Molecular Basis of Inheritance

1. Chapter at a Glance

In the previous chapter, you learnt the inheritance patterns and the genetic basis of such patterns. At the time of Mendel, the nature of those "factors" regulating the pattern of inheritance was not clear. Over the next hundred years, the nature of the putative genetic material was investigated, culminating in the realisation that DNA — deoxyribonucleic acid — is the genetic material, at least for the majority of organisms. In Class 11 you learnt that nucleic acids are polymers of nucleotides. DNA and RNA are the two types of nucleic acids found in living systems. DNA acts as the genetic material in most organisms. RNA, though it also acts as a genetic material in some viruses, mostly functions as a messenger. This chapter covers the structure and packaging of DNA, the experiments that proved DNA to be the genetic material, the RNA world, replication, transcription, the genetic code, translation, regulation of gene expression (lac operon), the Human Genome Project and DNA fingerprinting. It is the highest-weightage chapter of Class 12 biology — NEET asks four to six questions every year.

2. The DNA

DNA is a long polymer of deoxyribonucleotides. The length of DNA is usually defined as the number of nucleotides (or a pair of nucleotides referred to as base pairs) present in it. This also is characteristic of an organism.

Structure of polynucleotide chain

A nucleotide has three components — a nitrogenous base, a pentose sugar (ribose in case of RNA, and deoxyribose for DNA), and a phosphate group. There are two types of nitrogenous bases — purines (adenine and guanine) and pyrimidines (cytosine, uracil and thymine). Cytosine is common for both DNA and RNA, and thymine is present in DNA. Uracil is present in RNA at the place of thymine. A nitrogenous base is linked to the pentose sugar through an N-glycosidic linkage to form a nucleoside, such as adenosine or deoxyadenosine, guanosine or deoxyguanosine, cytidine or deoxycytidine and uridine or deoxythymidine. When a phosphate group is linked to the 5'-OH of a nucleoside through a phosphoester linkage, a corresponding nucleotide (or deoxynucleotide depending upon the type of sugar present) is formed. Two nucleotides are linked through a 3'-5' phosphodiester linkage to form a dinucleotide. More nucleotides can be joined in such a manner to form a polynucleotide chain. A polymer thus formed has at one end a free phosphate moiety at the 5'-end of the sugar, which is referred to as the 5'-end of the polynucleotide chain. Similarly, at the other end of the polymer the sugar has a free 3'-OH group, which is referred to as the 3'-end. The backbone in a polynucleotide chain is formed due to sugar and phosphates. The nitrogenous bases linked to the sugar moiety project from the backbone.

In RNA, every nucleotide residue has an additional –OH group present at the 2'-position in the ribose. Also, in RNA the uracil is found at the place of thymine (5-methyl uracil, another chemical name for thymine).

Double helix

Friedrich Meischer in 1869 first identified DNA as an acidic substance present in the nucleus and named it "Nuclein". However, due to technical limitation in isolating such long polymers intact, the elucidation of the structure of DNA remained elusive for a very long period of time. It was only in 1953 that James Watson and Francis Crick, based on the X-ray diffraction data produced by Maurice Wilkins and Rosalind Franklin, proposed a very simple but famous Double Helix model for the structure of DNA. One of the hallmarks of their proposition was base pairing between the two strands of the polynucleotide chains. However, this proposition was also based on the observation of Erwin Chargaff that for a double-stranded DNA, the ratios between adenine and thymine and guanine and cytosine are constant and equal one.

• It is made of two polynucleotide chains, where the backbone is constituted by sugar–phosphate, and the bases project inside.
• The two chains have anti-parallel polarity. It means, if one chain has the polarity 5'→3', the other has 3'→5'.
• The bases in two strands are paired through hydrogen bonds (H-bonds), forming base pairs (bp). Adenine forms two hydrogen bonds with thymine from the opposite strand and vice versa. Similarly, guanine is bonded with cytosine with three H-bonds. As a result, always a purine comes opposite to a pyrimidine. This generates approximately uniform distance between the two strands of the helix.
• The two chains are coiled in a right-handed fashion. The pitch of the helix is 3.4 nm (a nanometre is one billionth of a metre, that is 10⁻⁹ m) and there are roughly 10 bp in each turn. Consequently, the distance between a bp in a helix is approximately equal to 0.34 nm.
• The plane of one base pair stacks over the other in a double helix. This, in addition to H-bonds, confers stability of the helical structure.

The proposition of a double helix structure for DNA and its simplicity in explaining the genetic implication became revolutionary. Very soon, Francis Crick proposed the Central Dogma in molecular biology, which states that the genetic information flows from DNA → RNA → Protein. In some viruses the flow of information is in the reverse direction, that is, from RNA to DNA (reverse transcription).

Packaging of DNA helix

Taken the distance between two consecutive base pairs as 0.34 nm (0.34 × 10⁻⁹ m), if the length of DNA double helix in a typical mammalian cell is calculated (simply by multiplying the total number of bp with the distance between two consecutive bp, that is, 6.6 × 10⁹ bp × 0.34 × 10⁻⁹ m/bp), it comes out to be approximately 2.2 metres — a length that is far greater than the dimension of a typical nucleus (approximately 10⁻⁶ m). How is such a long polymer packaged in a cell?

