- 1 Histones & Importance of DNA Packaging – Nucleosomes and Chromosomes
- 1.1 Discovery of Nucleic Acids
- 1.2 General Composition of Nucleic Acid
- 1.3 Nucleotides
- 1.4 Formation of Nucleotide
- 1.5 Formation of Polynucleotide Chain
- 1.6 DNA (Deoxyribonucleic Acid)
- 1.7 Size of DNA
- 1.8 Forms of DNA
- 1.9 Properties of DNA
- 1.10 RNA (Ribonucleic Acid)
- 1.11 Structure of RNA
- 1.12 DNA Packaging
- 1.13 Chromosome and Nucleosome Organization
- 1.14 Nucleosome
- 1.15 Organization of the Nucleosome
- 1.16 DNA in the Nucleosome
Advances in technology have expanded the scope of Biology Topics we can investigate and understand.
Histones & Importance of DNA Packaging – Nucleosomes and Chromosomes
In cellular organisms, DNA and RNA are two important complex organic compounds. In general terms, they are called nucleic acids. A virus contains either DNA or RNA as the core component in the particle. However, in the prokaryotic or eukaryotic cell, both components are found to be present. In plant or animal cells, DNA is present in the chromatin component of the nucleus. However, in some cytoplasmic organelles (e.g., mitochondria, plastid, centriole, etc.) the presence of DNA may also be observed. On the other hand, though RNA is present in the nucleus, they are present in the cytoplasm in a major amount. Of the two chemical organic components, DNA acts as the genetic material and RNA assists in the expression of genetic materials. All the RNA materials present in a cell are produced from the cellular DNA. The information stored in the DNA comes to the RNA in the form of a message and the message in coded form in RNA is translated in the form of polypeptide. The polypeptide in turn gives a functional protein.
Discovery of Nucleic Acids
In 1969, F. Meischer first developed a technique to isolate the nucleus from pus cells and he first described the presence of nuclein as the nuclear component. In the later period, his student Altmann (1989) named nucleic acid for nuclein. During the early period of the 20th century, Kossel observed the ingredients like nitrogenous base, 5-carbon sugar, and phosphoric acid in the formation of nucleic acids. In the contemporary period, Ascolin, Levine, and Jones discovered the presence of nucleic acids in two forms i.e., DNA and RNA. Feulgen (1924) developed a technique to stain the DNA present in a cell and he could be able to show that DNA is present principally in the cell nucleus. The discoveries on the structural organization of nucleic acids are the events of the later period and * a clear idea of the molecular organization of DNA came from Watson and Crick in the year 1953.
General Composition of Nucleic Acid
In general, DNA or RNA may be stated as a polymer of unit components named nucleotides. On hydrolysis by acids, the polymer in the nucleic acid is broken down into nucleotides. The nucleotides in DNA or RNA are ligated sequentially one after another to produce a long chain called polynucleotide. The polynucleotide gives a structure to either RNA or DNA.
One nucleotide is formed of three basic components namely Nitrogenous base, Pentose sugar, and Phosphoric acid.
Nitrogenous bases in the nucleic acids are of two principal categories namely Purine and Pyrimidine. The purine molecules are double-ring components and the pyrimidines are single-ring components.
(i) Purine: Purines found in the nucleic acids are of two types, namely Adenine and Guanine. Adenine and Guanine are available in both DNA and RNA. However, adenine is chemically known as 6-amino purine and guanine is known as 2-amino 6 oxy purine. Hence the structure of the two purines of nucleic acids may be given by the following diagram.
(ii) Pyrimidine: Pyrimidines in nucleic acids are available in three forms, namely Uracil, Thymine, and Cytosine. All the molecules are single-ring components. Uracil is chemically known as 2, 4-di-oxy pyrimidine, thymine is known as 2,4-di-oxy, 5-methyl pyrimidine and cytosine is known as 2-oxy 4-amino pyrimidine. The structure of three pyrimidines may be given in the following diagram.
5 Carbon Sugar:
5 carbon (Penta) sugar of the nucleic acid is also known as pentose sugar. The sugar in its ring configuration contains the 5th carbon outside the ring. Other 4 carbon atoms are present within the ring. In the pentose sugar present in DNA, it lacks one oxygen at the 2′ position, therefore, the sugar in DNA is called deoxyribose sugar.
C. Phosphoric Acid:
Phosphoric acid or H3PO4 is an important part of a nucleotide. It helps the nucleotides to be joined together. The structure of phosphoric acid may be indicated in the following manner.
Formation of Nucleotide
Cells’ extracellular fluids in living organisms contain small quantities of nucleosides. Nucleosides consist of a base, a sugar without a phosphate. Nucleotides are nucleosides that have one, two or three phosphate groups esterified at 5′ hydroxyl. However, the mechanism of bond formation of the three ingredients is different. The nitrogenous base first combines with the pentose sugar to form a nucleoside. The linkage is established between the 1′ carbon of the pentose sugar and the nitrogen of the ring of purine or pyrimidine. If the nitrogenous base is a purine then the nitrogen present at the 9′ position is involved in the formation of linkage.
On the other hand, if it is a pyrimidine then the 1′ Nitrogen is involved in the bond formation. This form of linkage between the nitrogenous base and the pentose sugar is called a glycosidic bond. A nucleoside formed with adenine is known as adenosine, and that formed by guanine is known as guanosine. The nucleoside of uracil is called uridine, that of thymine is called thymidine and that formed of cytosine is called cytidine. Therefore,
Adenine + Pentose sugar = Adenosine
Guanine + Pentose sugar = Guanosine
Thymine + Pentose sugar = Thymidine
Cytosine + Pentose sugar = Cytidine
Uracil + Pentose sugar = Uridine
The nucleoside combines with phosphoric acid to form a nucleotide. The combination of phosphoric acid with the nucleoside involves the – OH group of the phosphoric acid and the OH group at the 5′ carbon of sugar on the nucleoside. The bond formed between the nucleoside and the phosphoric acid is known as the ester bond. With one nucleoside as many as three phosphoric acids may be attached with the formation of ester bonds between the phosphoric acids as indicated in the diagram and thereby each of the triphosphonucleosides becomes an energy-rich component for their utilization in various metabolic events.
