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Section A

Subsection 1

Nucleic acids :-first isolated in 1868 F.Miescher and called nuclein in 1889 Altman renamed nucleic acid. The nucleic acids are linear unbranched polymer of purine and pyrimidine nucleotides linked in a chain through phosphodiester bonds. The backbone of a nucleic acid is made of alternating sugar and phosphate molecules bonded together in a long chain , each of the sugar groups in the backbone is attached to a third type of molecule called a nucleotide base, as shown:

Long chains of nucleotides are called polynucleotides, and short chains of nucleotides are called oligonucleotides. The whole nucleic acid chain is usually synthesized by RNA polymerase or DNA polymerase.

A important feature of all nucleic acids is that they have two distinctive ends: the 5' (5-prime) bears a phosphate,and 3' (3-prime) ends a hydroxyl group. A nucleic acid chain (phophodiester linkages) have a direction : called polarity; unless otherwise indicated, the convention is 5' to 3' going from left to right. Synthesis of a nucleic acid chain always proceeds from 5' to 3' (left to right).The arrangement (or order) of specific nucleotides along the chain is called the sequence. The sequence is genetic information.

Nucleotides are small ,complex molecules :-

  • Which is made up of C, H, O,N and P .
  • Each nucleotide made up of a 5- carbon sugar (pentose), a nitrogenous base and phosphoric acid. The acidic character of the nucleic acids is due to presence of the phosphoric acid .
  • The pentose sugar is either ribose or deoxyribose. Ribose- In ribose ,the carbon atoms 1' , 2' ,3' and 5' has hydroxyl group. Ribose sugar occurs in nucleotides of RNA as well as nucleotides present free in cytoplasm or inside coenzymes. Deoxyribose- In deoxyribose the hydroxyl groups are found on 1’, 3’ , and 5’ and 2’ group is replaced by hydrogen, the deoxyribonucleotides, are the monomers of DNA.
  • The two bicyclic (two ring structure) bases Adenine (A) and guanine (G) are major purines and monocyclic bases (single ring structure) are thymine (T) , Uracil (U exclusively found in RNA) and cytosine ( C ) are major pyrimidines.

 

  • Erwin Chargaff (1950) found that in any DNA molecule, purine and pyrimidines are present in equal amount i. e. A + G = T +C. Amount of adenine (A) always equal to the amount of thymine (T) i.e A=T , and the amount of guanine (G) always equal to the amount of cytosine (C) i.e.G = C. They are all polyfunctional bases, and may exist in tautomeric forms.
  • Nucleotides form a part of the information transfer system and participate in energy transfer system.
  • In cells, a free nucleotide may contain one (mono), two (di) or three (tri) phosphate groups. The energy carrier AMP (adenosine monophosphate) has one; ADP (adenosine diphosphate) has two; ATP (adenosine triphosphate) has three phosphate groups.
  • These are mono -, di-, and triphosphate of adenosine , when more than one phosphate group present in the nucleotides it is known as higher nucleotides ( e. g. ATP and ADP).
  • Individual nucleotides play important roles in cellular activity of cell. In a molecule of ATP , two terminal chemical bonds that link the O and P atoms are known as high energy phosphate bonds . The replacement of one of these bonds by H-O bonds liberates two or three times as much energy as most chemical bonds. In living cells , the energy is made available by the hydrolytic splitting of the ATP molecule at the outermost high-energy phosphate bond. When one phosphate group is removed the adenosine troposphere becomes adenosine- di-phosphate and energy is released:-

ATP ---.> ADP + (P) + energy (equivalent to 7000 calorie)

The production and utilisation of ATP molecules is an essential characteristic of all living organisms .

  • Purines and pyrimidines nucleotides polymerise to give rise nucleic acids.
  • Nucleotides of vitamins nicotinamide - Nicotinamide adenine dinucleotide (NAD+) and Nicotinamide adenine dinucleotide phosphate (NADP+)
  • And the nucleotides of riboflavin - Flavin mononucleotide (FMN) and Flavin adenine dinucleotide (FAD) play a role in oxidation reactions occurring in the cell.