In prokaryotes, such as E. coli, though they do not have a defined nucleus, the DNA is not scattered throughout the cell. DNA (being negatively charged) is held with some proteins (that have positive charges) in a region termed the "nucleoid". The DNA in the nucleoid is organised in large loops held by proteins. In eukaryotes, this organisation is much more complex. There is a set of positively charged, basic proteins called histones. A protein acquires charge depending upon the abundance of amino acid residues with charged side chains. Histones are rich in the basic amino acid residues lysine and arginine. Both the amino acid residues carry positive charges in their side chains. Histones are organised to form a unit of eight molecules called histone octamer. The negatively charged DNA is wrapped around the positively charged histone octamer to form a structure called nucleosome. A typical nucleosome contains 200 bp of DNA helix. Nucleosomes constitute the repeating unit of a structure in the nucleus called chromatin, thread-like stained (coloured) bodies seen in the nucleus. The nucleosomes in chromatin are seen as "beads-on-string" structure when viewed under the electron microscope.

Theoretically, how many such beads (nucleosomes) do you imagine are present in a mammalian cell? The beads-on-string structure in chromatin is packaged to form chromatin fibres that are further coiled and condensed at metaphase stage of cell division to form chromosomes. The packaging of chromatin at higher level requires an additional set of proteins that collectively are referred to as Non-histone Chromosomal (NHC) proteins. In a typical nucleus, some region of chromatin is loosely packed (and stains light) and is referred to as euchromatin. The chromatin that is more densely packed and stains dark is called heterochromatin. Euchromatin is said to be transcriptionally active chromatin, whereas heterochromatin is inactive.

3. The Search for Genetic Material

Transforming principle (Griffith, 1928)

In 1928, Frederick Griffith, in a series of experiments with Streptococcus pneumoniae (bacterium responsible for pneumonia), witnessed a miraculous transformation in the bacteria. During the course of his experiment, a living organism (bacteria) had changed in physical form. When Streptococcus pneumoniae (pneumococcus) bacteria are grown on a culture plate, some produce smooth shiny colonies (S) while others produce rough colonies (R). This is because the S strain bacteria have a mucous (polysaccharide) coat, while R strain does not. Mice infected with the S strain (virulent) die from pneumonia infection, but mice infected with the R strain do not develop pneumonia.

He concluded that the R strain bacteria had somehow been transformed by the heat-killed S strain bacteria. Some "transforming principle", transferred from the heat-killed S strain, had enabled the R strain to synthesise a smooth polysaccharide coat and become virulent. This must be due to the transfer of the genetic material. However, the biochemical nature of genetic material was not defined from his experiments.

Biochemical characterisation of transforming principle

Prior to the work of Oswald Avery, Colin MacLeod and Maclyn McCarty (1933–44), the genetic material was thought to be a protein. They worked to determine the biochemical nature of the "transforming principle" in Griffith's experiment. They purified biochemicals (proteins, DNA, RNA etc.) from the heat-killed S cells to see which ones could transform live R cells into S cells. They discovered that DNA alone from S bacteria caused R bacteria to become transformed. They also discovered that protein-digesting enzymes (proteases) and RNA-digesting enzymes (RNases) did not affect transformation, so the transforming substance was not a protein or RNA. Digestion with DNase did inhibit transformation, suggesting that the DNA caused the transformation. They concluded that DNA is the hereditary material, but not all biologists were convinced.

The genetic material is DNA (Hershey and Chase, 1952)

The unequivocal proof that DNA is the genetic material came from the experiments of Alfred Hershey and Martha Chase (1952). They worked with viruses that infect bacteria called bacteriophages. The bacteriophage attaches to the bacteria and its genetic material then enters the bacterial cell. The bacterial cell treats the viral genetic material as if it was its own and subsequently manufactures more virus particles. Hershey and Chase worked to discover whether it was protein or DNA from the viruses that entered the bacteria.

They grew some viruses on a medium that contained radioactive phosphorus (³²P) and some others on a medium that contained radioactive sulphur (³⁵S). Viruses grown in the presence of radioactive phosphorus contained radioactive DNA but not radioactive protein, because DNA contains phosphorus but protein does not. Similarly, viruses grown on radioactive sulphur contained radioactive protein but not radioactive DNA, because DNA does not contain sulphur. Radioactive phages were allowed to attach to E. coli bacteria. Then, as the infection proceeded, the viral coats were removed from the bacteria by agitating them in a blender. The virus particles were separated from the bacteria by spinning them in a centrifuge. Bacteria which were infected with viruses that had radioactive DNA were radioactive, indicating that DNA was the material that passed from the virus to the bacteria. Bacteria that were infected with viruses that had radioactive proteins were not radioactive. This indicates that proteins did not enter the bacteria from the viruses. DNA is therefore the genetic material that is passed from virus to bacteria.

