7. Return of dna Segment to Original Form.

As the RNA chain grows, the transcribed region of the DNA molecule gets hydrogen bonded to the

missense strand and the two are spirally coiled to assume the original double helical form. When the last ribonucleotide is added, the RNA polymerase and RNA chain are fully released from the DNA, which also completes its winding into a double helix. The protective protein coat in added again to the DNA duplex.

Transcription Unit. The bases from the promoter to the terminator sites form a transcrip­tion unit. It may include one or more genes. A transcription unit is transcribed into a single RNA chain.

8. Processing (Maturation) of RNAs. The

forms of RNAs originally transcribed from DNA are called primary transcripts. These undergo ex­tensive changes, termed processing, or maturation, or post-transcriptional modification of RNAs,

before they can become functional in both prokaryotes and eukaryotes.

RNA Processing. In RNA processing, (/) larger RNA precursors are cut into smaller RNAs by a ribonuclease-P enzyme (cleaving), (ii) unwanted nucleotides are removed by enzymes named nucleases (splicing), (Hi) useful regions are rejoined by ligase enzyme (union), (iv) certain nucleotides are added at the ends enzymatically (terminal addition), (v) molecule may fold on itself to assume proper shape (folding), and (w) some nucleotides may be modified (nucleotide modification). RNA editing may also occur before translation starts.

Equation for RNA Transcription. The entire process of RNA transcription may be summed up in the equation— (ATP + GTP + CTP + UTP)„

DNA Template , Mg²⁺ or Mn²⁺

» RNA Chain + n PPi

RNA Polymerase

RNA Types. The three different types of RNA, namely, messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA) are transcribed from different regions of the DNA molecule. Three different RNA polymerases: I, II and III catalyse the transcription of rRNA, mRNA and tRNA respectively in eukaryotes. Transcription of RNA also occurs in the 5' — 3' direction like the replication of DNA.

In prokaryotes, a single RNA polymerase composed of different subunits does this work.

the A site one after another, and the peptide chain grows. Thus, the amino acids are linked up into a polypeptide in a sequence communicated by the DNA through the mRNA. The four-letter language of DNA (A, T, G, C) is transcribed into a four-letter language of mRNA (A, U, G, C), which is translated into a twenty- letter language of polypeptides. A polypeptide chain in the process of synthesis is often called a nascent polypeptide.

The growing polypeptide chain always remains attached to its original ribosome, and is not transferred from one ribosome to another. Only one polypeptide chain can be synthesized at a time on one ribosome.

(c) Termination and Release of Polypeptide Chain (Fig. 25.25). At the end of mRNA chain there is a "stop", or "terminator" codon (UAA, UAG or UGA). It reaches the A site but is not read and joined by the anticodon of any tRNA-amino acid complex. Hence, there can be no further addition of amino acids to the polypeptide chain. A protein called release factor (RF^ RF₂ and RF₃) joins the stop codon in the A site. It adds a water molecule to the poly peptide chain. This hydrolyses (cleaves) the completed polypeptide from the tRNA that is at the P site. The polypeptide is now free from the ribosome. The ribosome jumps off the mRNA chain

at the "stop" codon and dissociates into its two subunits.

The ribosomes and the tRNAs on release from the mRNA can function again in the formation of another polypeptide.

(v) Modification of Released Polypeptide. The just released polypeptide has primary struc­ture, i.e., it is a straight, linear molecule. It may lose some amino acids from the end with the help of an exopeptidase enzyme, and then coil and fold on itself to acquire secondary and tertiary structure. It may combine with other polypeptides to have quaternary structure.

A gene determines the primary structure of a protein, and the primary structure, in turn, deter-

mines the conformation. Chaperone proteins may help; polypeptide fold correctly.

Proteins for Use Inside and Outside the Cell. The proteins synthesized on free polysomes are released into the cytoplasm and function as structural and en­zymatic proteins. The proteins formed on the polysomes attached: to ER pass into the ER channels and are exported as cell secretions by exocytosis after packaging in the Golgi apparatus.