Nucleoside :- When all phosphate groups of a nucleotide are removed , a nucleotide becomes a nucleoside The combination of a base and a pentose is called a nucleoside. Example adenosine is made up of ribose + adenine.

The following table lists various nucleosides and their mono- NMP (nucleoside monophosphate) or dNMP (deoxynucleoside monophosphate), di- NDP (nucleoside diphosphate) or dNDP (deoxynucleoside diphosphate) , and triphosphates -NTP (nucleoside triphosphate) or dNTP (deoxynucleoside triphosphate). For DNA, these are dATP, dCTP, dGTP, and dTTP. For RNA, these are ATP, CTP, GTP, and UTP.):-

Nucleosides Composition (pentose sugar +base ) Symbol Nucleotides (= Nucleosides + Phosphate) Composition (pentose sugar +base + phosphate) Symbol
RNA
Adenosine Ribose + Adenine (A) A Adenosine Monophosphate , Diphosphate and Triphosphate Ribose + Adenine (A) + phosphate AMP , ADP, ATP
Guanosine Ribose + Guanine(G) G Guanosine Monophosphate , Diphosphate and Triphosphate Ribose + Guanine (G)+ phosphate GMP, GDP, GTP
Cytidine Ribose + Cytosine(C) C Cytidine Monophosphate , Diphosphate and Triphosphate Ribose + Cytosine(C) + phosphate CMP, CDP, CTP
Uridine Ribose +Uracil (U) U Uridine Monophosphate , Diphosphate and Triphosphate Ribose + Uracil (U) + phosphate UMP, UDP, UTP
DNA
Deoxyadenosine Deoxyribose + Adenine (A) dA Deoxyadenosine Monophosphate , Diphosphate and Triphosphate Deoxyribose + Adenine (A) + phosphate dAMP, dADP, dATP
Deoxyguanosine Deoxyribose + Guanine (G) dG Deoxyguanosine Monophosphate , Diphosphate and Triphosphate Deoxyribose + Guanine (G) + phosphate dGMP, dGDP, dGTP
Deoxycytidine Deoxyribose + Cytosine (C) dC Deoxycytidine Monophosphate , Diphosphate and Triphosphate Deoxyribose + Cytosine (C)+ phosphate dCMP, dCDP, dCTP
Deoxythymidine Deoxyribose +Thymine (T) dT Deoxythymidine Monophosphate , Diphosphate and Triphosphate Deoxyribose + Thymine (T) + phosphate dTMP, dTDP, dTTP

Subsection 2

Types of nucleic acids There are two main types of nucleic acids Deoxyribonucleic acid (DNA) occurs mainly in the nucleus, but also in chloroplasts and Ribonucleic acid (RNA) though also found in nucleus, stored in the nucleolus but moves out into the cytoplasm.In DNA or RNA, a nucleic acid chain is also called a strand. DNA (DeoxyriboNucleic Acid) is considered as the repository of the genetic information and a DNA molecule typically contains two strands whereas most RNA molecules contain a single strand and RNAs (RiboNucleic Acids) may be regarded as vectors and translators of the information.

The length of a nucleic acid chain is represented by the number of bases. In the case of a double-stranded nucleic acid, bases are paired between two strands.

DNA or Deoxyribonucleic acid :- J. Watson & S. Crick ( 1953) determined the double helix structure of DNA, owing to the important X-ray diffraction works made by Wilkins, R. Franklin and Astbury.

  • The DNA Molecule consist of two polynucleotides chains (strands), held together by the hydrogen bonding between their bases. 
  • The two Strands are wrapped plectonemically to form a double helix.
  • Each strand forms a right handed helix , with ten base pairs per spiral turn.
  • The two strands of the DNA double helix run in opposite directions, one in the 5' to 3' direction, the other in the 3' to 5' direction. is known as antiparallel. .