Properties of genetic material (DNA vs RNA)

From the foregoing discussion, it is clear that the debate between proteins versus DNA as the genetic material was unequivocally resolved from the Hershey–Chase experiment. It became an established fact that it is DNA that acts as genetic material. However, it subsequently became clear that in some viruses, RNA is the genetic material (for example, tobacco mosaic viruses, QB bacteriophage, etc.). A molecule that can act as a genetic material must fulfil the following criteria:

1. It should be able to generate its replica (replication).
2. It should chemically and structurally be stable.
3. It should provide the scope for slow changes (mutation) that are required for evolution.
4. It should be able to express itself in the form of "Mendelian characters".

The 2'-OH group present at every nucleotide in RNA is a reactive group and makes RNA labile and easily degradable. RNA is also now known to be catalytic, hence reactive. Therefore, DNA chemically is less reactive and structurally more stable when compared to RNA. Therefore, among the two nucleic acids, DNA is a better genetic material. In fact, the presence of thymine at the place of uracil also confers additional stability to DNA. Both DNA and RNA are able to mutate. In fact, RNA, being unstable, mutates at a faster rate. Consequently, viruses having RNA genomes and having shorter life span mutate and evolve faster. RNA can directly code for the synthesis of proteins, hence can easily express the characters. DNA, however, is dependent on RNA for synthesis of proteins. The protein-synthesising machinery has evolved around RNA. The above discussion indicates that both RNA and DNA can function as genetic material, but DNA, being more stable, is preferred for storage of genetic information. For the transmission of genetic information, RNA is better.

4. RNA World

From the above discussion, it is evident that there are some processes in the cell that seem to have evolved around RNA. RNA was the first genetic material. There is now enough evidence to suggest that essential life processes (such as metabolism, translation, splicing, etc.) evolved around RNA. RNA used to act as a genetic material as well as a catalyst (there are some important biochemical reactions in living systems that are catalysed by RNA catalysts and not by protein enzymes). But RNA being a catalyst was reactive and hence unstable. Therefore, DNA has evolved from RNA with chemical modifications that make it more stable. DNA, being double-stranded and having a complementary strand, further resists changes by evolving a process of repair.

5. Replication

While proposing the double helical structure for DNA, Watson and Crick had immediately proposed a scheme for replication of DNA. To quote their original statement: "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material". The scheme suggested that the two strands would separate and act as a template for the synthesis of new complementary strands. After the completion of replication, each DNA molecule would have one parental and one newly synthesised strand. This scheme was termed semi-conservative replication.

Experimental proof (Meselson and Stahl, 1958)

It is now proven that DNA replicates semi-conservatively. It was shown first in Escherichia coli and subsequently in higher organisms, such as plants and human cells. Matthew Meselson and Franklin Stahl performed the following experiment in 1958:

1. They grew E. coli in a medium containing ¹⁵NH₄Cl (¹⁵N is the heavy isotope of nitrogen) as the only nitrogen source for many generations. The result was that ¹⁵N was incorporated into newly synthesised DNA (as well as other nitrogen-containing compounds). This heavy DNA molecule could be distinguished from the normal DNA by centrifugation in a cesium chloride (CsCl) density gradient. (Please note that ¹⁵N is not a radioactive isotope, and it can be separated from ¹⁴N only based on densities.)
2. Then they transferred the cells into a medium with normal ¹⁴NH₄Cl and took samples at various definite time intervals as the cells multiplied, and extracted the DNA that remained as double-stranded helices. The various samples were separated independently on CsCl gradients to measure the densities of DNA.
3. Thus, the DNA that was extracted from the culture one generation after the transfer from ¹⁵N to ¹⁴N medium (that is after 20 minutes; E. coli divides in 20 minutes) had a hybrid or intermediate density. DNA extracted from the culture after another generation (that is after 40 minutes, II generation) was composed of equal amounts of this hybrid DNA and of "light" DNA.

Very similar experiments involving use of radioactive thymidine to detect distribution of newly synthesised DNA in the chromosomes were performed on Vicia faba (faba beans) by Taylor and colleagues in 1958. The experiments proved that the DNA in chromosomes also replicates semi-conservatively.

The machinery and the enzymes

In living cells, such as E. coli, the process of replication requires a set of catalysts (enzymes). The main enzyme is referred to as DNA-dependent DNA polymerase, since it uses a DNA template to catalyse the polymerisation of deoxynucleotides. These enzymes are highly efficient enzymes, as they have to catalyse polymerisation of a large number of nucleotides in a very short time. E. coli, which has only 4.6 × 10⁶ bp (compare it with human, whose diploid content is 6.6 × 10⁹ bp), completes the process of replication within 18 minutes; that means the average rate of polymerisation has to be approximately 2000 bp per second. Not only do these polymerases have to be fast, but they also have to catalyse the reaction with high degree of accuracy. Any mistake during replication would result in mutations. Furthermore, energetically replication is a very expensive process. Deoxyribonucleoside triphosphates serve the dual purposes: in addition to acting as substrates, they provide energy for the polymerisation reaction (the two terminal phosphates in deoxynucleoside triphosphates are high-energy phosphates, same as in case of ATP).

