Lecture 4

Topic: Structure and function of Nucleic acids (DNA and RNA). Genetic code. Protein synthesis.

Plan of the lecture:

1. Structure of nucleic acids.

2. Peculiarities of structural organization of DNA.

3. Peculiarities of structure of mitochondrial DNA.

4. Properties and functions of DNA.

1. Synthesis of DNA, stages of replication.

2. Characteristic of replication complex.

3. Peculiarities of synthesis of leading strand and lagging strand.

4. General characteristic of transcription.

5. Mechanism of transcription, basic steps: initiation, elongation, termination.

6. Ripening (processing) of mRNA and mechanism of splicing.

History. Nucleic acid was first isolated in: by a Swiss physician Friedrich Miescher from nuclei of pus cells. He called it nuclein. In 1 Oskar Hertwig wrote "nuclein is the substs responsible for the transmission of hereditary c acters". Nuclein was renamed nucleic acid Altaian in 1889. He discovered the existence of types of nucleic acids.

Location. The nucleic acids are strong a and were originally found in the nucleus, hence t name. They are now known to occur in mitochondria, plastids and cytoplasm as well. T also occur in bacteria that lack nucleus anc viruses too.

Composition. The nucleic acids are gi molecules having complex structure and very h molecular weights. The nucleic acid moleculi composed of a large number of nucleot molecules joined into a linear, unbranched ch; The nucleic acids are, thus, nucleotide polymer! polynucleotides. In a nucleic acid, the phosph component of one nucleotide is joined by a ph phodiester bond to the sugar component of adjacent nucleotide at the 3' carbon atom (I 20.11). The alternating sugar and phosphate co ponents form the "backbone" of the nucleic a molecule, and the nitrogenous base compone project on one side from the sugar compone (Figs. 20.10 and 20.14). Nucleic acids have two tyj of nitrogenous bases : double-ringed purines a single-ringed pyrimidines (Fig. 21.21) and two types of pentose sugars : ribose and deoxyribose.

Function. The nucleic acids store and transfer information needed for the synthesis of specific proteins that determine the structure and functions of the cells.

Types. There are two types of nucleic acids : deoxyribonucleic acid or DNA, and ribonucleic acid, or RNA. Each type has subtypes.

1. Deoxyribonucleic Acid (dna)

DNA is the genetic material and forms molecular basis of heredity in all organisms. In cer a given species. Just before cell division, however, the amount of DNA is doubled. The gametes have half the amount of DNA as they contain half the number of chromosomes.

Chemical Composition. Chemical structure of DNA was explained by PA. Levene. DNA is the largest macromolecule in the organisms. It is a long, double* chain of deoxyribonucleotide, or deoxyribotide ,units. The two deoxyribonucleotide chains are twisted around a common axis to form a right-handed double helix (spiral) that encloses a cylindrical space in it. Each deoxyribonucleotide unit, in turn, consists of 3 different molecules : phosphate, (PO|~), a 5-carbon deoxyribose sugar (C^HjqO^) and a nitrogenous base. Nitrogen gives the base its basic nature. The nitrogenous base may be a 9-membered, double-ringed purine, i.e., adenine (A) or guanine (G); or a 6-membered, single-ringed pyrimidine, viz., thymine (T) or cytosine (C) (Fig. 21.20). Thus, there are 4 types of deoxyribonucleotides in DNA, namely, adenine-deoxyribose-phosphate, or adenosine monophos­phate (AMP); guanine-deoxyribose-phosphate, or guanosine monophosphate (GMP); cytosine-deoxyribose-phosphate, or cytidine monophos­phate (CMP); and thymine-deoxyribose-phosphate, or thymidine monophosphate (TMP). In each chain, the phosphate component carried by the carbon atom at position 5' of the sugar in one nucleotide unit is joined by phosphodiester bond to the hydroxyl component of the carbon atom at posi­tion 3' of the sugar in the next nucleotide unit. These 3', 5'-phosphodiester bonds two deoxyribonucleotide chains are held together by;ii hydrogen bonds between ji; paired bases, and by van der | Waals attractions between the | stacked bases (Fig. 20.11). § Adenine of one chain is always % joined to thymine of the other I chain by 2 hydrogen bonds (A \ = T). Cytosine of one chain is \ always linked to guanine of the % other chain by 3 hydrogen | bonds(C = G). Thus, there are \ only four possible base pairs : A-T, T-A, C-G and G-C in the DNA molecule.

