must be formed on the old (parent) strands in opposite directions. One new strand is formed in a con­tinuous stretch in the 5' —3' direction. This strand is called leading strand (Fig. 25.14). On the other parent strand, short DNA segments are formed in the 5' —3' direction, starting from RNA primers. These DNA segments are known as Okazaki¹ frag­ments. A separate RNA primer is formed for the synthesis of each Okazaki fragment. The Okazaki fragments are later joined together, forming a con­tinuous lagging strand. The Okazaki fragments are linked up by the enzyme DNA ligase (DNA syn­thetase) after replacing the RNA primers with deoxyribonucleotides.

Semidiscontinuous Replication. DNA

replication is said to be semidiscontinuous because the leading strand is synthesized continuously and

the lagging strand is formed discontinuously in short pieces that later join.

7. Editing (Proof-reading) and DNA Repairs. The specificity of base-pairing ensures accurate replication. However, sometimes wrong bases do get in. These are noted and removed by DNA polymerase, which can go back for this purpose.

The abnormal regions of DNA resulting from mutation are cleaved by enzymes termed nucleases. The DNA polymerase resynthesizes the missing segments of DNA strand, using the intact DNA strand as the template. The DNA ligase joins the new and old segments of the strand under repair. This makes the damaged DNA strand normal.

Inspite of proof-reading, errors creep in though rarely.

8. Helix Formation. Each daughter double DNA molecule becomes spirally coiled to form a double helix.

Equation for DNA Replication. The entire process of DNA synthesis may be summed in the equation —

(deATP + deGTP + deCTP + deTTP)„

DNA Template, Mg*+ or Mn²⁺

^; * DNA Chain + nPPi

DNA Polymerase I and III

DNA Repair. The DNA may get damaged by agents like body's own heat, and aqueous environ­ment within the cell. The DNA repair enzymes identify and repair the damaged DNA strand by using the undamaged strand as a template. If both strands are altered, repair is not possible.

C. STRUCTURE AND ROLE OF RNA

Structure. RNA molecule is a long, un-branched, single-stranded polymer of ribonucleotides (Fig. 25.15). Each nucleotide unit is composed of three smaller molecules : a phosphate group, a 5- carbon ribose sugar, and a nitrogen-containing base. The bases in RNA are adenine, guanine, uracial and cytosine. The various com­ponents are linked up as in DNA.

Types. There are three types of RNA in every cell: messenger RNA or mRNA, ribosomal RNA or rRNA and transfer RNA or tRNA. The three types of RNAs are transcribed from different regions of DNA templete. RNA chain is com­plementary to the DNA strand which produces it. All the three kinds of RNAs play a role in protein synthesis.

(a) mRNA. The DNA, that controls protein synthesis, is located in the chromosomes within the nucleus, whereas the ribosomes, on which the protein synthesis actually occurs, are placed in the cytoplasm. Therefore, some sort of agency must exist to carry instructions from the DNA to the ribosomes. This agency does exist in the form of mRNA. The mRNA carries the message (informa­tion) from DNA about the sequence of particular amino acids to be joined to form a polypeptide, hence its name. It is also called informational RNA or template RNA. The mRNA forms about 5% of

the total RNA of a cell. Its molecule is linear and the longest of all the three RNA types. Its length is related to the size of the polypeptide to be syn­thesized with its information. There is a specific mRNA for each polypeptide. Because of the varia­tion is size in mRNA population in a cell, the mRNA is often called heterogeneous nuclear RNA, or hn RNA.

A fully processed mRNA is shown in fig. 25.16. It has at its 5' end a cap of methylated guanine followed successively by an initiation codon (AUG or GUG), a long coding region, a termination codon (UAA or UAG or UGA) and a poly-A tail of many adenine-containing nucleotides at 3' end. A small non-coding region, called leader segment, follows the cap and another noncoding region, termed trailer segment, precedes the tail. The cap protects the mRNA from degradation by hydrolytic enzymes. It also functions as a part of an "attach here" sign for the small ribosomal subunit. Tail also checks degradation of mRNA and helps the ribosome attach to it. It also helps exit of mRNA from the nucleus.

