Biochemistry NUCLEOSIDES, NUCLEOTIDES and TYPE of NUCLEIC ACIDS

Home , Guanosine monophosphate

Paper : 03 Structure and Function of Biomolecules II Module: 02 Nucleosides, Nucleotides and type of Nucleic Acids Principal Investigator Prof. Sunil Kumar Khare, Professor, Department of Chemistry, IIT-Delhi

Paper Coordinators Prof. Sunil Kumar Khare, Department of Chemistry, IIT-Delhi & Prof. M.N. Gupta, Department of Biochemical Engineering and Biotechnology, IIT-Delhi

Content Writer Prof. Sunil Kumar Khare, Department of Chemistry, IIT-Delhi

Content Reviewer Prof. Prashant Mishra, Professor, Department of Biochemical Engineering and Biotechnology, IIT-Delhi

STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry NUCLEOSIDES, NUCLEOTIDES AND TYPE OF NUCLEIC ACIDS

DESCRIPTION OF MODULE

Subject Name Biochemistry

Paper Name STRUCTURE AND FUNCTION OF BIOMOLECULES II

Module Name/Title Nucleosides, Nucleotides and type of Nucleic Acids

Dr. Vijaya Khader Dr. MC Varadaraj

STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry NUCLEOSIDES, NUCLEOTIDES AND TYPE OF NUCLEIC ACIDS

1. Objectives

 Nucleic acids and their components  What are Nucleosides?  What are Nucleotides?  Polynucleotides

2. Concept Map

Nucleic acids

What are nucleic Components of Nucleosides Nucleotides Polynucleotides acids? nucleic acids

Nitrogenous Cyclic Discovery Sugars Phosphate DNA and RNA bases nucleotides

Purines and Function pyrimidines

Properties of purines Types and pyrimidines

Base pairing concept

Chargaff's rule

STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry NUCLEOSIDES, NUCLEOTIDES AND TYPE OF NUCLEIC ACIDS

3. What are Nucleic Acids

Nucleic acids constitute the most important biomolecules of the cell and are critical entities for all known forms of life. Discovery: Nucleic acids were discovered by Friedrich Miescher in 1869. He reported that he had found a substance within the nuclei of human white blood cells, which was weakly acidic in nature and whose function was unknown. He had named this material as "nuclein". A few years later, Miescher was successfully able to separate nuclein into protein and nucleic acid components.

Nuclein was later named as nucleic acid in 1889 by Richard Altmann. They were so named because of their initial discovery from within the nucleus (~nucle), and due to the presence of phosphate groups in their molecules (phosphoric acid ~ ic acid).

Function: Nucleic acids are present in all living beings as well as in bacteria, archaea, mitochondria, chloroplasts, viruses and viroids. Nucleic acids are involved in the storage and transfer of genetic information in living organisms.

Types: There are two types of nucleic acids in cells, Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA). Both DNA and RNA are the molecular repositories of genetic data.

3.1 Components of nucleic acid

The basic components of a nucleic acid include three different entities, namely a nitrogenous base, a sugar moiety and a phosphate group. These combine to give one unit of a nucleotide (discussed later), which are stacked in a nucleic acid molecule (Fig. 1).

Fig. 1 Components of nucleic acid

The basic components of a nucleic acid are discussed in detail in sections below:

3.1.1 Nitrogenous Bases

STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry NUCLEOSIDES, NUCLEOTIDES AND TYPE OF NUCLEIC ACIDS

The nitrogenous bases are nitrogen-containing bases, which are derivatives of two heterocyclic compounds: purine and pyrimidine.

 Pyrimidines are monocyclic, whereas purines are bicyclic.  These bases are all polyfunctional in nature.  Purine bases are composed of a 9‐membered double‐ring structure with four nitrogens and five carbons while pyrimidine bases are composed of a 6‐membered ring with two nitrogens and four carbons.  The carbon and nitrogen atoms in purines and pyrimidines are numbered based on convention. The basic structures of purines and pyrimidines with appropriate numbering are shown in Fig. 2 below.

Fig. 2 Basic structures of purines and pyrimidines Nitrogenous bases found inside cells

Inside the cells, five major nucleobases or nitrogenous bases are found. The derivatives of purine are called adenine (A) and guanine (G) bases, while the derivatives of pyrimidine are called thymine (T), cytosine (C ) and uracil (U) bases. The DNA contains A, G, C and T, whereas RNA contains A, G, C and U bases. The chemical structures of the principal bases in nucleic acids are shown in Fig. 3.

STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry NUCLEOSIDES, NUCLEOTIDES AND TYPE OF NUCLEIC ACIDS

Fig. 3 Chemical structures of the principal bases in nucleic acids. Properties of purines and pyrimidines . Shape: Purines and pyrimidines differ in their shape. The shape of the pyrimidine ring is planar, whereas the shape of the purine rings is nearly planar but exhibits some amount of puckering. . Solubility: Purine and pyrimidine molecules are hydrophobic in nature and have a relatively low solubility in water near neutral pH. However, at acidic or alkaline pH, the purines and pyrimidines become charged, and their solubility therefore increases. . Chemical properties: They are conjugated molecules and weakly basic in nature.