Difference between Nucleoside and Nucleotide:
|1. Nucleoside is chemically composed of pentose sugar and a nitrogenous base.||1. Nucleotide is chemically composed of Pentose Sugar, a nitrogenous base, and a phosphate.|
|2. It is slightly basic in nature.||2. It is acidic in nature.|
|3. E.g.: Adenosine.||3. E.g.: AMP (Adenosine Mono Phosphate).|
Nucleoside = Pentose Sugar + Nitrogenous Base
Nucleotide = [Pentose Sugar + Nitrogenous Base] + Phosphoric Acid = Nucloside + Phosphoric Acid
Nucleotides formed of different nitrogenous bases are named in the following manner:
Adenosine + Phosphoric acid = Adenylic Acid or Adenylate
Guanosine + Phosphoric acid = Guanylic acid or guanylate
Uridine + Phosphoric acid = Uridylic acid or uridylate
Thymidine + Phosphoric acid = Thymidylic acid or Thymidylate
Cytidine + Phosphoric acid = Cytidylic acid or Cytidylate
|Nucleosides and Nucleotide Nucleobases||Purines (Double Ring Pyrimidin ring fused with imidazole)||Pyrimidines (Single ring member)|
|Adenine (A)||Guanine (G)||Cytosine (C)||Uracil (U) Thymine (T)|
|Deoxynucleoside mono, di, and triphosphates||dAMP, etc.||dGMP, etc.||dCMP, etc.||dTMP, etc.|
However, in the DNA the different nucleotides are pre-fixed with a d- for the presence of deoxyribose sugar in the molecule (e.g., d-adenylic acid, d-cytidylic acid, etc.).
Formation of Polynucleotide Chain
In nucleic acids, many nucleotides are joined one after another with the formation of ester bonds between two successive nucleotides. In this ester bond formation – OH groups of the phosphoric acid moiety are combined with the 5’ carbon of one nucleotide and the 3′ OH group of the sugar of the other nucleotide. Polymerization reaction involving the – OH group of the nucleotides liberates one molecule of water at each ester bond formation. A polynucleotide chain formed in this way may be featured in the following manner. The two terminal ends of a polynucleotide chain may be marked as 5′ end and 3′ end.
DNA (Deoxyribonucleic Acid)
DNA is the double-stranded macromolecule that acts as genetic material in the cell and in which each strand represents polynucleotides formed of nitrogenous bases, deoxyribose sugars, and phosphoric acids (forming nucleotide units).
The DNA Double Helix Model:
The proposed DNA double helix model by Watson and Crick may be described by the following points:
1. Each DNA molecule is macromolecular in organization having two polynucleotide chains.
2. Two polynucleotide strands in DNA are aligned side by side maintaining a specific distance and they remain oriented in antiparallel fashion.
3. The polynucleotide strands are organized in the form of a right-handed helix surrounding an imaginary central axis which may be comparable to a spiral staircase.
4. Of the two polynucleotide strands, one is complementary to the other, because the sequence of purine and pyrimidine of one strand determines the sequence of pyrimidine and purine of the other strand. The number of purine bases on one strand equals the number of pyrimidine on the other strand (i.e., A + G = T + C). This equivalence of purine and pyrimidine of DNA is known as Chargaff’s rule.
5. In the alignment of purine and pyrimidine of two polynucleotides of the DNA polynucleotide strands, adenine of one strand pairs with thymine of the other strand by two hydrogen bonds, and guanine of one strand pairs with cytosine of the other strand by three hydrogen bonds. The pattern of hydrogen bonding between purine and pyrimidine in the DNA molecule is called Watson-Crick’s base pairing rule.
6. The nucleotides in the polynucleotide strand are stacked one above the other and in this organization, the nitrogenous bases are directed towards the inner side of the helix and the sugar-phosphate backbone remains at the outer side of the helix.
7. The helix diameter is about 20Å and the nucleotide strand remains 10Å away from the imaginary axis. One complete turn of the helix covers 34Å and within this helix segment, there may be 10 base pairs being present equidistantly. Therefore, the distance between two adjacent base pairs is about 3.4Å.
8. Along the whole length of the helix, two grooves may be observed – one is called the major groove and the other is called the minor groove. The major groove is directed towards the inner face of the helix and the minor groove is directed towards the outer side of the helix. The major groove is 11.7Å wide and 8.8Å deep, while the minor groove is 5.7Å wide but 7.5Å deep.
9. In paired condition, guanine and cytosine span a distance of about 13.8Å and adenine and thymine span a distance of about 11.1Å.
10. The bases remain at a right angle to the helix axis, but on the horizontal line, each of them remains in a slightly tilted condition forming a definite angle. In this respect, the bases form the angle as G : 52°, C : 50°, T : 50° and A : 51°.
Size of DNA
The size of DNA differs in different types of organisms. The circular DNA of the polyoma virus measures about 1.7 pm and it contains about 5100 base pairs. The number of genes in this DNA is six. The DNA of T2 phage is linear having a length of about 55 pm. In this DNA there are 160000 base pairs giving the existence of about 100 genes. The E.coli cell contains a single circular DNA that measures about 1 mm with 40 lakh base pairs. In this DNA there are 4000 genes. The eukaryotic cells contain only linear DNA and they are present within the chromosomes. Normally one chromosome contains one DNA molecule. In comparison to the DNA of the prokaryotic cell, the DNA present in the eukaryotic cell is longer. All the DNA present in 23 chromosome pairs of man are joined end to end, its length comes to about 1.9 meters.