Levels of Structure. A DNA molecule shows primary, secondary and tertiary structure like a protein molecule, being coiled in three orders for accommodation in a small space.

Polarity. The polynucleotide chains show polarity (direction). One end of each DNA strand is called 5' end. The last deoxyribose unit at this end has the carbon at position 5 free. The other end of the strand is termed 3' end. The last deoxyribose unit at this end has the carbon at position 3 free.

Variety. Although onlyfour types of base pairs are involved in the formation of DNA molecule, these base pairs may occur in any sequence, and there may be any number of sequences in a molecule. This gives an infinite variety to the DNA molecule (Fig. 20.10). However, each individual has a specific sequence of base pairs in its DNA.

Types. DNA molecules are of 2 types : linear and circular. Linear DNA is found in the nuclei of eukaryotic cells. It is associated with proteins in about 1 : 1 ratio. Circular DNA is found in many viruses, all prokaryotic cells, and in the mitochondria and chloroplasts (plastids) of eukaryotic cells. It is associated with very little protein.

Complementarity of Base Pairs. The two nucleotide chains of DNA molecule are not identi­cal, but complementary to each other with respect to base pairs. This is due to restrictions on base pairing. A base pair must consist of a purine and a pyrimidine. There are two reasons for this limita­tion.

(/) The space available between the two sugar-phosphate chains of DNA, i.e., 20A (2 nm), can accommodate one purine and one pyrimidine, but not two purines, which would be too large, and not wo pyrimidines, which would not be close enough form proper hydrogen bonds. It may be noted that purine bases, with two carbon rings, are about * twice as wide as pyrimidine bases (Figs. 21.21 and 20.11).

(if) A specific purine pairs with a specific pyrimidine because of a perfect match between hydrogen donor and acceptor sites on the two bases. Only adenine and thymine and also guanine and sine have the proper spatial arrangements to form correct hydrogen bonding. Adenine and sine, and also guanine and thymine do not form proper hydrogen bonds and cannot pair.

The concept of complementary base pairing provides that an adenine in one chain must be natched with a thymine in the other chain; and a guanine in one chain must have cytosine opposite to i in the other chain. Thus, the two chains are com­plementary to each other. Hence, from the base sequence in one DNA chain, the base sequence in us complementary chain can be easily predicted. ^: r example, if the base sequence in one chain is A, T. C, G, G, C, T, A that in the other chain will be T, A. G, C, C, G, A, T.

Base Pair. The term ''base pair" refers to two bases, one in each chain of DNA molecule, joined together by hydrogen bonds. Each base pair consists of one 2-ringed purine and one 1-ringed pyrimidine. Therefore, all the base pairs have equal vidth, and the DNA helix has a constant diameter.Antiparallel Direction. The two chains of DNA molecule run in opposite or antiparallel direc­tions, fhis means that the carbon atom at position 5 in the sugar component is in one direction in one chain and in the opposite direction in the other chain between paired bases, and by van der Waals attractions between the stacked bases (Fig. 20.11). Thus, the two chains are parallel but their 5' -» 3 directions are opposite. This is analogous to a 2-lane road, where the lanes run parallel but carry traffic in opposite directions.

BaSiis Eor''DNA Replication. The complemen­tarity of bases in DNA molecule and antiparallel directions of flie two chains of DNA molecule pro­vide the basis for the precise replication of DNA.