Polysome Formation. When the ribosome has moved sufficiently down the mRNA chain towards 3' end, another ribosome takes up position at the initiator codon of mRNA, and starts synthesis of a second copy of the same polypeptide chain. At any given time, the mRNA chain will, therefore, carry many ribosomes over which are similar polypeptide chains of varying length, shortest near the initiator codon and longest near the stop codon. A row of ribosomes joined to the mRNA molecule, as already mentioned, is

called a polyribosome, or simply a polysome (Fig. 25.26).

Translational Amplification. Synthesis of many molecules of the same polypeptide simul­taneously from one mRNA molecule by a polysome is called translational amplification.Energy Used. One GTP is hydrolysed to GDP as each successive amino acid-tRNA complex at­taches to the A site of the ribosome. A second GTP is broken down to GDP as the ribosome moves to each new codon in the mRNA. One ATP is hydrolysed to AMP during amino acid activation. Thus, the formation of each peptide bond uses 3 high-energy molecules, one ATP and two GTP.

Formation of

ATP + 2 GTP » AMP + 2 GDP

Peptid Bond + 4 H₂PO|~

Why are DNA and ribosomes located at dif­ferent sites ? Location of instruction centre (DNA) and manufacturing centre (ribosomes) at different sites in a cell is I away from the energy I sources and raw I materials ; and if both § were in the cytoplasm, the | information centre would |be exposed to respiratory % breakdown. The nuclear advantageous. If both were in the nucleus, the manufacturing centre would be far

I envelope preserves

I stability of the DNA by ■:i protecting it from ;;: respiratory destruction. |The genes (DNA) are, _ I thus, permanent, authen­tic "master documents" | from which "working I copies" are prepared in | the form of mRNAs.

Inhibition of | Protein Synthesis in | Prokaryotes. Protein syn­ thesis in bacteria is in-| hibited by certain | antbiotics at various |stages. Such antibiotics I include tetracycline,

| streptomycin, chloram-| phenicoi, Erythromycin land neomycin. Inhibition | of protein synthesis clears *the bacterial infection

without harming the humans. This indicates that there is some difference in the molecular structure of prokaryotic and eukaryotic ribosomes.

F. GENE EXPRESSION

Meaning of Gene Expression

Gene expression refers to the molecular mechanism by which a gene shows its potential in the phenotype of an organism.

Mechanism of Gene Expression (Fig, 25.28)

A gene contains the blue print or code for a particular polypeptide in the form of a specific se­quence of its base pairs. It transfers its code to mRNA transcribed from it. The process is called

FIGURK 25.28. Mechanism of gene expression.

transcription. Only the sense strand of DNA is capable of transcription. Other genes transcribe rRNA and tRNAs. The mRNA binds to ribosomes and with the help of suitable tRNAs selects the required amino acids and links them in a proper sequence received from the gene to form a polypep­tide. This process occurs on the ribosomes and is called translation. The polypeptide chain so formed may act as a structural protein and form some organelle, or form proteinaceous biochemical such as haemoglobin, insulin, etc., or serve as an enzyme and catalyze some chemical reaction, or have a motile role like that of myosin, or act as a membrane receptor. In other words, the polypep­tide may contribute to a morphological or a func­tional trait (phenotype) of the cell and organism. Thus, the molecular structure of a gene is expressed in a phenotype. This process may be briefly stated as under —

Transcription rKINA Translation

One Gene » mRNA >

tRNA

Organelle Expression Enzvme

One Polypeptide > 4 _ / .

^{JC r} Proteinaceous

Biochemical There is a definite relationship between the genes and proteins present in a cell or an organism. A change in the gene would change the code and give rise to a different polypeptide.

Colinearity of Gene (DNA) and Polypeptide Structure. The nucleotides are arranged in the genes (DNA) in a linear manner and so are the amino acids in the polypeptides (proteins). The sequence of amino acids in the polypeptide chain is dictated by the order of the nucleotide bases in the messenger RNA, and this, in turn, it; dictated by the sequence of the nucleotide bases in the gene (DNA). Thus, the sequence of amino acids in the

polypeptide chain corresponds to the sequence of | the nucleotide bases in the gene that codes for it. 1 The gene and the polypeptide it codes for are said 1 to be colinear, and their correspondence is called | colinearity of gene and polypeptide structure.