Nature of DNA in Some Organisms:
|Organisms||Group||Length of DNA (mm)||Number of Base Pair (Thousand)||Nature/Shape of DNA|
|Polioma (SV 40)||Virus||1.7||5.1||Circular|
|φ × 174||Virus||1.8||5.39||Circular single-stranded|
|T2, T4, T6||Virus||55||166||Linear|
|Yeast||Eukaryote||4600||13500||Linear, 17 haploid chromosomes|
|Drosophila Melanogaster||Eukaryote||56000||16,500,00||Linear, 4 haploid chromosomes|
|Man||Eukaryote||990,000||29,00,000||Linear, 23 haploid chromosomes|
Forms of DNA
The DNA model as proposed by Watson and Crick is said to be the structure of B-DNA. DNA in this form is available in the cell with normal physiological conditions at 92% humidity with normal salt concentration. If the physiological condition is changed and humidity is reduced the B-DNA may attain A-DNA conformation. On the other hand at high salt concentration, it attains the Z-DNA conformation. Altogether six DNA conformations may be available and they are A, B, C, D, T, and Z DNA. These different forms of DNA differ by nature of the helix, number of base pairs per turn, base pair rise on the helix, and width and depth of the major and minor groove.
Different forms of DNA and their Characteristics:
|Base pair per turn||11||10||9.33||8||8||12|
|Rise per base pair||2.56Å||33 – 3.4Å||3.31Å||3.03Å||3.4Å||3.7Å|
|Major groove width||27Å||11.7Å||10.5Å||8.9Å||Wide||8.8Å|
|Major groove depth||13.5Å||8.8Å||7.5Å||5.8Å||Shallow||5.7Å|
|Minor groove width||11Å||5.7Å||4.8Å||1.3Å||Narrow||2Å|
|Minor groove depth||2.8Å||7.5Å||7.9Å||6.7Å||Deep||13.8Å|
Controversy persists with respect to C and D DNA, but there is ample evidence with regard to A, B, and Z DNA. At specific conditions, B DNA may be converted into A or Z DNA. All these structural organizations indicate that DNA is actually a dynamic chemical component with a certain degree of flexibility. Out of several forms of DNA, A, B, and Z DNA configurations are biologically significant. Only Z DNA exhibits left-handed coiling. Within the B DNA configuration, the Z configuration may be available in small stretches. In the Z DNA Sugar-phosphate backbone remain in a zig-zig orientation. Each complete turn of Z DNA contains 12 base pairs and its length is about 46A.
In this case, the diameter of the helix is about 18Å. In normal conditions, the stability of Z DNA is comparatively short. Alexander Rich and his colleagues in 1979 from X-ray crystallographic analysis of self-complementary oligonucleotides (d: CGCGCG) revealed the presence of Z DNA configuration. When DNA achieves -ve supercoiling or when in a DNA molecule, cytosine bases are methylated B DNA may achieve Z DNA configuration. From such observation, it has been predicted that Z DNA may have some role in the regulation of gene activity. However, there is no clear-cut idea of the real function of Z DNA.
Properties of DNA
Properties of DNA may be categorized into three types namely physical properties, chemical properties, and biological properties.
1. Physical Properties:
(a) Polymorphism at different physiological states, especially in the presence of excess salt and low humidity, DNA shows structural alterations. For example, B DNA at 75% humidity and in the presence of sodium salt is altered into A DNA. At high salt concentrations, B DNA may be converted into Z DNA.
(b) Change of Ionic Property: Being negatively charged DNA is attracted towards the anode in the electrical field. For this reason, DNA of variable dimensions may be separated by agarose gel electrophoresis.
(c) Solubility of DNA: DNA is soluble in water and alcohol. However, in chilled alcohol DNA becomes insoluble and becomes visible.
(d) Denaturation and Renaturation: The hydrogen bonds between two nucleotide chains of DNA may disappear at high temperatures and higher alkaline pH. Therefore, the polynucleotide strands of a DNA may be separated by applying heat or increasing the pH of a medium in which the DNA is present. Such separation of DNA strands is called denaturation. If the temperature of the solution is decreased slowly, the separated strands may be realigned again to achieve the normal state. This phenomenon is called renaturation or annealing.
The temperature at which half the DNA gets denatured is known as melting temperature (Tm). On the basis of the quantity of A-T and G-C in a DNA, the melting temperature may vary. The DNA that is higher in G-C content shows a higher melting temperature.
(e) Response of DNA to UV Light: DNA can absorb UV light of 260 nm. In comparison to double-stranded DNA molecules, single-stranded DNA may absorb more UV radiation. For this reason when at high temperatures denaturation of DNA occurs absorption of UV light is increased. This is called hyperchromic shift. Besides, depending on the variation in the quantity of G and C residues in DNA the absorbance of UV rays also increases.
(f) Buoyant Density: Every DNA molecule has a definite buoyant density. In most cases, it is 1.7 g/cm3, which is equivalent to the 6 molar concentration of CsCl solution. Depending on the variation of the G-C content in DNA, its buoyant density varies. DNAs taken from different species of organisms when exposed to density gradient ultracentrifugation in 6M CsCl solution, they localize at different regions of the centrifuge tube depending on the variation in G-C content in the DNA. Normally DNA having higher G-C content exhibits higher buoyant density. Therefore, the DNA which is having highest G-C content localizes towards the bottom of the centrifuge tube.
This sort of variable distribution is due to the development of different gradients of CsCl in the solution of the tube during ultracentrifugation. In these gradients, CsCl concentration becomes highest at the bottom of the centrifuge tube and it gradually decreases less towards the upper part of the centrifuge tube. The buoyant density of DNA is denoted by ρ and it is expressed by the equation ρ = 1.66 + 0.098% (G + C)
2. Chemical Properties:
(a) DNA is an acidic organic compound formed of deoxyribonucleotides of adenine, guanine, cytosine, and thymine. Being acidic in nature it may be stained by basic dyes.