In addition to DNA-dependent DNA polymerases, many additional enzymes are required to complete the process of replication with high degree of accuracy. For long DNA molecules, since the two strands of DNA cannot be separated in its entire length (due to very high energy requirement), the replication occurs within a small opening of the DNA helix, referred to as the replication fork. The DNA-dependent DNA polymerases catalyse polymerisation only in one direction, that is 5'→3'. This creates some additional complications at the replicating fork. Consequently, on one strand (the template with polarity 3'→5'), the replication is continuous (leading strand), while on the other (the template with polarity 5'→3'), it is discontinuous (lagging strand). The discontinuously synthesised fragments (Okazaki fragments) are later joined by the enzyme DNA ligase.

The DNA polymerases on their own cannot initiate the process of replication. Also, the replication does not initiate randomly at any place in DNA. There is a definite region in E. coli DNA where the replication originates. Such regions are termed origin of replication. It is because of the requirement of the origin of replication that a piece of DNA, if needed to be propagated during recombinant DNA procedures, requires a vector. The vectors provide the origin of replication. Further, not every detail of replication is understood well. In eukaryotes, the replication of DNA takes place at the S-phase of the cell cycle. The replication of DNA and cell division cycle should be highly coordinated. A failure in cell division after DNA replication results in polyploidy (a chromosomal anomaly).

6. Transcription

The process of copying genetic information from one strand of the DNA into RNA is termed transcription. Here also, the principle of complementarity governs the process of transcription, except that adenine now forms base pair with uracil instead of thymine. However, unlike in the process of replication, which once set in, the total DNA of an organism gets duplicated, in transcription only a segment of DNA and only one of the strands is copied into RNA. This necessitates defining the boundaries that would demarcate the region and the strand of DNA that would be transcribed. Why both the strands are not copied during transcription has a simple answer. First, if both strands act as a template, they would code for RNA molecules with different sequences (remember complementarity does not mean identical), and in turn, if they code for proteins, the sequence of amino acids in the proteins would be different. Hence, one segment of the DNA would be coding for two different proteins, and this would complicate the genetic information transfer machinery. Second, the two RNA molecules if produced simultaneously would be complementary to each other, hence would form a double-stranded RNA. This would prevent RNA from being translated into protein, and the exercise of transcription would become a futile one.

Transcription unit

A transcription unit in DNA is defined primarily by three regions in the DNA: (i) a promoter, (ii) the structural gene, and (iii) a terminator. There is a convention in defining the two strands of the DNA in the structural gene of a transcription unit. Since the two strands have opposite polarity and the DNA-dependent RNA polymerase also catalyses the polymerisation in only one direction, that is, 5'→3', the strand that has the polarity 3'→5' acts as a template, and is also referred to as the template strand. The other strand, which has the polarity (5'→3') and the sequence same as RNA (except thymine at the place of uracil), is displaced during transcription. Strangely, this strand (which does not code for anything) is referred to as the coding strand. All the reference point while defining a transcription unit is made with the coding strand.

The promoter and terminator flank the structural gene in a transcription unit. The promoter is said to be located towards the 5'-end (upstream) of the structural gene (the reference is made with respect to the polarity of the coding strand). It is a DNA sequence that provides a binding site for RNA polymerase, and it is the presence of a promoter in a transcription unit that also defines the template and coding strands. By switching its position with the terminator, the definition of coding and template strands could be reversed. The terminator is located towards the 3'-end (downstream) of the coding strand and it usually defines the end of the process of transcription. There are additional regulatory sequences that may be present further upstream or downstream to the promoter.

Transcription unit and the gene

A gene is defined as the functional unit of inheritance. Though there is no ambiguity that the genes are located on the DNA, it is difficult to literally define a gene in terms of DNA sequence. The DNA sequence coding for tRNA or rRNA molecule also defines a gene. However, by defining a cistron as a segment of DNA coding for a polypeptide, the structural gene in a transcription unit could be said to be monocistronic (mostly in eukaryotes) or polycistronic (mostly in bacteria or prokaryotes). In eukaryotes, the monocistronic structural genes have interrupted coding sequences — the genes in eukaryotes are split. The coding sequences or expressed sequences are defined as exons. Exons are said to be those sequences that appear in mature or processed RNA. The exons are interrupted by introns. Introns or intervening sequences do not appear in mature or processed RNA. The split-gene arrangement further complicates the definition of a gene in terms of a DNA segment. Inheritance of a character is also affected by promoter and regulatory sequences of a structural gene. Hence, sometimes the regulatory sequences are loosely defined as regulatory genes, even though these sequences do not code for any RNA or protein.