In eukaryotes, mRNA carries information for one polypeptide only. It is monocistronic (monogenic) because it is transcribed from a single cistron (gene) and has a single initiator codon and a single terminator codon.

Bacterial mRNA often carries information for more than one polypeptide chains. Such an mRNA is said to be polycistronic (polygenic) because it is transcribed from many contiguous (adjacent) genes. A polycistronic mRNA has an initiator codon and a terminator codon for each polypeptide to be formed by it.

(b) tRNA. The tRNA has many varieties. Each variety carries a specific amino acid from the amino acid pool to the mRNA on the ribosomes to form a polypeptide, hence its name. The tRNAs from about 15% of the total RNA of a cell. Its molecule is the smallest of all the RNA types. A tRNA

molecule, as proposed by R.W. Holley in 1965, has the form of a clover leaf that results from self-folding and base pairing, creating paired stems and unpaired loops (Fig. 25.17). It has four regions—

(/) Carrier End. This is the 3' end of the molecule. Here a specific amino acid joins it. It, in all cases, has a base triplet CCA with — OH at the tip. The — COOH of amino acid joins the — OH.

(h) Recognition End. It is the opposite end of the molecule. It has 3 unpaired ribonucleotides. The bases of these ribonucleotides have complementary bases on the mRNA chain. A base triplet on mRNA chain is called a codon, and its complementary base

triplet on tRNA molecule is termed an anticodon. Anticodon reads its appropriate codon and tem­porarily joins it by hydrogen bonds during protein synthesis.

(Hi) Enzyme Site. It is on one side of the molecule. It is meant for a specific charging enzyme which catalyses the union of a specific amino acid to tRNA molecule.

(iv) Ribosome Site. It is on the other side of the molecule. It is meant for attachment to a ribosome.

The tRNA has almost similar structure in prokaryotes and eukaryotes.

(c) rRNA. The rRNA molecule is greatly coiled. In combination with proteins, it forms the small and large subunits of the ribosomes, hence its name. It forms about 80% of the total RNA of a cell. A eukaryotic ribosome is 80S ; its large 60S subunit consists of 28S, 5-8 S and 5S rRNAs and over 45 different basic proteins ; its small 40S subunit comprises 18S RNA and about 33 different basic proteins. A prokaryotic ribosome is 70S ; its large 50S subunit consists of 23S and 5S rRNAs and about 34 different basic proteins; its small 30S subunit comprises 16S rRNA and about 21 different basic proteins (Fig. 25.18). The 3' end of 18S rRNA (16S rRNA in prokaryotes) has a binding site for the mRNA cap. The 5S rRNA has a binding site for tRNA. The rRNA also seems to play some general role in protein synthesis.

Additional RNA types. There are two more minor types of RNAs : (iv) small nuclear RNA (snRNA) that helps in processing of /-RNA and mRNA in the nucleus ; and (v) small cytoplasmic RNA (sc RNA) which helps in binding the ribosomes to ER in the cytoplasm.

RNA serves as genetic material in some viruses, e.g., tobacco mosaic virus (TMV). This sixth type of RNA is called genetic RNA.

D. TRANSCRIPTION : SYNTHESIS OF RNA

Meaning. DNA contains information for the synthesis of cell's specific proteins. DNA is located in the nucleoid (prokaryotes) or nucleus (eukaryotes) and protein synthesis occurs in the cytoplasm. DNA does not move to the site of protein synthesis (ribosomes) to directly guide the process. Instead, it transfers its information to mRNA molecules which move to the ribosomes to direct protein synthesis. The process of the formation of RNA from DNA template is called transcription (written across). It involves rewriting the genetic message coded in DNA into an RNA molecule.

Site and Time of Occurrence. Transcription occurs in the nucleus during the Gj and G₂ phases of cell cycle. DNA has promoter and terminator sites. Transcription starts at the promoter site and stops at the terminator site.