That the gene and the polypeptide it codes for | are colinear had been presumed earlier but Charles | Yanofsky and associates have now proved it in 1965. I They have shown that an alteration in a specific codon in DNA leads to an alteration of amino acid in a corresponding location in the polypeptide syn­thesized by the mutant individual of E. coli (Fig. 25.29).

Since each amino acid is coded by a set of three consecutive nucleotide bases, a polypeptide chain of 500 amino acids will be coded by a gene of 1500 nucleotide bases.

G. CENTRAL DOGMA : FLOW OF INFORMATION

The chromosomal DNA is the primary infor­mation centre. It contains complete information

about all the specific proteins to be synthesized in the cells for their needs. This information is coded in the DNA as the particular sequences of bases called genetic code. The unit of genetic information is called a codon. It is a group of three adjacent deoxyribonucleotides that specify a single amino acid in a polypeptide chain. The DNA is a double strand of deoxyribonucleotides, whereas polypep­tide is a single chain of amino acids. Thus, one of the two strands of DNA, i.e., the sense strand, contains information to guide the synthesis of polypeptide, and a single stranded nucleic acid molecule can carry information from the DNA to the site of protein synthesis (ribosomes). This nucleic acid is mRNA. The information is passed on by the DNA of the gene located in the sense strand to the mRNA transcribed from it as a single chain of complemen­tary bases. The mRNA, with the help of tRNAs and ribosomes, then directs the specific amino acids to

come together and join in a sequence communi­cated by DNA. By this arrangement, the informa­tion initially coded as a specific sequence of deoxyribonucleotides in the DNA (gene) is transcribed as a specific sequence of complemen­tary ribonucleotides in the mRNA, and is finally translated into a specific order of the amino acids in the polypeptide chain. Transcription does not change the genetic code as it forms the mRNA which is complementary to the DNA producing it, except that T is replaced by U. Translation changes the code from the nucleotide sequence to the amino acid sequence. The flow of information from DNA to mRNA and thence to a polypeptide is called the central dogma* of molecular biology. This concept was advanced by Crick in 1958. It emphasizes that flow of information is unidirectional in the cells. Only DNA self-replicates. DNA is not made on RNA or polypeptide template. Similarly, mRNA

sequence is never copied on polypeptide template. The information that has passed from DNA to mRNA and thence to polypeptide has reached the end of the line and is not reversed. Three processes are involved in the inheritance of genetic informa­tion and its expression in the form of proteins.

/ ^ Transcription

Replication DNA * mRNA

V^ _-/ (In the nucleus) Translation * Polypeptide (In the cytoplasm)

Central Dogma Reverse. In 1970, H.M. Temin and D. Baltimore reported that RNA, which func­tions as the genetic material in the cancer-causing chicken virus, first synthesizes a DNA copy of it, and the DNA then transfers information to mRNA, which finally passes it on to a polypeptide. This discovery in molecular biology introduced the con­cept of "central dogma reverse", or "teminism". It states that the information flow is not necessarily from DNA to RNA but may be from RNA to DNA also. Reverse transcription is aided by an RNA-de-pendent DNA polymerase enzyme called reverse transcriptase. The viruses capable of reverse transcription are termed retroviruses. Human im-muno-deficiency virus (HIV), that causes AIDS, is also a retrovirus. DNA

JI Transcription Translation ^DNA -> RNA * Polypeptide Reverse Transcription

Restatement of Central Dogma. Pending fur­ther research, the central dogma of molecular biol­ogy may be tentatively restated as the flow of information from nucleic acids to polypeptides.

Some RNA viruses, such as paramyxo virus, which causes measles or mumps, can synthesize new genomic RNA on RNA template itself without re­quiring a DNA intermediate.