Types of RNA and the process of transcription

In bacteria, there are three major types of RNAs: mRNA (messenger RNA), tRNA (transfer RNA) and rRNA (ribosomal RNA). All three RNAs are needed to synthesise a protein in a cell. The mRNA provides the template, tRNA brings amino acids and reads the genetic code, and rRNAs play structural and catalytic roles during translation. There is a single DNA-dependent RNA polymerase that catalyses transcription of all types of RNA in bacteria. RNA polymerase binds to the promoter and initiates transcription (initiation). It uses nucleoside triphosphates as substrate and polymerises in a template-dependent fashion following the rule of complementarity. It somehow also facilitates opening of the helix and continues elongation. Only a short stretch of RNA remains bound to the enzyme. Once the polymerase reaches the terminator region, the nascent RNA falls off, so also the RNA polymerase. This results in termination of transcription.

An intriguing question is how the RNA polymerases are able to catalyse all the three steps, which are initiation, elongation and termination. The RNA polymerase is only capable of catalysing the process of elongation. It associates transiently with initiation-factor (σ) and termination-factor (ρ) to initiate and terminate the transcription, respectively. Association with these factors alters the specificity of the RNA polymerase to either initiate or terminate. In bacteria, since the mRNA does not require any processing to become active, and also since transcription and translation take place in the same compartment (there is no separation of cytosol and nucleus in bacteria), many times the translation can begin much before the mRNA is fully transcribed. Consequently, the transcription and translation can be coupled in bacteria.

Transcription in eukaryotes

In eukaryotes, there are two additional complexities: (i) There are at least three RNA polymerases in the nucleus (in addition to the RNA polymerase found in the organelles). There is a clear-cut division of labour. The RNA polymerase I transcribes rRNAs (28S, 18S and 5.8S), whereas RNA polymerase III is responsible for transcription of tRNA, 5S rRNA and snRNAs (small nuclear RNAs). The RNA polymerase II transcribes precursor of mRNA, the heterogeneous nuclear RNA (hnRNA). (ii) The second complexity is that the primary transcripts contain both the exons and the introns and are non-functional. Hence, it is subjected to a process called splicing, where the introns are removed and exons are joined in a defined order. hnRNA undergoes two additional processes called capping and tailing. In capping, an unusual nucleotide (methyl guanosine triphosphate) is added to the 5'-end of hnRNA. In tailing, adenylate residues (200–300) are added at the 3'-end in a template-independent manner. It is the fully processed hnRNA, now called mRNA, that is transported out of the nucleus for translation.

The significance of such complexities is now beginning to be understood. The split-gene arrangements represent probably an ancient feature of the genome. The presence of introns is reminiscent of antiquity, and the process of splicing represents the dominance of RNA-world. In recent times, the understanding of RNA and RNA-dependent processes in the living system have assumed more importance.

7. Genetic Code

During replication and transcription, a nucleic acid was copied to form another nucleic acid. Hence, these processes are easy to conceptualise on the basis of complementarity. The process of translation requires transfer of genetic information from a polymer of nucleotides to a polymer of amino acids. Neither does any complementarity exist between nucleotides and amino acids, nor could any be drawn theoretically. There existed ample evidence, though, to support the notion that change in nucleic acids (genetic material) were responsible for change in amino acids in proteins. This led to the proposition of a genetic code that could direct the sequence of amino acids during synthesis of proteins.

If determining the biochemical nature of genetic material and the structure of DNA was very exciting, the proposition and deciphering of the genetic code were most challenging. In a very true sense, it required involvement of scientists from several disciplines — physicists, organic chemists, biochemists and geneticists. It was George Gamow, a physicist, who argued that since there are only 4 bases and if they have to code for 20 amino acids, the code should constitute a combination of bases. He suggested that in order to code for all the 20 amino acids, the code should be made up of three nucleotides. This was a very bold proposition, because a permutation combination of 4³ (4 × 4 × 4) would generate 64 codons, generating many more codons than required. Providing proof that the codon was a triplet was a more daunting task. The chemical method developed by Har Gobind Khorana was instrumental in synthesising RNA molecules with defined combinations of bases (homopolymers and copolymers). Marshall Nirenberg's cell-free system for protein synthesis finally helped the code to be deciphered. Severo Ochoa enzyme (polynucleotide phosphorylase) was also helpful in polymerising RNA with defined sequences in a template-independent manner (enzymatic synthesis of RNA).

Salient features of the genetic code

1. The codon is a triplet. 61 codons code for amino acids and 3 codons do not code for any amino acids, hence they function as stop codons.
2. One codon codes for only one amino acid; hence, it is unambiguous and specific.
3. Some amino acids are coded by more than one codon; hence the code is degenerate.
4. The codon is read in mRNA in a contiguous fashion. There are no punctuations.
5. The code is nearly universal: for example, from bacteria to human UUU would code for phenylalanine (phe). Some exceptions to this rule have been found in mitochondrial codons and in some protozoans.
6. AUG has dual functions. It codes for methionine (met), and it also acts as the initiator codon.
7. UAA, UAG, UGA are stop terminator codons.

tRNA — the adapter molecule

From the very beginning of the proposition of code, it was clear to Francis Crick that there has to be a mechanism to read the code and also to link it to the amino acids, because amino acids have no structural specialities to read the code uniquely. He postulated the presence of an adapter molecule that would on one hand read the code and on the other hand would bind to specific amino acids. The tRNA, then called sRNA (soluble RNA), was known before the genetic code was postulated. However, its role as an adapter molecule was assigned much later. tRNA has an anticodon loop that has bases complementary to the code, and it also has an amino acid acceptor end to which it binds to amino acids. tRNAs are specific for each amino acid. For initiation, there is another specific tRNA that is referred to as initiator tRNA. There are no tRNAs for stop codons. In the figure, the secondary structure of tRNA has been depicted that looks like a clover-leaf. In actual structure, the tRNA is a compact molecule which looks like an inverted L.