. Tautomerism: Both purines and pyrimidines exhibit keto-enol tautomerism. The keto tautomer is known as a lactam ring, whereas the enol tautomer is known as a lactim ring. At neutral pH, the keto-tautomer remains the more predominanting form. Upon interaction with other molecules, ring nitrogens in the lactam serve as donors of hydrogen bond (H-bond), and the keto oxygens behave as H-bond acceptors.

Fig. 4 Keto-enol tautomerism in uracil.

STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry NUCLEOSIDES, NUCLEOTIDES AND TYPE OF NUCLEIC ACIDS

. Absorption: As a consequence of aromatic ring structure and associated resonance, pyrimidine and purine bases absorb ultraviolet light (UV light), with an absorption maxima at a wavelength 260 nm (Fig.5). The measurement of the concentration of DNA or RNA in a given sample is therefore performed by measuring the UV absorbance at this wavelength.

Fig. 5 An absorption spectra of purified DNA sample.

Base pairing of Purines and Pyrimidines

Purines and pyrimidines, being complementary bases, can participate in base pairing, based on the specific shapes and hydrogen bond properties.

Guanidine, being a complement of cytosine, pairs with cytosine through three hydrogen bonds. Adenine (A) is the complement of thymine (T) in DNA and uracil (U) in RNA. Adenine base pairs with thymine and uracil through two hydrogen bonds.

The pairings of the bases are as follows (Fig. 6):

STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry NUCLEOSIDES, NUCLEOTIDES AND TYPE OF NUCLEIC ACIDS

Fig. 6. Base pairing in purines and pyrimidines

Chargaff’s Rule

Erwin Chargaff (1905-2002), an Austrian-American biochemist gave the Chargaff's rule, according to which DNA always contains equal amounts of certain base pairs.

Fig. 7. Erwin Chargaff

He observed that the amount of adenine (A) always equalled with the amount of thymine (T), and the amount of guanine (G) always equalled the amount of cytosine (C), regardless of the DNA source.

%A=%T and %C=%G

The ratio of (A+T) to (C+G) varied from 2.70 to 0.35 in various organisms.

Table 1. Nucleoside Base Distribution in DNA

Organism Base Composition (mole %) Base Ratios Ratio (A+T)/(G+C)

STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry NUCLEOSIDES, NUCLEOTIDES AND TYPE OF NUCLEIC ACIDS

A G T C A/T G/C Human 30.9 19.9 29.4 19.8 1.05 1.00 1.52 Chicken 28.8 20.5 29.2 21.5 1.02 0.95 1.38 Yeast 31.3 18.7 32.9 17.1 0.95 1.09 1.79 Clostridium perfringens 36.9 14.0 36.3 12.8 1.01 1.09 2.70 Sarcina lutea 13.4 37.1 12.4 37.1 1.08 1.00 0.35

3.1.2 Sugars Two types of pentose sugars are found in nucleic acids, namely ribose and 2-deoxy ribose. The carbons in the ribose sugar are numbered according to convention. Ribose differs from deoxyribose in the presence of a hydroxyl group at the 2’C. The structures of both ribose and deoxyribose are shown in Fig. 8. The D-ribose and D-deoxyribose are found in RNA and DNA respectively, in their furanose (closed five-membered ring) forms.

Fig.8. Ribose and deoxyribose sugar

3.1.3 Phosphates

Phosphate is another important component of the nucleic acid molecule. It gets attached to C-5’ OH group of the sugar and gets incorporated into nucleic acid (both DNA and RNA).

3.2 Nucleosides A nucleoside consists of a combination of a nitrogenous base and a sugar (ribose or deoxyribose).

Nucleosides = nitrogenous base + sugar

STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry NUCLEOSIDES, NUCLEOTIDES AND TYPE OF NUCLEIC ACIDS

The bond between them is called the beta-glycosidic linkage. The position of attachment is shown below.

Fig.9. Nucleoside

Examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine

Fig.10. Examples of nucleosides

3.3 Nucleotides

Nucleotides comprises of a nitrogenous base linked to a 5-carbon sugar and one or more phosphate group. The

phosphate is attached to 5’ CH2OH group of sugar part of nucleoside. They function as the building blocks of nucleic acids. Nucleotides = nitrogenous base + sugar + phosphate Nucleotides = Nucleosides + phosphate

The position of attachment is shown below in Fig. 11

STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry NUCLEOSIDES, NUCLEOTIDES AND TYPE OF NUCLEIC ACIDS

Fig.11. Nucleotides

Formation of nucleotide: The base of a nucleotide (position N-1 of pyrimidines or N-9 of purines) is forms a covalent N—glycosyl bond with the 1’ carbon of the pentose, by removal of a water molecule. The phosphate is esterified to the 5’ carbon (Fig. 12).

Fig.12. Formation of nucleotides

Examples of nucleotides include deoxyadenosine monophosphate, deoxycytidine monophosphate, deoxyguanoside monophosphate, deoxythymidine monophosphate (Fig. 13).

STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry NUCLEOSIDES, NUCLEOTIDES AND TYPE OF NUCLEIC ACIDS

Fig.13. Examples of nucleotides

Nucleotide di- and tri-phosphates: The term "nucleotide" generally refers to a nucleoside monophosphate, But in case additional phosphoric acid groups are present, they can link to the existing phosphate (in nucleotide monophosphates) to produce nucleotide diphosphates and nucleotide tri-phosphates (Fig. 14).

Fig.14. Nucleoside mono, di and triphosphates

3.3.1 Cyclic nucleotides

Nucleoside monophosphates can sometimes form two ester bonds with the phosphoric acid, at the 5' and 3' hydroxyl groups of the ribose sugar. This therefore results in the formation of cyclic nucleotides. These are designated as cNMP where “c” stands for cyclic and “N” stands for the respective nucleoside.

STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry NUCLEOSIDES, NUCLEOTIDES AND TYPE OF NUCLEIC ACIDS

Fig.15. 3’, 5’-Cyclic guanosine monophosphate (3’, 5’-GMP)

Figure 15 above shows a cyclic guanosine monophosphate (cGMP). Cyclic AMP (cAMP) and cyclic GMP (cGMP) are the two of the well-studied cyclic nucleotides. These are found in all cells and play important role in the regulation of cell metabolism.

3.4 Summary of nucleobase derivatives

Table 2. Summary of nucleobase derivatives

Base Symbol Nucleoside Nucleotide (Base+ (Base+ (Base+ deoxyribose (Base+ ribose sugar+

deoxyribose sugar) ribose sugar) sugar + phosphate ) phosphate) A Adenosine 2'-Deoxyadenosine-5'- Adenosine Adenine 2'-Deoxyadenosine monophosphate (dAMP) monophosphate (AMP) C Cytidine 2'-Deoxycytidine-5'- Cytidine Cytosine 2'-Deoxycytidine monophosphate monophosphate (CMP) (dCMP) T Guanosine 2'-Deoxyguanosine-5'- Guanosine Guanine 2'-Deoxyguanosine monophosphate (dGMP) monophosphate (GMP) G Thymidine 2'-Deoxythymidine-5'- Thymidine Thymine 2'-Deoxythymidine monophosphate (dTMP) monophosphat (TMP) Uracil U Uridine 2'-Deoxyuridine-5'- Uridinemonophosphate 2'-Deoxyuridine monophosphate (dUMP) (UMP)

3.5 Polynucleotides A polynucleotide formation is initiated when many nucleotides continue to join together by phosphodiester linkages. The formation of polynucleotide is catalyzed by polymerase enzymes (DNA polymerase in case of DNA or RNA polymerase in case of RNA). The -OH group on the 3’-carbon of sugar in one nucleotide reacts with the

STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry NUCLEOSIDES, NUCLEOTIDES AND TYPE OF NUCLEIC ACIDS

phosphate attached to the 5’-carbon of another to form phosphate ester bonds and a dinucleotide. Repeated formation of such bonds leads to further elongation of the polynucleotide chain.

Fig.16. Formation of polynucleotides

DNA and RNA are examples of polynucleotides (Fig. 17), where the nucleotides are arranged in linear way and proceeds in the 5' ----> 3' direction. A common representation of polynucleotide is given below:

5'pApTpGpC OH3'

While RNA is single stranded, DNA is double stranded and contains two such polynucleotide chains spiralling round each other to form a double helical structure. The two chains in the double helix are held together by hydrogen bonds by complementary bases on different chains.

STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry NUCLEOSIDES, NUCLEOTIDES AND TYPE OF NUCLEIC ACIDS

Fig.17. DNA and RNA as polynucleotides

Summary

In this lecture we learnt:

 Nucleic acids are important cellular biomolecules involved in the storage and transfer of genetic information in all living organisms.  There are two types of nucleic acids in cells, Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA).  Nucleic acids include three different entities, namely a nitrogenous base, a sugar moiety and a phosphate group. o Bases are nitrogen-containing molecules, which are derivatives of purine and pyrimidine. Five major bases found in cells are adenine (A) and guanine (G) (purine bases), thymine (T), cytosine (C ) and uracil (U) bases (pyrimidine bases). o The purines and the pyrimidines are complementary bases. Base pairing occurs between G and C, A and T. o Chargaff’s rule: Amount of adenine (A) equals the amount of thymine (T), and amount of guanine (G) equals the amount of cytosine (C). o Ribose and 2-deoxy ribose are two types of pentose sugars found in nucleic acids.  Nucleotides = nitrogenous base + sugar + phosphate.  Nucleotides = Nucleosides + phosphate; Nucleosides = nitrogenous base + sugar.

STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry NUCLEOSIDES, NUCLEOTIDES AND TYPE OF NUCLEIC ACIDS

 When nucleotides join together by phosphodiester linkages, a polynucleotide formation is initiated.

STRUCTURE AND FUNCTION OF BIOMOLECULES II Biochemistry NUCLEOSIDES, NUCLEOTIDES AND TYPE OF NUCLEIC ACIDS