Discovery. The mechanism of RNA synthesis was worked out in the late 1950s by the American investigators Jerard Hurwitz, Samuel B. Weiss, and Audrey Stevens by independent in vitro experi­ments.

Materials Required. RNAtranscription requires —(i) The enzyme RNA polymerase

(ii) A DNA template (tii) All four types of ribonucleoside triphosphates (ATP, CTP,GTPandUTP).

(iv) Divalent metal ions Mg²⁺ or Mn²⁺ as a cofactor. No primer in needed for RNA synthesis Procedure. RNA transcription takes placas follow(Fig. 25.19).

1. Binding of RNA Polymerase to DNDuplex. On a signal from the cytoplasm, the histone coat protecting the DNA double helix at the gene to be transcribed is removed, exposing the polynucleotide sequences in this region of DNA. The RNA polymerase enzyme binds to a specific site, called promoter, in the DNA double helix. The enzyme recognizes the promoter by its sigma (a) subunit in prokaryotes and by many transcription factors in eukaryotes.

The promoter site is located on the 5' side of the gene to be transcribed, and signals where to start RNA synthesis. The promoter also determines which DNA strand is transcribed.

Transcription factors and RNA polymerase bond to the promoter together, forming a transcrip­tion initiation complex.

2. Exposure of RNA Bases. The RNA

polymerase moves along the DNA and causes local unwinding and splitting of the DNA duplex into two chains in the region of the gene to be transcribed. This exposes the A, T, C and G bases that project into the karyoplasm from the phosphate — deoxyribose sugar backbone. Only one strand, called sense strand¹, of DNA functions as a template, the other strand is complementary, it is called antisense or non-coding strand. The mechanism which selects the template is not known.

3. Base Pairing. The ribonucleoside triphos­ phates, namely, adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphos­ phate (CTP) and uridine triphosphate (UTP), floating free in the nucleus, serve as the raw material for RNA synthesis. They are formed by activation (phosphorylation) of ribonucleoside monophos­phates, viz., adenosine monophosphate (AMP), guanosine monophosphate (GMP), cytidine monophosphate (CMP) and uridine monophos­phate (UMP) through union with ATP. The enzyme phosphorylase catalyses the activation process. The ribonucleotide triphosphates get joined to the bases of the DNA template chain one by one by hydrogen bonding according to the base pairing rule of Watson and Crick, /. e., A — U, U—A, C — G, G-C. This is brought about by the RNA polymerase.

4. Conversion to Ribonucleoside Monophos­ phates. Thevariousribonucleosidetriphosphates on linking to the DNA template chain break off their highenergy bonds. This changes them to ribonucleoside monophosphates that are the nor­ ma components of RNA, and sets free pyrophos­ phate groups (P ~ P). Pyrophosphate contains a high energy bond (~). It undergoes hydrolysis bythe enzyme pyrophosphatase, releases energy andsets free inorganic phosphate PP;. The first ribonucleotide triphosphate retains all the three phosphates d is, thus, chemically distinct from the another nucleotides added after it.

Pyrophosphatase P ~ P + H₂0 > 2 Pi + Energy

5. Formation of RNA Chain. With the energy so released, each ribonucleoside monophosphate joined to DNA template chain then joins the ribonucleotide arrived earlier, making the RNA chain longer. The process is catalyzed by the en­zyme RNA polymerase already referred to, and requires a divalent ion Mg⁺"fc or Mn⁺⁺.

5. Separation of RNA Chain. As transcription proceeds, the hybrid DNA —RNA molecule dis­sociates, partly freeing the RNA molecule under synthesis. When the polymerase reaches a ter­minator signal on the DNA, it leaves the DNA. The fully formed RNA chain is now released. One gene forms several molecules of RNA, which are released from the DNA template one after the other on completion.

Termination of Transcription. In some cases, such as E. coli, a specific chain terminating protein, called rho factor (P), stops the synthesis of RNA chain. In most cases, the RNA polymerase can stop transcription.