Chargaff s Rules. In 1950, E.E. Chargaff for­mulated important generalizations about DNA structure. These generalizations are called ChargafFs rules in his honour. They are sum­marized below—

(i) The DNA molecule, irrespective of its source, always has the A —T base pairs equal in number to the G — C base pairs.

(ii) The purines and pyrimidines are always in equal amounts, i.e., A + G = T + C

(Hi) The amount of adenine is always equal to that of thymine, and the amount of guanine is always equal to that of cytosine, i.e., A = T and G = C.

(iv) The base ratio A + T/G + C may vary from one species to another, but is constant for a given species. This ratio can be used to identify the source of DNA, and can help in classification.

(v) The deoxyribose sugar and phosphate components occur in equal proportions.

Denaturation and Renaturation. If DNA

molecule is exposed to high temperature or titration with an acid or an alkali, the two strands unwind and separate by breakdown of hydrogen bonds between the base pairs. This process is called denaturation¹, or melting. When denatured DNA is incubated at a low temperature, the two separated strands reas-sociate to form a DNA duplex. This procss is termed renaturation².

Physical Structure. Astbury, Wilkins and Franklin have suggested 3-dimensional, helical configuration for DNA molecule by X-ray diffraction studies. The purines and pyrimidines are flat rings stacked one above the other, and arranged at

angles to the sugar-phosphate backbones. The adenine-thymine and guanine-cytosin base pairs are precisely of the same size and shape. Therefore, the double helix has a constant diameter of 20 A along its entire length. Its one complete turn is 34 A long and has 10 base pairs. The successive base pairs are 3 • 4 A apart. These investigations helped Watson and Crick to design a model of DNA molecule.

Watson-Crick Model. J.D. Watson, an American biologist, and F.H.C. Crick, an English chemist, in 1953 suggested a model of DNA molecule to explain its structure (Fig. 20.12). Their paper consisted of just 2 pages but it became a cornerstone of the molecular biology. Their model got them the 1962 Nobel Prize. According to Wat-son-Crickmodel, the DNA molecule consists of two long, parallel chains (strands) which are joined together by short crossbars at regular intervals. The two chains are spirally coiled around a common axis i in a regular manner to form a double helix. The double helix is of a constant diameter and has a major groove about 22 A wide and a minor groove i about 12 A wide alternately. The bases face the ■ interior of the double helix whereas the sugar and phosphate components form backbones on the out­side. The helixis generally right- handed, that is, the turns run clockwise looking along the helical axis.

In other words, the DNA molecule has the form of a twisted ladder (Fig. 20.13). The vertical bars of the ladder are formed of alternating phos- ■ phate and deoxyribose sugar components. The horizontal rungs of the ladder are joined to the sugar components of the vertical bars, and are com­posed of purines and pyrimidines linked by hydrogen atoms.

DNA Forms. Five forms of DNA have been reported : A, B, C, D and Z. The A, B, C and D forms are right-handed double helices. The B-DNA occurs under the physiological conditions prevail­ing in the living cells. It has the specification given \ in the foregoing description of DNA. The A-DNA has 11 base pairs per turn of the helix. THe C-DNA has 9 base pairs per turn of the helix, and D-DNA has only 8 base pairs. The Z-DNA is left-handed, contains 12 base pairs per turn of the helix, each turn is 45 A long, and its sugar-phosphate backbones follow a zig-zagpath along the helix. The last feature gives the Z-DNA its name.

Genetic Code. The particular sequence of bases in each polynucleotide chain is known as the primary sequence of DNA. This primary sequence forms the genetic code, i.e., the information re­quired by the cell to synthesize all the specific proteins it needs. The primary sequence of DNA shows unlimited variation, and this is l $j basis of huge variety seen in the living systems.

A DNA molecule includes many genes, each with a specific sequence of nucleotides. A sequence AGGTAACCT codes for one protein and the se­quence CGCCTTAAC codes for a different protein (real genes are very long, may have hundreds to thousands of nucleotides).