(b) In a double-stranded DNA total number of purine is equivalent to the total number of pyrimidine. Therefore, the number of A + T = Number of G + C. In the double-stranded DNA, adenine (A) of one strand pairs with thymine (T) of the other strand. Similarly guanine (G) of one strand pairs with cytosine (C) of the other stand. Because of this in the double-stranded DNA A/T or G/C is equal to 1.
The ratio of Purine and Pyrimidine in the DNA of Some Organisms:
|Name of the Organism||Number of Bases||Ratio|
(c) On hydrolysis DNA is broken down into fragments and may even produce unit nucleotides. At high temperatures i.e., at 100°C in perchloric acid DNA is completely hydrolyzed to produce components like base, sugar, and phosphates. At 60°C in 1N HCl solution DNA gets partially hydrolyzed producing apurinic acid, when the purine bases are detached from sugar moieties and at the sugars aldehyde group (- CHO) may be generated. Such aldehydes respond to Fuelgen reaction to bind with leucofuchsin producing coloured compound.
(d) In acidic or alkaline pH, DNA gets denatured i.e., hydrogen bonds between the polynucleotide strands are dissolved to separate the polynucleotide stands. Besides this in a natural solution if the concentration of NaCl (< 10-5) becomes insufficient, DNA exhibits denaturation.
(e) At a state of higher physiological pH (i.e., above 7.8) the nitrogenous bases show tautomeric shift. At neutral pH, the bases like thymine and guanine remain in keto form, but at alkaline pH, they may undergo a tautomeric shift producing their enol forms.
(f) Some chemical substances may trigger the denaturation of DNA. Two such chemicals are urea (H2NCONH2) and formamide (HCONH2). The concentrated solution (7M) of these chemicals may break the hydrogen bonds between the nitrogenous bases of the DNA strands and cause denaturation.
(g) Separated polynucleotide strands due to denaturation if encounters complementary bases they may be renatured with the formation of hydrogen bonds between purine and pyrimidine bases. Complementarily, DNA polynucleotide pair with a polynucleotide of some other DNA or RNA, is known as hybridization.
3. Biological Properties:
DNA obtained from either living or dead cells is very much dynamic and is capable of exhibiting various biological properties or activities.
(a) The DNA material present in a cell acts as genetic material. Each DNA molecule represents the storehouse of a number of information in the form of codes. A gene represents a segment of this information-bearing macromolecule. The coded information may be expressed via the production of RNA or protein. Therefore, DNA is the key component in a cell for the control and expression of all sorts of characteristic features of living organisms. The diversity observed from organism to organism or from species to species is based on the difference in the composition of DNA molecules among the concerned organisms. The inheritance of characters from the parents to the offspring becomes possible when DNAs from both parents accumulate in the progeny by the gametic fusion from the parents.
(b) DNA molecule is capable of producing its own copy by way of replication. Before cell division almost every cell may double its DNA quantity through the process of replication and due to this the mother cell may contribute its pool of DNA to each of the daughter cells equally. During such duplication of cellular DNA, replication occurs in a semi-conservative fashion.
(c) Under favourable conditions the genic part of DNA may synthesize single-stranded RNA and this process of RNA synthesis is known as transcription. In the genic part of DNA, only one stand plays as the template to produce the RNA strand having complementary base sequences.
(d) The DNA of all organisms as well as cells is quite stable. However, in certain situations, this stability goes under attack due to specific causes. Mutation and recombination are the two events in the life of organisms, that may hamper the DNA stability resulting change in DNA organization. Due to such alterations new features even new combinations of characters may appear in the organisms.
Functions of DNA
- DNA acts as the genetic material.
- DNA controls directly or indirectly all the activities of a cell.
- DNA promotes RNA as well as protein synthesis.
- DNA can form its own copy by replication.
- DNA remains the important constituent material of chromatin.
Biological Significance of DNA
- During the growth and division of cells DNA is replicated very faithfully.
- In any organism, DNA is very stable and occasionally undergoes mutation by some external influence.
- DNA is inherently potent to control cellular activities.
- DNA is a storehouse of biological information for an organism.
- Characters of an organism are transmitted to the progeny via the DNA molecule.
- DNA promotes the synthesis of RNA and protein for the expression of characters.
Difference between Prokaryotic and Eukaryotic DNA:
|Prokaryotic DNA||Eukaryotic DNA|
|1. Circular and usually remain in super-coiled form.||1. Linear and remain in the chromosome.|
|2. DNA remains is the cytoplasm.||2. DNA remains in the nucleus.|
|3. DNA is G = C rich.||3. DNA is A = T rich.|
|4. DNA is with exon sequence only.||4. DNA contains both introns and exons.|
|5. DNA contains one ori C Replication origin.||5. DNA contains many replication origins.|
RNA (Ribonucleic Acid)
RNA is a single-stranded nucleic acid that represents a polynucleotide chain formed of nitrogenous bases, ribose sugars, and phosphoric acids, and that helps in the expression of genes in the cell.
Structure of RNA
Identically with DNA, RNA is also the cellular organic component formed of many nucleotides. However, RNA is not so large in dimension as DNA is. The largest RNA contains only several thousands of nucleotides. Being developed from the DNA of the cell, the RNA molecules may participate in many cellular functions. However, principally RNA molecules play as the mediators in the transfer t of information from DNA to phenotype. In the living body, the flow of information may occur by different ways and this information flow is represented diagrammatically in the following way and this is known as Central Dogma.
In the central dogma, RNA is placed between the DNA and protein. In certain cases, RNA may act as genetic material. For all these reasons RNA is considered an important biomolecule.