8. Translation

Translation refers to the process of polymerisation of amino acids to form a polypeptide. The order and sequence of amino acids are defined by the sequence of bases in the mRNA. The amino acids are joined by a bond which is known as a peptide bond. Formation of a peptide bond requires energy. Therefore, in the first phase itself, amino acids are activated in the presence of ATP and linked to their cognate tRNA — a process commonly called charging of tRNA or aminoacylation of tRNA, to be more specific. If two such charged tRNAs are brought close enough, the formation of peptide bond between them would be favoured energetically. The presence of a catalyst would enhance the rate of peptide bond formation.

The cellular factory responsible for synthesising proteins is the ribosome. The ribosome consists of structural RNAs and about 80 different proteins. In its inactive state, it exists as two subunits — a large subunit and a small subunit. When the small subunit encounters an mRNA, the process of translation of the mRNA to protein begins. There are two sites in the large subunit, for subsequent amino acids to bind to and thus be close enough to each other for the formation of a peptide bond. The ribosome also acts as a catalyst for the formation of peptide bond. For example, 23S rRNA in bacteria is the enzyme — ribozyme.

A translational unit in mRNA is the sequence of RNA that is flanked by the start codon (AUG) and the stop codon and codes for a polypeptide. An mRNA also has some additional sequences that are not translated and are referred to as untranslated regions (UTR). The UTRs are present at both 5'-end (before start codon) and at 3'-end (after stop codon). They are required for efficient translation process.

• Initiation: the ribosome binds to the mRNA at the start codon (AUG) that is recognised only by the initiator tRNA.
• Elongation: the ribosome proceeds to the elongation phase of protein synthesis. During this stage, complexes composed of an amino acid linked to tRNA sequentially bind to the appropriate codon in mRNA by forming complementary base pairs with the tRNA anticodon. The ribosome moves from codon to codon along the mRNA. Amino acids are added one by one, translated into polypeptide sequences dictated by DNA and represented by mRNA.
• Termination: at the end, a release factor binds to the stop codon, terminating translation and releasing the complete polypeptide from the ribosome.

9. Regulation of Gene Expression

Regulation of gene expression refers to a very broad term that may occur at various levels. Considering that gene expression results in the formation of a polypeptide, it can be regulated at several levels. In eukaryotes, the regulation could be exerted at (i) transcriptional level (formation of primary transcript), (ii) processing level (regulation of splicing), (iii) transport of mRNA from nucleus to the cytoplasm, (iv) translational level.

The genes in a cell are expressed to perform a particular function or a set of functions. For example, if an enzyme called beta-galactosidase is synthesised by E. coli, it is used to catalyse the hydrolysis of a disaccharide, lactose, into galactose and glucose; the bacteria use them as a source of energy. Hence, if the bacteria do not have lactose around them to be utilised for energy source, they would no longer require the synthesis of the enzyme beta-galactosidase. Therefore, in simple terms, it is the metabolic, physiological or environmental conditions that regulate the expression of genes. The development and differentiation of embryo into adult organisms are also a result of the coordinated regulation of expression of several sets of genes.

In prokaryotes, control of the rate of transcriptional initiation is the predominant site for control of gene expression. In a transcription unit, the activity of RNA polymerase at a given promoter is in turn regulated by interaction with accessory proteins, which affect its ability to recognise start sites. These regulatory proteins can act both positively (activators) and negatively (repressors). The accessibility of promoter regions of prokaryotic DNA is in many cases regulated by the interaction of proteins with sequences termed operators. The operator region is adjacent to the promoter elements in most operons, and in most cases the sequences of the operator bind a repressor protein. Each operon has its specific operator and specific repressor. For example, the lac operator is present only in the lac operon, and it interacts specifically with the lac repressor only.

The lac operon

The elucidation of the lac operon was also a result of a close association between a geneticist, Francois Jacob, and a biochemist, Jacque Monod. They were the first to elucidate a transcriptionally regulated system. In lac operon (here lac refers to lactose), a polycistronic structural gene is regulated by a common promoter and regulatory genes. Such arrangement is very common in bacteria and is referred to as operon. To name a few such examples, lac operon, trp operon, ara operon, his operon, val operon, etc.