RNA -* RNA -* mRNA -* Polypeptide

In view of this, the central dogma may be said to state that the nucleic acids store and transfer information, and the transfer of information into proteins is irreversible. Replication

f J Transcription C \

NDNA > ^RNA*< Reverse Transcription Translation * Polypeptide

H. RELATIONSHIP BETWEEN GENES AND ENZYMES

Inborn Errors of Metabolism A close re.^on

between genes and enzymes was known lc-: . :c the mechanism of gene expression was fully worked out. This relationship was found in 1902 by Ar­chibald Garrod, a British physician. He held tha: the genes operate by producing enzymes. He sugg: that each of the diseases inherited by offspring from the parents is caused by the lack of an enzyme which catalyzes the change of one metabolic substance to another. He called such diseases inborn errors of metabolism. Lack of enzyme is due to the absence of the normal form of the gene that controls the synthesis of that enzyme. The defect is inherited as a simple recessive trait.

Beadle and Tatum's One Gene-One Enzyme Hypothesis. The concept that the genes contain information for the synthesis of enzymes, which are proteins in nature, was put on firm footing by Beadle and Tatum in 1948 with the help of their experi­ments on the pink bread mould Neurospora crassa. This mould can grow normally in a simple minimal medium containing salts and sugar, preparing all other materials such as amino acids, purines, pyrimidines, etc., by enzyme-catalysed reactions. Such a normal or wild type individual is called a prototroph. They exposed the mould to X-rays, which were known to cause heritable gene muta­tions by changing the base sequence of DNA. They found that the mutants were unable to grow in the minimal medium unless additional nutrients were added to the medium. Such nutritional mutants are knjwn as auxotrophs. Different mutants needed the amino acids ornithine or citrulline or arginine for their growth (Fig. 25.30). It is known that a cell can sequentially change ornithine to citrulline and the latter to arginine, using a specific enzyme for each conversion, Beadle and Tatum held that the mutant which needed ornithine for growth lacked an ornithine-producing enzyme due to mutation in gene that coded for this enzyme. Similarly a muta­tion in another gene caused lack of the enzyme which catalysed the change of ornithine to citrulline. This mutant would need citrulline for growth. They thought that each gene had the information for producing a single enzyme, and proposed their famous one gene-one enzyme hypothesis in 1948. This hypothesis states that each gene controls the synthesis of a specific enzyme, or a particular protein. This hypothesis got them 1958 Nobel prize

in Medicine. Their work laid the foundation of biochemical genetics.

Modification of Beadle and Tatum's Hypothesis. It has been now found that (/) all genes do not produce enzymes and that (ii) certain proteins contain more than one polypeptide chain, for example, the haemoglobin molecule consists of two a and two ft polypeptide chains. Similarly, the enzyme tryptophan synthetase of bacterium Es­cherichia coli consists of two polypeptides: A and B (Yanofsky et al, 1965). These cases show that more than one gene may control the synthesis of a protein. Hence, it is now held that one gene is responsible for the formation of one polypeptide chain. One gene-one enzyme hypothesis has been replaced by one gene-one polypeptide chain principle.

Mendelian Principles and Enzymic Role of Genes. Mendelian principles can be explained on the basis of enzyme-controlling role of the genes. The dominant genes code for the synthesis of active enzymes and the recessive genes code for the inac-

tive or defective enzymes. This i; enables the dominant genes to I produce their phenotypes even | in the presence of their recessive |alleles, i.e., in heterozygous con-|dition also. In homozygous I recessive persons, both the genes | direct the synthesis of inactive gform of the enzyme. Hence, such 1 persons suffer from abnormality. A specific case, that of | alkaptonuria, is cited. In 1 homozygous dominant persons, fboth the genes direct the fsysnthesis of active alkapton or Ihomogentisate oxidase enzyme. I Therefore, such persons are nor-;::mal. In heterozygous persons, 1 one (dominant) gene directs the I synthesis of active alkapton ;:! oxidase enzyme and the other | (recessive) gene directs the syn­thesis of an inactive form of this | enzyme. Such persons are also normal. In homozygous reces­sive persons, both the genes direct the synthesis of inactive ;form of the enzyme. Hence, such :: persons suffer from alkap­tonuria.