Sense and Missense DNA Chains. The genetic information exists in the base sequence of one of the two chains of DNA molecule. This chain is often called sense chain. Its complementary chain is termed missense chain, or antisense chain. The missense chain is important in the replication of DNA molecule in cell division, but does not take part directly in transcription. It is copied from the sense chain.

Noncoding DNA. Greater part of DNA in eukaryotic cells does not code for RNAs. This "extra", or noncoding DNA, seems t.o \vasie, tvo i\Hvc-\AOtv.\t Ws two special forms :

(i) Repetitious DNA. The noncoding DNA has many base sequences repeated several times. The repeated sequences are collectively called repetitious DNA.

(ii) Jumping Genes. Some repetitive DNA sequences are not found at fixed sites in the DNA of different individuals of the same species. Such "mobile" DNA segments are often referred to as "jumping genes". They cause mutations and, thus, have a role in evolution. However, they usually have no role in the life of the individual.

The existence of nonfunctional DNA indi­cates that the eukaryotes use only a small fraction of their total DNA.

Bacteria have little nonfunctional DNA.

Satellite DNA. Part of DNA having long stretches of repetitive base pairs is called satellite DNA. A satellite DNA having a few (1 — 6) base pair repeats is termed microsatellite, and that with more (10 — 60) base pair repeats is known as minisatel-lite. The minisatellites are highly variable and are specific for each individual. These help in DNA matching for identification of persons. Minisatel­lites were first discovered by Jaffreys et al in 1985.

Palindromic DNA. It is a part of DNA in which the base sequence of one strand is opposite to that of the other strand when read from opposite directions. DNA regions that transcribe rRNA are often palindromic. However, the true significance of palindromic DNA is not clear

3'_C-G-G-A-A-T-T-C-C-G-5' 5'~G-C-C-T-T-A-A-G-G-C-3'

Human body cells normally have 46 chromosomes with a total of T 6 billion base pairs. The DNA of 46 chromosomes of a single cell has a total length of about 2 metres. The chromosomes of a dividing cell are ten thousand times shorter than this. The proteins cause the packing of DNA molecules so tightly.

In Vitro Synthesis of DNA. In 1961, Kornberg and his coworkers synthesised DNA from a mixture of all the 4 types of deoxyribonucleoside triphos­phates, DNA polymerase enzyme, metal ions and a single strand of viral DNA. The latter was separated from its complementary strand by denaturation. It acted as a template.

Functions. The DNA plays a multiple role — 1. It is the genetic material and carries hereditary ckara.c\&x% itom parens to t\ve young ones. This is achieved through its unique property of 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 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 the postnatal development through adulthood to death by its "internal clock."

Thus, DNA is the very basis of life. DNA molecule is a double chain of deoxyribonucleotide units and has a uniform diameter.The successive units are joined by phos-phodiester bonds in each chain (strand).

The sugar-phosphate-sugar-phosphate backbones are located on the outside of the molecule, and nitrogenous bases project inward.

The bases project inward at planes ap­proximately perpendicular to the long axis of the molecule, and are, therefore, stacked one above the other. The two chains are spirally coiled around a common axis to form a regular, right-handed double helix.

5. The helix has a major groove and a minor groove alternately.

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

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

6. 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' direction.

10. 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 proportion.

10. The DNA molecule is remarkably stable due to hydrogen bonding and hydrophobic reac­tions.

11. The DNA molecule can replicate itself and can also transcribe RNAs.

14. The base sequence of one chain (sense chain) of DNA serves as the genetic code. The amount of DNA per nucleus is con­stant in all the body cells of a given species.

15. DNA can easily undergo denaturation and renaturation.

16. DNA can be synthezized in vitro.

17. Only a small fraction of DNA is functions in eukaryotes.

18. Eukaryotic DNA has many repeated ba sequences, some of which are "mobile".

19. DNA is dextrorotatory.

20. DNA code is discontinuous, having non-coding segments (introns) between coding sqj ments (exons).

20. Eachstrand of DNA molecule has polar with 3' and 5' ends.