In most cases, RNA is single stranded polynucleotide molecule, but in some viruses, double-stranded RNA may be encountered. Primarily single-stranded RNA in the form of the linear chain may attain secondarily double-stranded helical configuration at least segmentally due to the presence of complementary base sequences at certain regions of the polynucleotide chain. In the cell, different species of RNA may be available and they differ in their number of bases and in their secondary organization. The major types of RNA species found in the cells are mRNA, tRNA, rRNA, hnRNA, snRNA, and scRNA.
This type of RNA is called messenger RNA. The presence of this type of RNA was first detected by Francois Jacob and Jacques Monod in 1961. This class of RNAs though differ in size and dimension, their average molecular weight and sedimentation coefficient are 500000 and 8S respectively.
mRNA is always single-stranded and is produced from the functional genetic site of DNA. In eukaryotic cells, the mature functional mRNA is different in dimension from the nascent mRNA from which it comes after processing. However, in prokaryotic cells, mRNA prior to its final production may be engaged in protein synthesis and therefore, there is no difference between the mature functional mRNA and the nascent mRNA molecule in a prokaryotic organism.
The mature eukaryotic mRNA is composed of several specific segments or parts, these are the proximal G5’PPP5′-N-3’P at the 5′ end known as a cap, the rear poly A tail formed of about 100-200 adenine residues and the medial protein coding region, and non-coding regions at its both sides.
The cap at the proximal 5′ end of the mRNA may give it protection from exonuclease digestion. The guanine residue in the cap is usually methylated. The medial cod¬ing region helps in the polymerization of amino acids during translation. The region frontal to the coding segment is called the leader sequence while the distal part of it is known as the trailer sequence. These regions are not involved in translation, the poly-A tail in one mRNA is formed usually after the RNA is synthesized from the DNA. The poly-A tail is known to be the transporter of mature mRNA from the nucleus to the cytoplasm. In the mRNA of the prokaryotic cells, both the cap and tail are wanting. The eukaryotic mRNA is usually monocistronic, i.e., the mRNA is the product of a single gene only. In prokaryotic cells, several genes may give rise to a single large mRNA molecule. Hence, prokaryotic mRNA molecules may be polycistronic in nature.
This type of RNA is available in the ribosome. Primarily rRNA is single-stranded, but 70% of the region in one rRNA molecule is secondarily double-stranded. rRNA within one ribosome may be of various dimensions. In the prokaryotic 70S ribosome, there are three different rRNA fractions, namely 16S rRNA, 23S rRNA, and 5S rRNA. of these, the 16S rRNA is present in a small 30S ribosomal sub-unit and both the 23S rRNA and 5S rRNA fractions are present in the 50S ribosomal sub-unit.
In eukaryotic cells, there are four types of rRNA molecules, namely, 18S rRNA, 28S rRNA, 5.8S rRNA, and 5S rRNA fractions. 18S rRNA is present in the small 40S ribosomal subunit, while the others are present in the large 60S ribosomal subunit. All the different rRNA fractions differ in their molecular organization. The rRNA molecules being bound with the protein subunit give the structure of the ribosome. Besides, they take a role in protein synthesis in some ways or other. The region of DNA that participates in rRNA synthesis is known as rDNA.
This RNA is called transfer RNA. Alternatively, transfer RNA is also known as adapter RNA as well as soluble RNA. In dimension, this type of RNA is smaller in comparison to mRNA or rRNA molecules. Usually, this type of RNA is formed of 73 to 93 nucleotides. Primarily they are single-stranded but due to the presence of complementary bases at different locations, double-stranded stems and loops are formed at different segments of the molecule. tRNA, besides having adenine, guanine, cytosine, and uracil, contains some unusual and modified bases. Several such unusual modified bases are methyl guanine, dimethyl guanine, methylcytosine, ribothymine, pseudo-uracil, dihydrouracil, and inosine.
In the cell, there are more than twenty different tRNA molecules. However, investigation on cells starting from bacteria to higher eukaryotic cells reveals the presence of about 75 types of tRNA molecules. These tRNA molecules primarily differ by their base composition and the base sequence in a tRNA molecule represents its primary organization. Robert Holley and his colleagues in 1965, first discovered the primary structural organization of yeast alanine tRNA molecule. While describing the secondary structure of this tRNA molecule, they proposed the Clover Leaf Model to feature the structure of the tRNA molecule. The^ secondary configuration of the tRNA molecule as described by Holley et.al. is called the cloverleaf model.
According to the clover leaf model, one tRNA molecule contains four or five arms and three to four loops. The loops are known as the DHU loop (dihydrouridine loop), anticodon loop, T pseudouracil C loop (T ψ C loop), and a variable loop. The arms are accordingly called the acceptor arm, D arm, anticodon arm, T pseudouracil C arm (T ψ C arm), and variable arm. The variable arm and loop may not be present together. The arm or stem represents the double-stranded segments along the tRNA molecule and that remains stabilized by the formation of hydrogen bonds between the complementary bases. Between the two complementary sequences, the region with a unique base sequence develops the loop configuration at the terminal end of an arm.
The acceptor stem comprises a region with 7 base pair sequences in which seven bases start from the 5′ end of the tRNA molecule and pair with seven bases at the other end by about four bases away from the 3’terminal end. It means that at the 3′ end of the tRNA, a single-stranded short segment with four bases is present. In this single-stranded segment, the last three bases are 5’CCA3′. The CCA end can receive amino acid for its transportation to the ribosome and therefore, the arm attached to this segment is called the acceptor arm. In the DHU arm, three to four nucleotides form the paired segment, while seven to eleven nucleotides in a unique sequence develop the DHU loop region. Due to the presence of dihydrouridine in excess, the arm is named the DHU arm. Alternatively, this arm is also known as D arm and the loop is designated as a loop no. I.