  • The sugar-phosphate backbone is on the outside of the helix, and the bases are on the inside, stacked on top of each other like the steps of a spiral staircase, whereas the bases in the middle form the rungs of the ladder.
  • Each rung is composed of two base pairs. Either an adenine-thymine pair that form a two-hydrogen bond together, or a cytosine-guanine pair that form a three-hydrogen bond. The base pairing is thus restricted.
  • Due to restriction when the DNA is being copied: the DNA-helix is first "unzipped" in two long stretches of sugar-phosphate backbone with a line of free bases sticking up from it, like the teeth of a comb. Each half will then be the template for a new, complementary strand.
  • dA-dT and dG-dC base pairs are the same length, and occupy the same space within a DNA double helix. Therefore the DNA molecule has a uniform diameter.
  • dA-dT and dG-dC base pairs can occur in any order within DNA molecules.
  • The N-glycosic bonds (sugar-base) are not directly opposite one strand another, therefore two alternating grooves are evident, a wide and deep major groove, and a shallow and narrow minor groove, between ribose-phosphate chains on the surface of the molecule.Which may facilitate binding with specific proteins.
  • The double helix diameter is 2.0 nanometers (there are one billion nanometers in a meter).
  • The distance between two neighboring base pairs is 0.34 nmnanometers and the helix makes a turn every 3.4 nm (distance along the axis per 360 degree turn) .
  • Denaturation (also known as melting):- The conversion of double helical DNA into single strands(uncoil and separate) due to presence of heat or alkali is called denaturation . The conversion of single strands back to the double-stranded structure is called renaturation. or annealing.
  • There are three natural forms of DNA (A, B and Z). The origin of these different forms are related to the conformation of the sugar (C2'-endo/ C3'-endo) and the orientation of the base relative to the sugar (syn/anti):-
  • B-form is the common natural form, found at a very high degree of hydration and low ionic strength . B-DNA arranges 10 nucleotides per helix tour, all of conformation C2'-endo/anti . The plane of the bases is nearly perpendicular to the helix axis and the helix surface exhibits two prominent grooves (major and minor).
  • A-form is sometimes found in some parts of natural DNA in presence of high concentration of cations or at a lower degree of hydration ( <65%). A-DNA possess 11 nucleotides per tour (all C3'-endo/anti) and two grooves (a narrow deep major and a wide shallow minor).
  • C-form and D-form are unusual subclasses of B-type. C-DNA is sometimes observed under 45% of hydration while D-DNA is only found in artificial DNA.
  • Z-form (Zigzag chain) is observed in DNA G-C rich local region. Z-DNA is longer, thinner and possess an unusual left-handed helix (of 12 bases pairs/tour) with a single narrow deep groove. These Zigzag form mainly results from the alternation of purines (C3'-endo/syn) and pyrimidines (C2'-endo/anti).

Subsection 3

DNA Replication Before a cell divides into two daughter cells, all the DNA molecule must be duplicated. In eukaryotes, this occurs during S phase of the cell cycle. Duplication of an old DNA molecule into two new DNA molecules is called replication.

The double-stranded DNA molecule has the unique ability that it can make exact copies of itself, or self-replicate. During replication, the hydrogen bonds between the nucleotide bases break and the two single strands of DNA separate.Then each DNA strand serves as a template for the synthesis of a new complementary strand. New complementary bases are brought in by the cell and paired up with each of the two separate strands, thus forming two new, identical, double-stranded DNA molecules. Each daughter DNA molecule is identical copy of its parent molecule, consisting of one old and one new DNA strand. Thus the replication is semi-conservative.

Mechanism of replication :- The replication mechanisms in both bacteria and eukaryotes are similar. However, eukaryotic DNA polymerases do not contain a subunit similar to the E. coli b subunit. They use a separate protein called proliferating cell nuclear antigen (PCNA) to clamp the DNA.