The lac operon consists of one regulatory gene (the i gene — here the term i does not refer to inducer, rather it is derived from the word inhibitor) and three structural genes (z, y and a). The i gene codes for the repressor of the lac operon. The z gene codes for beta-galactosidase (β-gal), which is primarily responsible for the hydrolysis of the disaccharide, lactose into its monomeric units, galactose and glucose. The y gene codes for permease, which increases permeability of the cell to β-galactosides. The a gene encodes a transacetylase. Hence, all the three gene products in lac operon are required for metabolism of lactose. In most other operons as well, the genes present in the operon are needed together to function in the same or related metabolic pathway.

Lactose is the substrate for the enzyme beta-galactosidase and it regulates switching on and off of the operon. Hence, it is termed as inducer. In the absence of a preferred carbon source such as glucose, if lactose is provided in the growth medium of the bacteria, the lactose is transported into the cells through the action of permease (remember, a very low level of expression of lac operon has to be present in the cell all the time, otherwise lactose cannot enter the cells). The lactose then induces the operon in the following manner.

The repressor of the operon is synthesised (all the time — constitutively) from the i gene. The repressor protein binds to the operator region of the operon and prevents RNA polymerase from transcribing the operon. In the presence of an inducer, such as lactose or allolactose, the repressor is inactivated by interaction with the inducer. This allows RNA polymerase access to the promoter, and transcription proceeds. Essentially, regulation of lac operon can also be visualised as regulation of enzyme synthesis by its substrate. Remember, glucose or galactose cannot act as inducers for lac operon. Regulation of lac operon by repressor is referred to as negative regulation. Lac operon is under control of positive regulation as well, but it is beyond the scope of discussion at this level.

10. Human Genome Project

In the preceding sections you have learnt that it is the sequence of bases in DNA that determines the genetic information of a given organism. In other words, genetic make-up of an organism or individual lies in the DNA sequences. If two individuals differ, then their DNA sequences should also be different, at least at some places. These assumptions led to the quest of finding out the complete DNA sequence of the human genome. With the establishment of genetic engineering techniques where it was possible to isolate and clone any piece of DNA and availability of simple and fast techniques for determining DNA sequences, a very ambitious project of sequencing the human genome was launched in the year 1990.

The Human Genome Project (HGP) was called a mega project. You can imagine the magnitude and the requirements for the project if we simply define the aims of the project: Human genome is said to have approximately 3 × 10⁹ bp, and if the cost of sequencing required is US $3 per bp (the estimated cost in the beginning), the total estimated cost of the project would be approximately 9 billion US dollars. Further, if the obtained sequences were to be stored in typed form in books, and if each page of the book contained 1000 letters and each book contained 1000 pages, then 3300 such books would be required to store the information of DNA sequence from a single human cell. The enormous amount of data expected to be generated also necessitated the use of high-speed computational devices for data storage and retrieval, and analysis. HGP was closely associated with the rapid development of a new area in biology called Bioinformatics.

Goals of HGP — some of the important goals of HGP were: (i) identify all the approximately 20,000–25,000 genes in human DNA; (ii) determine the sequences of the 3 billion chemical base pairs that make up human DNA; (iii) store this information in databases; (iv) improve tools for data analysis; (v) transfer related technologies to other sectors, such as industries; (vi) address the ethical, legal and social issues (ELSI) that may arise from the project.

The Human Genome Project was a 13-year project coordinated by the U.S. Department of Energy and the National Institute of Health. During the early years of the HGP, the Wellcome Trust (U.K.) became a major partner; additional contributions came from Japan, France, Germany, China and others. The project was completed in 2003. Knowledge about the effects of DNA variations among individuals can lead to revolutionary new ways to diagnose, treat and someday prevent the thousands of disorders that affect human beings. Besides providing clues to understanding human biology, learning about non-human organisms' DNA sequences can lead to an understanding of their natural capabilities that can be applied toward solving challenges in health care, agriculture, energy production, environmental remediation. Many non-human model organisms, such as bacteria, yeast, Caenorhabditis elegans (a free-living non-pathogenic nematode), Drosophila (the fruit fly), plants (rice and Arabidopsis), etc., have also been sequenced.

Methodologies: the methods involved two major approaches. One approach focused on identifying all the genes that are expressed as RNA (referred to as Expressed Sequence Tags (ESTs)). The other took the blind approach of simply sequencing the whole set of genome that contained all the coding and non-coding sequence, and later assigning different regions in the sequence with functions (a term referred to as Sequence Annotation). For sequencing, the total DNA from a cell is isolated and converted into random fragments of relatively smaller sizes (recall DNA is a very long polymer, and there are technical limitations in sequencing very long pieces of DNA) and cloned in suitable host using specialised vectors. The cloning resulted in amplification of each piece of DNA fragment so that it subsequently could be sequenced with ease. The commonly used hosts were bacteria and yeast, and the vectors were called as BAC (bacterial artificial chromosomes) and YAC (yeast artificial chromosomes). The fragments were sequenced using automated DNA sequencers that worked on the principle of a method developed by Frederick Sanger. These sequences were then arranged based on some overlapping regions present in them. This required generation of overlapping fragments for sequencing. Alignment of these sequences was humanly not possible. Therefore, specialised computer-based programmes were developed. These sequences were subsequently annotated and were assigned to each chromosome. The sequence of chromosome 1 was completed only in May 2006 (this was the last of the 24 human chromosomes — 22 autosomes and X and Y — to be sequenced).