The third anticodon stem or arm represents a segment with 5 base pairs region. At its terminal end, the anticodon loop contains seven nucleotides. Three bases in the intermediate position of this loop constitute the anticodon that can pair with the codon of mRNA. The anticodon loop is also called loop no. II. The T ψ C or T arm represents a stem with 5 base pairs. The loop at its terminal end contains a unique sequence of seven bases. This loop carries the T ψ C sequence in all tRNA molecules.
The additional arm is called the variable stem. In certain tRNA molecules, only a loop of 4-5 bases may be observed without any stem at all. However, the presence of both loop and stem in this region is the usual phenomenon. According to the organization of variable stem, tRNAs are classified into two categories. In about 75% of the tRNA, these arms are constituted of 3 to 5 base pairs and they are known as class I tRNA. On the other hand, in some tRNA molecules, this arm contains 13 to 21 base pairs. The tRNA of this category is known as class II tRNA. The secondary or two-dimensional structure of all tRNA molecules is more or less identical. They differ only on their overall base sequences, variable arm, and anticodon segment. The tRNAs which differ by their structural organization but carry the same amino acid are known as iso-acceptor tRNA.
Three-Dimensional Structure of tRNA: Kim et al., (1974) proposed a three-dimensional structure of tRNA. From the X-ray diffraction analysis of crystallized yeast phenylalanine tRNA, they could be able to identify the L-shaped configuration for this tRNAphe. As per this model, the tRNAphe is in the shape of an inverted L. The small arm of the L represents the acceptor arm and T ψ C arm together, while the long arm of the L is formed of DHU and anticodon arms. The model also suggests that besides the formation of hydrogen bonds between the complementary bases, the formation of some non-conventional pairing between bases is responsible for the attainment of such three-dimensional configuration in the tRNA. The overall function of the tRNA is to help the cell in the synthesis of the polypeptide chain. In this process, the tRNA molecules transport the amino acids from the cellular pool to the ribosomal site where the amino acid may be polymerized.
4. hn RNA:
This is called heterogenous nuclear RNA. This type of RNA is only available in the eukaryotic cell nucleus and these are far greater and longer than the mRNA molecules. In 1962, Scherrer and Darnell first detected the presence of this type of RNA. The average number of nucleotides in this RNA is about 4000, however, in certain cases, the number of nucleotides may reach upto 20,000 in a molecule. Comparatively, the average number of nucleotides in mRNA is about 1800.
The hn RNA species is considered to be the precursor of mRNA in the eukaryotic cell. The mRNA molecules cannot be directly synthesized from the DNA, rather via the production of hn RNA, mRNA is formed. The hn RNA molecules contain two types of sequences, namely introns, and exons. Introns are superfluous intervening sequences between exons that combine during processing to form the functional mRNA molecule. The exons are excised out by a process called splicing and they are then ligated to form the mRNA molecule.
5. sc RNA and sn RNA:
sc RNA and sn RNA are two small species of RNA found in eukaryotic cells, sn RNA is found in the nucleus, and sc RNA is found in the cytoplasm therefore, sn RNA and sc RNA are named as small nuclear RNA and small cytoplasmic RNA respectively. sn RNAs are available in both nucleolus and nucleoplasm. Because of the presence of more U residues in these molecules, they are also called Usn RNA. The length of an average sn RNA is approximately 150 nucleotides. There are seven types of U sn RNA in total. They are also many in number in the cell and are also quite stable.
Each of these RNA molecules being combined with 7 or 8 protein sub-units forms sn RNP molecule. These molecules may act as enzymes and therefore, they are called ribozymes. In the processing of large RNA species, they play an active role and carry out splicing. They are also called spliceosomes. sc RNAs are also small fractions present in the cytoplasm of the cell. Each molecule may remain combined with six protein sub-units. In a cell, the number of sc RNA may be about 5105. They help in protein transport within the endoplasmic reticulum. Alu 7S RNA is one type of sc RNA in the cell.
Some Special Categories of RNA
Besides the above-noted RNA molecules, there are several specific types of RNA fractions. These RNA molecules are named according to their functional properties.
- iRNA or Initiator RNA: This type of RNA is formed of a few nucleotides. During the replication of DNA, such RNA is formed on the lagging strand where it acts as a primer.
- Telomerase RNA: In the eukaryotic cell nucleus this type of RNA is available. This RNA remains part of the enzyme telomerase.
- gRNA or Guide RNA: In the kinetoplast of Trypanosomes, this RNA is available. This acts as a template in RNA editing.
- Antisense RNA: Being combined with mRNA, this type of RNA may prevent protein synthesis. This type of RNA, therefore, contains complementary bases with respect to one mRNA molecule. In the bacterial cell, such RNA is found most often.
Functions of RNA
According to central dogma, the primary role of RNA is to convert information stored in DNA into protein. But in reality, there are many more functions of RNA and the functions depend on the type of RNA. Thus the functions of RNAs may be enumerated in the following way.
- mRNA bears the message from the information stored in DNA in the coded form and proteins in the cell may be produced with the involvement of mRNA molecules.
- rRNAs participate in the formation of ribosomes and they also help in the process of protein synthesis in the cell.
- tRNA carries the amino acids from the cytoplasmic pool to the ribosome surface for their polymerization during protein synthesis. With the help of the anticodon sequence of it, the tRNA may incorporate the amino acid in the polypeptide chain for their polymerization. Hence, tRNA may act as an interpreter of the mRNA code.
- Some small fractions of RNA as primers initiate the process of RNA replication in the cell.
- Some RNAs may show catalytic functions and they act as enzymes. These RNA molecules are called ribozymes.
- In some viruses, RNA acts as genetic material. Some RNAs, called gRNA or guide RNA, carry out the editing of RNA at specific sites. So some small RNAs known as miRNAs can inhibit translation by base pairing with complementary sequences s of mRNA.
- Small nuclear RNAs or snRNAs catalyze the splicing of mRNA to produce functional mRNA molecules in the cell.