  1. Initiation: -The intertwined DNA starts separating from a particular point (origin of replication) eukaryotes have multiple replication bubbles(origin of replication ) on the same parent DNA, while prokaryotes have a single replication bubbles(origin of replication ) per DNA molecule.
  2. The splitting occurs in places of the chains which are rich in A-T. That is because there are only two bonds between Adenine and Thymine (there are three hydrogen bonds between Cytosine and Guanine).
  3. Enzyme helicase unwind the two strands and enzyme topoisomerases break and release one strand of DNA. The Y-shaped structure that is formed due to unwound of double stranded DNA is known as replication Fork (replication bubbles).The entire activity located in the replication fork is called a replisome.
  4. Each separate strand of DNA functions as a template to guide the insertion of a complementary set of bases on the strand being synthesized. .

  1. Elongation: - A free 3'OH group is required for replication, but when the two chains separate no group of that nature exists. RNA primers are synthesized with the help of enzyme primase, and the free 3'OH of the primer is used to begin replication.
  2. One of the most important steps of DNA replication is the binding of RNA Primase in the initiation point of the 3'-5' parent chain. RNA Primase can attract RNA nucleotides which bind to the DNA nucleotides of the 3'-5' strand due to the hydrogen bonds between the bases.
  3. The major activity is taken on by a group of enzymes termed DNA polymerases. The DNA polymerases recruit free nucleotides and match them by base pairing with the complementary nucleotides of the parent strand. In prokaryotes there are two main DNA polymerases : pol I and pol III. Though, the polymerase activity of eukariotic cells is a little more complex than those of prokaryotes, there is an equivalent of prokaryote pol III named pol-a (alpha) and one of pol I named pol-b (beta). The start point for DNA polymerase is a short segment of an RNA primer (to "prime" or start DNA synthesis at certain point).
  4. As replication of the separate strands occurs, the replication fork moves away unwinding additional lengths of DNA.The elongation process is different for the 5'-3' and 3'-5' template. 5'-3' Template:- Since the fork is moving toward the 5'-end of the strand, replication of this strand may take place in a continuous fashion (building the new strand in a 5' to 3' direction). This continuously formed new strand is called the leading strand. Because DNA Polymerase ä can "read" the template and continuously adds nucleotides (complementary to the nucleotides of the template, for example Adenine opposite to Thymine etc). 3'-5'Template:- In contrast, the replication fork moves toward the 3'-end of the original strand, preventing continuous polymerization of a complementary new strand. Short segments of complementary DNA, called Okazaki fragments, are produced, and these are linked together later by the enzyme ligase. This new DNA strand is called the lagging strand, in the lagging strand the RNA primase adds more RNA primers.The RNA primers are necessary for DNA polymerase a (alpha)to bind nucleotides to the 3' end of them.
  5. The lagging requires these repeated steps:
  • Primer synthesis
  • Elongation
  • Primer removal
  • Ligation (joining the pieces).
  1. The daughter strand is elongated with the binding of more DNA nucleotides. In the lagging strand the DNA pol I -exonuclease- reads the fragments and removes the RNA primers. The gaps are closed with the action of DNA polymerase (adds complementary nucleotides to the gaps) and DNA ligase (adds phosphate in the remaining gaps of the phosphate - sugar backbone).
  2. Termination:-The last step of DNA replication is the termination. This process happens when the DNA polymerase reaches to an end of the strands.
  3. In the last section of the lagging strand, when the RNA primer is removed, it is not possible for the DNA polymerase to seal the gap (because there is no primer). So, the end of the parental strand where the last primer binds isn't replicated. These ends of linear (chromosomal) DNA consists of noncoding DNA that contains repeat sequences and are called telomeres. As a result, a part of the telomere is removed in every cycle of DNA replicati Before the DNA replication is finally complete, enzymes are used to proofread the sequences to make sure the nucleotides are paired up correctly. If mistake or damage occurs, an enzyme called nuclease removes the incorrect DNA. DNA polymerase then fills in the gap.When the process is complete, two DNA molecules have been formed identical to each other and to the parent molecule.

Subsection 4

Speed of Replication :- Bacteria The single molecule of DNA that is the E. coli genome contains 4.7 x 106 nucleotide pairs. DNA replication begins at a single, fixed location in this molecule, the replication origin, proceeds at about 1000 nucleotides per second, and thus is done in no more than 40 minutes. Eukaryotes The average human chromosome contains 150 x 106 nucleotide pairs which are copied at about 50 base pairs per second, because there are many places on the eukaryotic chromosome where replication can begin. Replication begins at some replication origins earlier in S phase than at others, but the process is completed for all by the end of S phase. When replication nears completion, "bubbles" of newly replicated DNA meet and fuse, finally forming two new molecules.

RNA or Ribonucleic acid:-

  • RNA differs from DNA in that each RNA (Ribonucleic acid) molecule is usually consist of a single strand of ribonucleotides (or ribotides) and is much shorter. But in Reovirus and Rice dwarf virus it is double-stranded.

  • An RNA molecule is a linear polymer in which the monomers (nucleotides) are linked together by means of phosphodiester bridges, or bonds. These bonds link the 3' carbon in the ribose of one nucleotide to the 5' carbon in the ribose of the adjacent nucleotide.
  • The molecule folds on itself forming structures called hairpin loops. In the base paired region, the RNA molecule adopts a helical structure as in DNA.
  • RNA has four different bases: adenine, guanine, cytosine, and uracil.
  • In RNA, guanine and cytosine pair (GC) by forming a triple hydrogen bond, and adenine and uracil pair (AU) by a double hydrogen bond; additionally, guanine and uracil can form a single hydrogen bond base pair.
  • One of the major differences between DNA and RNA is the sugar, with 2-deoxyribose being replaced by the alternative pentose sugar ribose in RNA and the pyrimidine base uracil of RNA replaces the thymine base of DNA.
  • Ribose sugar is more reactive because of C-OH (hydroxyl) bonds. Not stable in alkaline conditions. RNA on the other hand has larger grooves which makes it easier to be attacked by enzymes
  • RNA does not follow Chargaff’s rules i.e.,1:1 ratio does not exist between purines and pyrimidines bases due to single -stranded nature and lack of complementarity.

The non-genetic RNA is synthesized on DNA template and is of three types :-

  • Messenger RNA (mRNA Jacob and Monod 1960)- messenger RNA , is produced in the nucleus and carries the information for protein synthesis.
  • Transfer RNA or soluble RNA or Adoptive RNA (s-RNA , t-RNA -- Hoagland 1955 ) It is smallest type of RNA which constitutes about 10-15% of total cellular RNA and, having on average 80 nucleotides per molecule. Each amino acid has its own tRNA molecule which transfers amino acids present in the cytoplasm to the ribosome, where they translate mRNA into amino acid sequences.

The 5’-end of the tRNA always ends in the base guanine while the 3’-end always ends in the base sequence CCA. The triplet base sequence at the anticodon is directly related to the amino acid carried by that tRNA by its own form of the enzyme amino-acyl-tRNA synthetase.

  • Ribosomal RNA ( rRNA) - It is the most stable and largest type of RNA .Constitutes about 80% of total cellular RNA and rRNA is a major structural component of ribosomes (site of protein synthesis).In prokaryotes, the ribosomal RNA (rRNA) has three types: 23S, 5S, and 16S. Eukaryotic ribosomes contain four different rRNA molecules: 18 s, 5.8 s, 28 s, and 5 s rRNA. In which three of the rRNA molecules are synthesized in the nucleolus, and one is synthesized elsewhere. The unit "S" stands for Svedberg, which is a measure of the sedimentation rate. After rRNA molecules are produced in the nucleus, they are transported to the cytoplasm, where they combine with tens of specific proteins to form a ribosome. In prokaryotes, the size of a ribosome is 70S, consisting of two subunits: 50S and 30S. The size of a eukaryotic ribosome is 80S, comprising a 60S and a 40S subunit. t-RNA molecule is folded to form a clover leaf -like structure.

snRNA:- Small nuclear RNA (snRNA) is the name used to refer to a number of small RNA molecules found in the nucleus. These RNA molecules are important in a number of processes including RNA splicing (removal of the introns from hnRNA) and maintenance of the telomeres, or chromosome ends. They are always found associated with specific proteins and the complexes are referred to as small nuclear ribonucleoproteins (SNRNP) or sometimes as snurps.