11. DNA Fingerprinting

As stated in the preceding section, 99.9 per cent of base sequence among humans is the same. Assuming human genome as 3 × 10⁹ bp, in how many base sequences would there be differences? It is these differences in sequence of DNA which make every individual unique in their phenotypic appearance. If one aims to find out genetic differences between two individuals or among individuals of a population, sequencing the DNA every time would be a daunting and expensive task. Imagine trying to compare two sets of 3 × 10⁶ base pairs. DNA fingerprinting is a very quick way to compare the DNA sequences of any two individuals.

DNA fingerprinting involves identifying differences in some specific regions in DNA sequence called repetitive DNA, because in these sequences a small stretch of DNA is repeated many times. These repetitive DNA are separated from bulk genomic DNA as different peaks during density gradient centrifugation. The bulk DNA forms a major peak and the other small peaks are referred to as satellite DNA. Depending on base composition (A:T rich or G:C rich), length of segment, and number of repetitive units, the satellite DNA is classified into many categories, such as micro-satellites, mini-satellites etc. These sequences normally do not code for any proteins, but they form a large portion of the human genome. These sequences show a high degree of polymorphism and form the basis of DNA fingerprinting. Since DNA from every tissue (such as blood, hair-follicle, skin, bone, saliva, sperm etc.) from an individual show the same degree of polymorphism, they become very useful identification tools in forensic applications. Further, as the polymorphisms are inheritable from parents to children, DNA fingerprinting is the basis of paternity testing, in case of disputes.

As polymorphism in DNA sequence is the basis of genetic mapping of the human genome as well as of DNA fingerprinting, it is essential that we understand what DNA polymorphism means in simple terms. Polymorphism (variation at genetic level) arises due to mutations. New mutations may arise in an individual either in somatic cells or in the germ cells. If a germ cell mutation does not seriously impair an individual's ability to have offspring who can transmit the mutation, it can spread to the other members of the population (through sexual reproduction). Allelic sequence variation has traditionally been described as a DNA polymorphism if more than one variant (allele) at a locus occurs in a human population with a frequency greater than 0.01. In simple terms, if an inheritable mutation is observed in a population at high frequency, it is referred to as DNA polymorphism. The probability of such variation is higher in non-coding DNA sequences, as mutations in these sequences may not have any immediate effect or impact on an individual's reproductive ability.

The technique of DNA fingerprinting was initially developed by Alec Jeffreys. He used a satellite DNA as a probe that shows a very high degree of polymorphism. It was called Variable Number of Tandem Repeats (VNTR). The technique, as used earlier, involved Southern blot hybridisation using radiolabelled VNTR as a probe. It included: (i) isolation of DNA; (ii) digestion of DNA by restriction endonucleases; (iii) separation of DNA fragments by electrophoresis; (iv) transferring (blotting) of separated DNA fragments to synthetic membranes, such as nitrocellulose or nylon; (v) hybridisation using labelled VNTR probe; and (vi) detection of hybridised DNA fragments by autoradiography.

The VNTR belongs to a class of satellite DNA referred to as mini-satellite. A small DNA sequence is arranged tandemly in many copy numbers. The copy number varies from chromosome to chromosome in an individual. The numbers of repeats show a very high degree of polymorphism. As a result, the size of VNTR varies in size from 0.1 to 20 kb. Consequently, after hybridisation with VNTR probe, the autoradiogram gives many bands of differing sizes. These bands give a characteristic pattern for an individual DNA. It differs from individual to individual in a population except in the case of monozygotic (identical) twins. The sensitivity of the technique has been increased by use of polymerase chain reaction (PCR). Consequently, DNA from a single cell is enough to perform DNA fingerprinting analysis. In addition to application in forensic science, it has much wider application, such as in determining population and genetic diversities. Currently, many different probes are used to generate DNA fingerprints.

12. Master Summary Table

13. Mind Map

14. Tips and Tricks

15. Important One-liners

• Friedrich Meischer named DNA "nuclein" in 1869.
• Histones are rich in lysine and arginine.
• TMV and QB bacteriophage have RNA as genetic material.
• RNA was the first genetic material (RNA world).
• Taylor proved semi-conservative replication in Vicia faba chromosomes.
• Replication in eukaryotes occurs in the S-phase.
• George Gamow proposed the triplet code.
• Khorana synthesised RNA with defined sequences; Nirenberg used a cell-free system.
• tRNA is clover-leaf in 2-D and inverted L in 3-D.
• 23S rRNA acts as a ribozyme in bacteria.
• Chromosome 1 was the last human chromosome sequenced (May 2006).
• VNTR sizes vary from 0.1 to 20 kb.

16. NEET-pattern Questions with Detailed Answers

These questions are original and based on concepts that NEET UG asks again and again.

17. Quick Revision Summary