Comparative Analysis of tRNA, rRNA, and nRNA of Eukaryotes:
|1. 32 or more types of tRNA. The no is based on the no of amino acids.||4 types 18S rRNA, 28S rRNA, 5.8S rRNA, and 5S rRNA.||Many, according to a number of genes.|
|2. Shape like a clover leaf. With 3 to 4 stems and 3 to 4 loops.||Partially double-stranded, but no specific shape.||Lineral with 5′ and 3′ end.|
|3. Presence of anticodon and anti-codon loop.||No such structure.||Contains a codon region and the codon may bind with the anticodon of tRNA.|
|4. 5′ and 3′ ends come together and segment like an acceptor stem.||No such acceptor stem in rRNAs. 18S rRNA are present in 40S RNA and 28S rRNA 5.8S rRNA and 5S rRNA are present in 60S rRNA.||5′ end and 3′ end are at two distal ends.|
|5. Carry amino acids or ribosomal sites.||Helps in binding of mRNA to the ribosome as well as tRNA on the ribosomal site.||Binds with the ribosomal site for translation with its codon allow the animoeyl tRNA to enter a ribosomal site.|
|6. 3′ end always contains the CCA sequence.||No such specification may be attributed.||3′ end always contains poly A tail and 5′ end contains 7 methyl guanosine cap.|
|7. Helps in amino acid transportation to the ribosomal site for their polymerization.||Helps in the binding of mRNA over the ribosome.||Helps in the formation of polypeptide chains during translation.|
Difference between tRNA, rRNA, and mRNA of Prokaryotes:
|1. 32 types||3 types 5S rRNA, 16S rRNA and 23S rRNA.||Many types are according to genes.|
|2. Single-stranded but in some regions partially double-stranded.||Secondarily double-stranded and about 70% of regions show double-stranded conditions.||Single-stranded and with 5′ and 3′ ends.|
|3. 5′ and 3′ ends are organized in the way that some sequences of 3′ and 5′ ends tend to form a stem.||No such orientation is found.||Separate 5′ and 3′ ends and mRNA is usually polycistronic in nature.|
Differences between DNA and RNA:
|1. DNA is present in the chromosomes of the nucleus.||1. RNA is mainly present in the cytoplasm of the cell.|
|2. DNA is double-stranded.||2. RNA is primarily single-stranded.|
|3. DNA exhibits its helical orientation.||3. RNA is basically linear.|
|4. Larger in size.||4. Smaller in size.|
|5. Pentose sugar in DNA is deoxyribose sugar.||5. Pentose sugar in RNA is ribose sugar.|
|6. In DNA nitrogen bases are adenine, guanine, cytosine, and thymine.||6. In RNA nitrogen bases are adenine, guanine, cytosine, and uracil.|
|7. Purine and pyrimidine in DNA are equal in amount.||7. Purine and pyrimidine in RNA may not be equal in amount.|
|8. DNA acts as genetic material.||8. RNA mostly helps the DNA for the expression of genetic material.|
Similarities between DNA and RNA
- Both DNA and RNA are nucleic acids.
- Both DNA and RNA are composed of nucleotides.
- Both DNA and RNA contain adenine and guanine as purines.
- Both DNA and RNA contain cytosine as pyrimidine.
Presence of DNA and RNA:
|1. Single-Stranded DNA||Bacteriophage M13, Parvovirus, φ × 174.|
|2. Double-Stranded DNA||Bacteriophage T2, T4, E. coli, animals & plants.|
|3. Single Stranded RNA||TMV, Poliovirus, Retrovirus.|
|4. Double-Stranded RNA||Reo virus.|
- The RNA World hypothesis was proposed by Nobel Laureate Walter Gilbert.
- The hypothesis suggests that the origin of life was preceded by a simple self-replicating RNA molecule i.e. RNA was the first genetic material.
- RNA molecules can perform dual functions like storage of information and also act as an enzyme. (Ribozyme)
- The RNA world hypothesis seems implausible in today’s world as RNA is unstable, fragile degraded into its constituent monomeric nucleotide.
The genetic material DNA is present in the nucleus of a cell which is significantly small in comparison to the DNA a cell contains. In human body cells, one nucleus contains about 2 meters of DNA and this DNA is accommodated in the nucleus which is about 3 pm in dimension. This has been possible due to the packaging of DNA in the chromosomes. The packaging of DNA in the chromosome may be elaborated with an example. The length of the chromosomes at metaphase ranges from 1-10 pm and a single chromosome contains a single DNA molecule. The largest human metaphase chromosome is about 10 x 0.5 pm in dimension and it contains 85 mm long DNA.
Therefore, the DNA of this chromosome has to undergo 104 times condensation to be accommodated in the 10 pm chromosome. Chromatin thread at an interphase stage when viewed under an electron microscope it appears as beads on a string-like configuration. DNA being complexed with proteins called histones develops this structure of chromatin. At the primary stage, the beads on the string are the primary chromatin structure in which the DNA undergoes the primary level of packaging. In this primary packaging, four histones namely H2A, H2B, H3, and H4 in pairs form a complex that is surrounded by 146 bp DNA forming the chromatin bead. Several such beads may be developed in successive fashion and the beads remain joined by linker DNA segment. At the point of entry of DNA in the histone bead, there exists another histone called HI which remains as sealing material. The bead-like structure containing DNA and histones is known nucleosome.
At this stage, DNA gets condensed to some extent and the packaging ratio is 1 : 6. This chromatin fiber is about 10 nm thick and the chromatin-forming solenoid forms 30 nm condensed chromatin fiber. At this stage packing ratio of DNA comes to 1 : 40. Following this, fiber forms radial loops surrounding a scaffold structure at the center when the DNA packing ratio comes to 1: 1000. At the metaphase stage DNA packaging in the chromatin becomes maximum when the packing ratio comes to about 1 : 1000. At metaphase stages DNA packaging in the chromatin becomes maximum and the packing ratio comes to about 1 : 10000. This condensation of DNA in the chromosome due to stepwise packaging may be elaborated with a description of nucleosome model and mode of organization of the nucleosome in the chromosomes.
Chromosome and Nucleosome Organization
A chromosome is principally formed of proteins (histones and non-histones), nucleic acid (DNA & RNA), and several metallic ions (Ca, Mg, and Fe). Primarily chromosome is constituted of 90% DNA and basic proteins and 10% RNA and acidic proteins. Basic proteins are usually histones, which may be arginine, histidine, and lysine-rich in nature. The acidic proteins are usually tryptophan and tyrosine rich. Histones are of five different types, namely HI, H2A, H2B, H3, and H4. Histones are combined with DNA from nucleohistone. One double-stranded DNA being combined with histones forms a chromatin. In each chromatid, there is a single DNA molecule in longitudinal orientation.
Different Types of Histones:
|Histones||Mol. Wt.||Nature||No. per 200 bp. of DNA|
|1. H1||21130||Lysine Rich||1|
|2. H2A||13,960||Slightly Lysine Rich||2|
|3. H2B||13,774||Slightly Lysine Rich||2|
|4. H3||15,273||Arginine Rich||2|
|5. H4||11,236||Arginine Rich||2|
In a large chromosome, DNA may be about 85 mm long, but a metaphase chromosome is microscopic. How such a long DNA molecule is housed in a very small mi¬croscopic metaphase chromosome, is a prominent question. To explain the chromosome organization, the nucleosome model was proposed by scientists. The nucleosome model states that the DNA thread having a dimension of about 2 nm wraps around several histones to form a nucleosome forming a basic thread of 10 nm diameter. Several nucleosomes being associated develop a chromatin thread of 30 nm dimension which again orients in the form of radial loops to form metaphase chromosomes having a dimension of about 0.5 pm.
Chemical Constituents of Chromosome
Lipid: in trace amount
Ca2+, Mg2+, Fe2+: In Trace Amount
Histones & Other Proteins: 50%
Nucleosome is a chromatin particle composed of five histones (HI, H2A, H2B, H3 & H4) and 200 nucleotide pair DNA. Wilkins and Luzzati (1960) and Olins (1964) gave some ideas on nucleosome organization. Initially, nucleosome was called nu bodies and Outdate (1975) used the term nucleosome. However, a detailed description of nucleosomes was given by A. Kornberg and J. T. Finch, and others in 1974. According to them, a chromosome is actually in the form of beads on a string. The beaded form of nucleosomes is arranged in linear order on the DNA fiber to give the structure of 10 nm chromatin fiber. The nucleosomes are considered units in chromosome organization. The 10 nm chromatin fiber then becomes organized in the form of the solenoid to give 30 nm chromatin fiber. This is the second order of chromosome organization. The tertiary level of organization develops with the configuration of about 1 μm thick metaphase chromosome.
Organization of the Nucleosome
Digestion of chromosomes with micrococcal nuclease converts a chromosome into beads on a string configuration. Along this beaded string, a part containing 200 bp DNA and five histones together forming a beaded structure is taken to be the unit in chromosome organization, called the nucleosome. Therefore, one nucleosome is formed of 200 bp DNA and five histones.
The histones remain in the core region of the nucleosome and DNA remains surrounding the core histones. Four types of histones, i.e., H2A, H2B, H3 and H4, each of two molecules together form the histone core as the histone octamer. The dimension of histone octamer is about 40Å × 80Å. A DNA segment having 146 bp wraps the octamer by 1 3/4 turn. The entry and exit points of DNA on the histone octamer remain at the same side and at this point one HI histone binds as the sealing agent for the DNA on the histone. The best part of the DNA of the nucleosome acts as the linker between 2 nucleosome beads. The linker region contains a DNA stretch of about 55 bp. However, the length of the linker DNA may vary in organisms ranging from 8-80 bp. The nucleosome core containing the wrapped DNA shows a dimension of 60Å × 110Å.
The nucleosome part containing 166 bp DNA, histone octamer and the H1 histone is called chromatosome. Digestion of chromatin with micococcal nuclease for a comparatively short period results in the appearance of chromatosome and further digestion may develop, the fragment with 146 bp DNA.
DNA in the Nucleosome
The DNA in the nucleosome may be divisible into two types:
- Core DNA: measuring 146 bp DNA that wraps the histone octamer.
- Linker DNA: measuring 8-80 bp connecting two nucleosome beads.
Difference between Nucleosome and Chromatosome:
|Basis of Difference||Nucleosome||Chromatosome|
|1. Part of||Chromosome||Nucleosome|
|2. Constitution||200 bp DNA histone protein taking H1 histone.||145 bp DNA and H2A, H2B, H3 and H4 histone, No H1 histone.|
|3. Organization of DNA||Double-stranded DNA wraps histone octamer by two complete turns with H1 histone outside the histone octamer as seating for DNA.||Double-stranded DNA wraps histone octamer by 1.65 turns without H1 histone.|
|4. Region beyond Octamer||Contains a linker DNA that joins two Nucleosomes.||Linker DNA is absent.|
Though a nucleosome contains a 200 bp DNA, this may vary in different organisms, e.g., 260 bp in sea urchin sperm. A minimum of 154 bp DNA in nucleosomes has been found in certain cases.
Solenoid Model: Finch and Klug (1976) proposed the solenoid model of chromatin organization. In the solenoid organization, the 10 nm chromatin fiber attains a 30 nm dimension. The solenoid organization appears due to the coiling of the 10 nm fiber. In each solenoid, there are about 6-7 nucleosomes.
Radial Loops: The solenoid structure when forms a 30 nm fiber, it then forms radial loops from a central core so that a thicker chromosome fiber may be formed. At this stage, it takes the form of a chromatid having a dimension of about 500 nm to 700 nm. The steps in chromosome organization may be shown by the following flow-chart: